WO2012049784A1 - Substrat pour la mesure de la migration cellulaire, et procédé d'examen de la migration cellulaire - Google Patents
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M39/00—Means for cleaning the apparatus or avoiding unwanted deposits of microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5029—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell motility
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5088—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above confining liquids at a location by surface tension, e.g. virtual wells on plates, wires
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1027—Determining speed or velocity of a particle
Definitions
- the present invention relates to a cell culture substrate and a cell migration test method using the cell culture substrate.
- Some cells have an adhesion dependency that grows by adhering to something, and cannot survive for a long time in a floating state in vitro.
- a carrier for cell adhesion is required, and generally, a plastic culture in which cell adhesion proteins such as collagen and fibronectin are uniformly applied. A dish is used. These cell adhesion proteins are known to act on cultured cells, facilitate cell adhesion, and affect cell morphology.
- cell migration is involved in various stages such as immune response, embryo morphogenesis after fertilization, tissue repair and regeneration. In addition, it plays an extremely important role in the progression of diseases such as cancer, atherosclerosis and arthritis. Specifically, cell migration through the vascular endothelium is an important phenomenon in the pathophysiology of conditions such as inflammation, atherosclerosis, and cancer metastasis. Therefore, methods for measuring cell migration in vitro have been developed for many years.
- Non-Patent Document 1 describes a technique for controlling cell adhesion and non-adhesion by applying a voltage on a conductive substrate whose entire surface is not patterned.
- the properties of the entire surface of the substrate change due to the application of voltage, so after attaching cells on the substrate, only a specific region is changed to cell adhesion, and only in that region. Cells cannot be controlled to migrate.
- Non-Patent Document 2 discloses that a cell adhesion-inhibiting film is formed on a substrate having a conductive region and an insulating region, and the cell adhesion-inhibiting film is attached to the cell by applying a voltage to a specific conductive region. A method is described in which the cells are cultured in such a manner that the cells are adhered to only the region after being modified to sex. However, the cell adhesive region and the cell adhesion inhibitory region are not adjacent to each other on the conductive region of the substrate of Non-Patent Document 2.
- An object of the present invention is to provide means for testing cell migration by causing cells adhered on a substrate to migrate to a specific region.
- the present inventors have formed a cell adhesion-inhibiting region in a region adjacent to the cell adhesive region by adopting two different types of means for modifying the cell adhesion-inhibiting membrane into a pattern-like shape. Furthermore, the present inventors have found that the cell adhesion-inhibiting region can be changed to a cell adhesion region, thereby allowing cells to be controlled and migrated to a specific region.
- the present invention includes the following inventions.
- Substrate having a conductive region and an insulating region, a cell adhesion region and a cell adhesion inhibitory region formed on the conductive region, and a cell adhesion inhibitor formed on the insulating region
- a cell culture substrate comprising a cell region, wherein the cell adhesive region and the cell adhesion inhibitory region are adjacent to each other on the conductive region.
- a substrate for cell culture comprising a base material having a conductive region and an insulating region, and a cell adhesive region and a cell adhesion inhibitory region formed on the conductive region and the insulating region, respectively.
- the cell adhesive region and the cell adhesion inhibitory region are adjacent to each other on the conductive region, and the cell adhesive region and the cell adhesion inhibitory region are adjacent to each other on the insulating region (1 )
- the substrate for cell culture according to the above.
- the cell adhesion region is a cell adhesion inhibitory region obtained by subjecting a cell adhesion inhibitory hydrophilic membrane containing an organic compound having a carbon-oxygen bond to oxidation treatment and / or degradation treatment to make it cell adhesive.
- a method for testing cell migration (I) a step of seeding cells on the cell culture substrate according to any one of (1) to (10) and allowing the cells to adhere to the cell adhesion region; (Ii) changing the cell adhesion-inhibiting region on the conductive region into a cell adhesive region by applying a voltage to the conductive region; and (iii) adhering to the cell adhesive region on the conductive region. And observing the movement of the cells to the region modified to the cell adhesion region in step (ii).
- the conductive region includes a linear conductive region, Insulating regions are arranged on both sides along the length direction of the linear conductive region, A cell adhesive region is formed so as to include all or a part of each position of the linear conductive region at one or two or more positions of the linear conductive region at different positions in the length direction.
- the cell adhesive region further includes a portion of an insulating region adjacent to the portion, The part not included in the cell adhesive region of the linear conductive region and the insulating region disposed on both sides along the length direction of the linear conductive region is a cell adhesion inhibitory region,
- the dimension of each cell adhesive region in the linear conductive region length direction is defined as the length of the cell adhesive region
- the dimension perpendicular to the length direction is defined as the width of the cell adhesive region
- each cell The length and width of the adhesive region are each independently 1 to 500 ⁇ m
- the line width of the portion not included in the cell adhesive region is 0.1 to 10 ⁇ m, and the line width is larger than the width of the cell adhesive region connected to the portion.
- a method for testing cells (I) a step of seeding cells on the cell culture substrate according to (14), and attaching the cells to the cell adhesion region; (Ii) a step of changing a cell adhesion-inhibiting region of the linear conductive region to a cell adhesive region by applying a voltage to the linear conductive region; and (iii) a cell adhered to the cell adhesive region. Observing the method.
- a method of forming a circuit-like cell construct (I) a step of seeding cells on the cell culture substrate according to (15) and attaching the cells to the cell adhesion region; (Ii) a step of modifying the cell adhesion-inhibiting region of the linear conductive region into a cell adhesive region by applying a voltage to the linear conductive region; and (iii) cell adhesion by culturing the cell.
- the present invention provides a system capable of easily assaying cell migration of cells that migrate while adhering to a carrier, and can be used for screening in the drug discovery industry.
- the substrate for cell culture of the present invention includes a base material having a conductive region (conductive portion) and an insulating region (insulating portion), and a cell adhesive region and a cell formed on the conductive region (conductive portion).
- a cell culture substrate comprising an adhesion-inhibiting region and a cell-adhesion-inhibiting region formed on the insulating region (insulating part), the cell-adhering region and the cell adhesion on the conductive region (conducting part) It is characterized in that the inhibitory region is adjacent.
- the cell adhesion region and the cell adhesion inhibition region are arranged in a pattern.
- a cell adhesive region and a cell adhesion inhibitory region are formed on the insulating region (insulating part), and the cell adhesive region and the cell adhesion inhibitory region are adjacent to each other on the insulating region (insulating part). In some cases, it is preferable.
- the cell adhesiveness means that cells adhere or cells adhere easily.
- Cell adhesion inhibitory means that cells are difficult to adhere or cells do not adhere. Therefore, when cells are seeded on a substrate on which a cell adhesion region and a cell adhesion inhibition region are patterned, cells adhere to the cell adhesion region, but cells do not adhere to the cell adhesion inhibition region.
- the cells are arranged in a pattern on the substrate surface.
- cell adhesion may vary depending on the cells to be adhered, cell adhesion means cell adhesion to certain types of cells. Therefore, on the cell culture substrate, there may be a plurality of cell adhesion regions for a plurality of types of cells, that is, there may be two or more levels of cell adhesion regions having different cell adhesion properties.
- Examples of the structure of the cell adhesion region and the cell adhesion inhibition region in the cell culture substrate of the present invention include the following two forms.
- the first form is a form in which the cell adhesion region is a cell adhesion region obtained by subjecting a cell adhesion-inhibiting hydrophilic membrane containing an organic compound having a carbon-oxygen bond to oxidation treatment and / or degradation treatment. is there.
- a cell adhesion-inhibiting hydrophilic film containing an organic compound having a carbon-oxygen bond is formed on the entire surface of the substrate, and then an oxidation treatment and / or a degradation treatment are performed on a region where cell adhesion is desired.
- the part not subjected to the treatment is a cell adhesion-inhibiting region.
- the second form is a form in which the cell adhesion region is formed of a hydrophilic film containing an organic compound having a carbon-oxygen bond at a low density.
- This form utilizes the fact that a hydrophilic film containing an organic compound having a carbon-oxygen bond at a high density has cell adhesion inhibitory properties, whereas a hydrophilic film containing the compound at a low density has a cell adhesion property. It is a thing.
- a first region where the compound is likely to bind to the substrate surface and a second region where the compound is difficult to bind are provided on the substrate surface, and a film of the compound is formed on the substrate surface, the first region becomes a cell adhesion inhibitory region, The region becomes a cell adhesive region.
- the cell adhesion-inhibiting region on the conductive region can be altered to cell adhesion by applying a voltage to the conductive region, preferably applying a positive voltage.
- a voltage preferably applying a positive voltage.
- cell adhesion obtained by subjecting a cell adhesion-inhibiting hydrophilic membrane containing an organic compound having a carbon-oxygen bond to oxidation treatment and / or degradation treatment is applied to the region modified to cell adhesion by application of voltage.
- the surface area may be different from the property region.
- the base material used for the cell culture substrate of the present invention is not particularly limited as long as it is formed of a material capable of forming a conductive region and an insulating region. It is preferable to form a conductive region and an insulating region by forming a conductive region on a base material made of an insulating material. Moreover, it is preferable that it is formed with the material which can form the film of the organic compound which has a carbon oxygen bond on the surface.
- glass, quartz glass, borosilicate glass, alumina, sapphire, ceramics, forsterite, photosensitive glass, ceramic, silicon, elastomer, plastic for example, polyester resin, polyethylene resin, polypropylene resin, ABS resin, nylon
- plastic for example, polyester resin, polyethylene resin, polypropylene resin, ABS resin, nylon
- the shape is not limited, for example, a flat shape such as a flat plate, a flat membrane, a film, a porous membrane, a three-dimensional shape such as a cylinder, a stamp, a multiwell plate, a microchannel, and irregularities are formed on the surface. Shape.
- its thickness is not particularly limited, but is usually 0.1 to 1000 ⁇ m, preferably 1 to 500 ⁇ m, more preferably 10 to 200 ⁇ m.
- the fine unevenness means a depth of 1 nm to 10 ⁇ m, a width of a line protrusion of 1 nm to 10 ⁇ m, and a width of a line recess of 1 nm to 10 ⁇ m.
- a known patterning technique can be used.
- Known patterning techniques include, for example, gravure printing methods, screen printing methods, offset printing methods, flexographic printing methods, contact printing methods and other printing methods, various lithography methods, ink jet methods, and the like.
- a three-dimensional shaping technique such as engraving a fine groove.
- a conductive material such as a metal film or a metal oxide film is formed on a base material made of an insulating material, such as a glass base material, and is patterned using a known technique such as a photolithography technique.
- a conductive region and an insulating region can be formed.
- the film formation of the conductive material on the substrate can be performed by a known method.
- microwave plasma CVD Chemical vapor deposition
- ECRCVD Electrocal cyclotron deposition
- ICP Inductive coupled plasma
- direct current sputtering method ICP (Inductive coupled plasma) method
- direct current sputtering method examples thereof include an arc type vapor deposition method, a laser vapor deposition method, an EB (Electron beam) vapor deposition method, and a resistance heating vapor deposition method.
- the film formation may be performed by coating. Spin coating and various printing methods can also be used.
- Examples of the conductive material film constituting the conductive region include a metal film or a metal oxide film, a film in which metal fine particles or metal nanofibers are dispersed in an insulator, a film made of a conductive organic material, and the like.
- Examples of the metal oxide include ITO (indium tin oxide) and IZO (indium zinc oxide).
- Examples of the metal fine particles include fine particles such as silver, gold, and copper.
- Examples of the metal nanofibers include carbon nanotubes.
- Examples of the material include polyethylene dioxythiophene (PEDOT).
- the conductive material film is not particularly limited, but is preferably a transparent film, and examples thereof include an ITO film, an IZO film, and a polyethylenedioxythiophene film of a conductive polymer. Further, it is preferable that the film is transparent even after voltage application. In the present invention, it is preferable to form the conductive region by forming an ITO film by sputtering and then patterning. A transparent membrane is advantageous in observing cells.
- the thickness of the conductive material film is usually from a monomolecular film to about 100 ⁇ m, preferably from 2 nm to 1 ⁇ m, more preferably from 5 nm to 500 nm.
- the conductive region is preferably patterned into a comb shape composed of a plurality of comb teeth and a base that supports each comb.
- the width of the comb teeth formed by the conductive region is preferably Is 0.1 to 500 ⁇ m, and preferably 5 to 500 ⁇ m, more preferably 10 to 200 ⁇ m for observing cell migration.
- the width of the comb teeth portion is too narrow, the electric resistance in the length direction of the comb teeth portion will increase, and when a constant voltage is applied, a voltage drop tends to occur in the length direction of the comb teeth portion, and the potential control is possible. It becomes difficult. Since the degree of the voltage drop varies depending on the conductivity of the conductive material to be used, the lower limit of the width of the comb tooth portion is appropriately determined according to the conductivity of the conductive material, but is generally preferably 5 ⁇ m or more.
- the width of the conductive region on the base material is narrowed to 5 ⁇ m or less, a wide electrode is provided on the substrate, a part of the surface of the electrode is covered with an insulating film, and a desired thickness of 5 ⁇ m or less is provided.
- a wide electrode is provided on the substrate, a part of the surface of the electrode is covered with an insulating film, and a desired thickness of 5 ⁇ m or less is provided.
- the width of the comb teeth portion When the width of the comb teeth portion is wide, the number of comb teeth entering the field of view when observing with a microscope is reduced, which is disadvantageous for statistical processing. Generally, since the field of view of a microscope is several mm or less, in order for at least one comb tooth part to enter the field of view, the width of the comb tooth part is preferably 500 ⁇ m or less. In addition, when the width of the comb-tooth portion is moderately narrow, the migration direction of the cell to be measured is limited, so that the migration distance after a lapse of a certain time is increased, which has an advantageous effect on measurement.
- the interval between the comb teeth is preferably 10 to 1000 ⁇ m, more preferably 50 to 500 ⁇ m.
- the distance between the comb teeth is 10 ⁇ m or more, more preferably 50 ⁇ m or more. If the interval between the comb teeth is too wide, the number of comb teeth entering the field of view when observing with a microscope is reduced, which is disadvantageous for statistical processing. In general, since the field of view of a microscope is several mm or less, the width of the comb teeth is preferably 1000 ⁇ m or less. Two comb-shaped patterns in which the comb teeth and the comb teeth are engaged with each other are particularly preferable. Such a pattern is preferable because a current flows effectively when a voltage is applied in the cell migration test.
- the patterning of the conductive region as described above can be performed by performing resist coating, exposure through a photomask, development and etching on the formed metal film or metal oxide film.
- the cell adhesion-inhibiting region is preferably formed by a hydrophilic film formed of an organic compound having a carbon-oxygen bond.
- the hydrophilic film is a thin film mainly composed of an organic compound having a carbon-oxygen bond, which has water solubility and water swellability, and has a cell adhesion inhibitory property before being oxidized and is oxidized and / or decomposed. There is no particular limitation as long as it has cell adhesion.
- the carbon-oxygen bond means a bond formed between carbon and oxygen, and is not limited to a single bond but may be a double bond.
- Examples of the carbon-oxygen bond include a C—O bond, a C ( ⁇ O) —O bond, and a C ⁇ O bond.
- main raw materials include water-soluble polymers, water-soluble oligomers, water-soluble organic compounds, surfactants, amphiphiles, etc., which are physically or chemically cross-linked with each other to form a physical bond with the substrate. Or it becomes a hydrophilic thin film by combining chemically.
- Specific water-soluble polymer materials include polyalkylene glycol and derivatives thereof, polyacrylic acid and derivatives thereof, polymethacrylic acid and derivatives thereof, polyacrylamide and derivatives thereof, polyvinyl alcohol and derivatives thereof, and zwitterionic polymers. , Polysaccharides, and the like. Examples of the molecular shape include a straight chain, a branched one, and a dendrimer.
- polyethylene glycol a copolymer of polyethylene glycol and polypropylene glycol, such as Pluronic F108, Pluronic F127, poly (N-isopropylacrylamide), poly (N-vinyl-2-pyrrolidone), poly (2- Hydroxyethyl methacrylate), poly (methacryloyloxyethylphosphorylcholine), copolymers of methacryloyloxyethylphosphorylcholine and acrylic monomers, dextran, and heparin, but are not limited thereto.
- Pluronic F108 Pluronic F127
- poly (N-vinyl-2-pyrrolidone) poly (2- Hydroxyethyl methacrylate
- poly (methacryloyloxyethylphosphorylcholine) copolymers of methacryloyloxyethylphosphorylcholine and acrylic monomers, dextran, and heparin, but are not limited thereto
- Specific water-soluble oligomer materials and water-soluble low molecular weight compounds include alkylene glycol oligomers and derivatives thereof, acrylic acid oligomers and derivatives thereof, methacrylic acid oligomers and derivatives thereof, acrylamide oligomers and derivatives thereof, saponified vinyl acetate oligomers and Derivatives thereof, oligomers composed of zwitterionic monomers and derivatives thereof, acrylic acid and derivatives thereof, methacrylic acid and derivatives thereof, acrylamide and derivatives thereof, zwitterionic compounds, water-soluble silane coupling agents, water-soluble thiol compounds, etc. be able to.
- ethylene glycol oligomer (N-isopropylacrylamide) oligomer, methacryloyloxyethylphosphorylcholine oligomer, low molecular weight dextran, low molecular weight heparin, oligoethylene glycol thiol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene Examples include, but are not limited to, glycol, 2- [methoxy (polyethyleneoxy) -propyltrimethoxysilane, and triethyleneglycol-terminated-thiol.
- the hydrophilic film has a high cell adhesion inhibitory property before treatment and exhibits cell adhesion after oxidation treatment and / or degradation treatment.
- the average thickness of the hydrophilic film is preferably 0.8 nm to 500 ⁇ m, more preferably 0.8 nm to 100 ⁇ m, more preferably 1 nm to 10 ⁇ m, and most preferably 1.5 nm to 1 ⁇ m. If the average thickness is 0.8 nm or more, it is preferable because protein adsorption and cell adhesion are not easily affected by the region not covered with the hydrophilic thin film on the substrate surface. Moreover, if the average thickness is 500 ⁇ m or less, coating is relatively easy.
- the hydrophilic film can be formed on the substrate surface by directly adsorbing the hydrophilic organic compound on the substrate, coating the hydrophilic organic compound directly on the substrate, or coating the hydrophilic organic compound on the substrate.
- a method of performing a crosslinking treatment later, a method of forming a hydrophilic thin film in a multi-stage manner in order to increase the adhesion to the substrate, a base layer is formed on the substrate in order to increase the adhesion to the substrate examples thereof include a method of coating a hydrophilic organic compound, a method of forming a polymerization initiation point on the substrate surface, and then polymerizing a hydrophilic polymer brush.
- particularly preferable methods include a method of forming a hydrophilic thin film in a multistage manner, and a base layer is formed on the substrate in order to improve adhesion to the substrate, and then a hydrophilic organic Mention may be made of a method of coating a compound. This is because using these methods makes it easy to improve the adhesion of the hydrophilic organic compound to the substrate.
- the term “bonding layer” is used.
- the bonding layer means a layer existing between the outermost hydrophilic thin film layer and the substrate in the case of forming a hydrophilic organic compound thin film in a multistage manner.
- the underlayer is meant.
- the binding layer is preferably a layer containing a material having a binding portion (linker).
- linker examples of combinations of the linker and the functional group at the end of the material to be bonded to the linker include epoxy group and hydroxyl group, phthalic anhydride and hydroxyl group, carboxyl group and N-hydroxysuccinimide, carboxyl group and carbodiimide, amino group and glutaraldehyde, etc. Is mentioned. In each combination, any may be a linker.
- a bonding layer is formed of a material having a linker on a substrate. The density of the material in the bonding layer is an important factor that defines the bonding force.
- the density can be easily evaluated using the contact angle of water on the surface of the bonding layer as an index.
- a silane coupling agent having an epoxy group at the end epoxysilane
- the water contact angle of the substrate surface to which epoxysilane is added is typically 45 ° or more, preferably 47 ° or more.
- a substrate having sufficient cell adhesion inhibitory property can be made by adding an ethylene glycol-based material or the like in the presence of an acid catalyst.
- the water contact angle refers to a water contact angle measured at 23 ° C.
- the cell adhesion region is formed by a cell adhesion-inhibiting region obtained by subjecting a cell adhesion-inhibiting hydrophilic membrane containing an organic compound having a carbon-oxygen bond to oxidation treatment and / or degradation treatment.
- oxidation means in a narrow sense, and means a reaction in which an organic compound reacts with oxygen and the oxygen content is higher than before the reaction.
- “decomposition” refers to a change in which a bond between organic compounds is broken to produce two or more organic compounds from one organic compound.
- “Decomposition treatment” typically includes, but is not limited to, decomposition by oxidation treatment and decomposition by ultraviolet irradiation. When the “decomposition treatment” is decomposition accompanied by oxidation (that is, oxidative decomposition), “decomposition treatment” and “oxidation treatment” indicate the same treatment.
- “Decomposition by ultraviolet irradiation” means that an organic compound absorbs ultraviolet rays and decomposes through an excited state.
- an organic compound when an organic compound is irradiated with ultraviolet rays in a system that contains molecular species containing oxygen (oxygen, water, etc.), the molecular species are activated in addition to being absorbed by the compound and causing decomposition. May react with organic compounds. The latter reaction can be classified as “oxidation”.
- a reaction in which an organic compound is decomposed by oxidation by an activated molecular species can be classified as “decomposition by oxidation” rather than “decomposition by ultraviolet irradiation”.
- oxidation treatment and “decomposition treatment” may overlap as operations, and the two cannot be clearly distinguished. Therefore, in this specification, the term “oxidation treatment and / or decomposition treatment” is used.
- Examples of the oxidation treatment and / or decomposition treatment methods include a method of subjecting the hydrophilic film to ultraviolet irradiation treatment, a method of photocatalytic treatment, and a method of treatment with an oxidizing agent.
- the hydrophilic film is partially oxidized and / or patterned. Or disassemble.
- a mask such as a photomask or a stencil mask or a stamp may be used.
- the oxidation treatment and / or the decomposition treatment may be performed by a direct drawing method such as a method using a laser such as an ultraviolet laser.
- a lamp that emits ultraviolet rays in the UV-C region from the VUV region such as a mercury lamp that emits ultraviolet rays with a wavelength of 185 nm or 254 nm or an excimer lamp that emits ultraviolet rays with a wavelength of 172 nm.
- a light source that emits ultraviolet light having a wavelength of 365 nm or less it is preferable to use a light source that emits ultraviolet light having a wavelength of 254 nm or less.
- the photocatalyst it is preferable to use a titanium oxide photocatalyst, a titanium oxide photocatalyst activated with a metal ion or a metal colloid.
- a titanium oxide photocatalyst a titanium oxide photocatalyst activated with a metal ion or a metal colloid.
- an organic acid or an inorganic acid can be used without any particular limitation. However, since a high-concentration acid is difficult to handle, it is preferable to dilute it to a concentration of 10% or less.
- the optimum ultraviolet treatment time, photocatalyst treatment time, and oxidant treatment time can be appropriately determined according to various conditions such as the ultraviolet light intensity of the light source used, the activity of the photocatalyst, the oxidizing power and concentration of the oxidant.
- the cell adhesive region may be formed by a hydrophilic film containing an organic compound having a carbon-oxygen bond at a low density.
- both the cell adhesive region and the cell adhesion inhibitory region are formed of a hydrophilic film containing an organic compound having a carbon-oxygen bond.
- the two regions differ in the density of the organic compound. The higher the density, the more difficult the cells adhere.
- the density of the organic compound is low enough to allow cells to adhere.
- the density of the organic compound is so high that cells cannot adhere.
- Examples of the method for controlling the density of the hydrophilic organic compound include a method in which a bonding layer is provided between the hydrophilic organic compound thin film and the substrate surface, and the bonding force of the bonding layer with the hydrophilic organic compound is adjusted. It is done.
- the “binding layer” is as defined above, and may be composed of the preferred materials described above.
- the bonding force of the bonding layer increases as the density of the material having the linker in the bonding layer increases, and decreases as the density decreases.
- the density of the material having a linker in the bonding layer can be easily evaluated using the water contact angle on the surface of the bonding layer as an index.
- the density of the material having a linker in the binding layer of the cell adhesive region is low.
- the water contact angle of the surface of the bonding layer before forming the hydrophilic organic compound thin film is taken as an example in the case of using a silane coupling agent having an epoxy group as a terminal as a material having a linker. And typically from 10 ° to 43 °, desirably from 15 ° to 40 °.
- a method for forming such a bonding layer a method of forming a coating (bonding layer) of a material having a linker on the surface of the substrate and then oxidizing and / or decomposing the surface of the bonding layer can be mentioned.
- Examples of the method for oxidizing and / or decomposing the bonding layer surface include a method of treating the bonding layer surface with ultraviolet irradiation, a method of treating with a photocatalyst, and a method of treating with an oxidizing agent.
- the entire surface of the bonding layer surface may be oxidized and / or decomposed or partially processed.
- the partial processing can be performed by using a mask such as a photomask or a stencil mask, or by using a stamp.
- the oxidation treatment and / or the decomposition treatment may be performed by a direct drawing method such as a method using a laser such as an ultraviolet laser.
- a cell adhesion region can be formed by forming a thin film of a hydrophilic organic compound on the binding layer thus formed.
- the density of the material having a linker in the binding layer of the cell adhesion-inhibiting region is high.
- the water contact angle on the surface of the bonding layer before forming a thin film of hydrophilic organic compound in the cell adhesion inhibitory region is an example of using a silane coupling agent having an epoxy group at the end as a material having a linker. When it is taken, it is typically 45 ° or more, desirably 47 ° or more.
- Such a bonding layer can be obtained by forming a coating of a material having a linker on the substrate surface. When the bonding layer surface is partially oxidized and / or decomposed, the remaining portion not subjected to the treatment becomes the bonding layer having the water contact angle.
- a cell adhesion-inhibiting region can be formed by forming a hydrophilic organic compound thin film on the binding layer thus formed.
- the amount of carbon in the cell adhesion region (including the bonding layer when a bonding layer is present) is lower than the amount of carbon in the cell adhesion inhibiting region (including the bonding layer when the bonding layer is present). It is preferable. Specifically, the amount of carbon in the cell adhesion region is preferably 20 to 99% with respect to the amount of carbon in the cell adhesion inhibitory region. Corresponding to this range is particularly suitable when the thickness of the hydrophilic film (the total of the thickness of the bonding layer and the hydrophilic film when a bonding layer is present) is 10 ⁇ m or less. “Carbon content (atomic concentration,%)” is as defined below.
- the value of the ratio (%) of the carbon bonded to oxygen in the cell adhesive region is the cell adhesion inhibitory region (the bonded layer is It is preferable that the value be smaller than the value of the ratio (%) of carbon bonded to oxygen among the carbon in the carbon (including the bonded layer if present).
- the value of the ratio (%) of carbon bonded to oxygen in the cell adhesion region is equal to the ratio of carbon bonded to oxygen among the carbon in the cell adhesion inhibitory region ( %) Is preferably 35 to 99%.
- the thickness of the hydrophilic film (the total of the thickness of the bonding layer and the hydrophilic film when a bonding layer is present) is 10 ⁇ m or less. “The proportion of carbon bonded to oxygen (atomic concentration,%)” is as defined below.
- Evaluation methods for the hydrophilic thin film of the present invention include contact angle measurement, ellipsometry, atomic force microscope observation, electron microscope observation, Auger electron spectroscopy measurement, X-ray measurement Photoelectron spectroscopy, various mass spectrometry, etc. can be used.
- X-ray photoelectron spectroscopy XPS / ESCA
- XPS / ESCA X-ray photoelectron spectroscopy
- the “carbon amount” of the hydrophilic thin film is defined as “the carbon amount obtained from the analytical value of the C1s peak obtained using an X-ray photoelectron spectrometer”.
- the “ratio of carbon bonded to oxygen” of the hydrophilic thin film is “the ratio of carbon bonded to oxygen determined from the analytical value of the C1s peak obtained using an X-ray photoelectron spectrometer. Is defined. Specific measurement can be performed as described in JP-A-2007-312736.
- the cell adhesion region and the cell adhesion inhibition region are arranged in a pattern.
- the shape of the pattern is not particularly limited as long as it is a two-dimensional pattern, and can be selected depending on the type of cell, the tissue to be formed, and the like.
- a line shape, a tree shape (dendritic shape), a mesh shape, a lattice shape, a circular shape, a quadrangular pattern, a pattern in which the inside of a graphic shape such as a circular shape and a quadrangular shape is a cell adhesive region or a cell adhesion inhibitory region. can be formed.
- the pattern of the cell adhesion region and the cell adhesion inhibition region is such that the cell adhesion region and the cell adhesion inhibition region are adjacent to each other on the conductive region of the substrate, and the cell adhesion region and the cell are separated on the insulation region. It is formed so that the adhesion-inhibiting region is adjacent. Since the cell adhesion region and the cell adhesion inhibitory region are adjacent to each other on the conductive region, the cells are first adhered to the cell adhesion region and cultured, and then the cell adhesion is performed by applying a voltage to the conductive region. When the inhibitory region is changed to the cell adhesion region, cells can migrate to the altered region. Therefore, the region to be altered to the cell adhesion region can be determined in advance by patterning, and the region and direction in which the cells migrate in the cell migration test can be controlled.
- the cell adhesion inhibitory region is an elongated shape orthogonal to the comb teeth portions, that is, conductive
- the cell migration along the comb-tooth portion can be tested by performing patterning so as to form an elongated shape, preferably a line-shaped region, straddling the conductive region and the insulating region.
- the width of the elongated shape, preferably the line shape, orthogonal to the comb tooth portion may be shorter than the length in which the comb tooth portion extends from the base and can be cultured in cells.
- the width of the line shape is usually 1 ⁇ m to 2 cm, preferably 50 to 1000 ⁇ m.
- the elongated region, preferably the line-shaped region is patterned so as to be orthogonal to the comb-tooth portion but away from the comb-shaped base portion, preferably as far away as possible. By doing so, the cells adhered outside the elongated shape, preferably the line-shaped region, can move in the region of the comb tooth portion that is altered to cell adhesion after application of voltage.
- the cell culture substrate of the present invention can be produced as described above, and in one embodiment, a cell containing an organic compound having a carbon-oxygen bond on the entire surface of a substrate having a conductive region and an insulating region.
- the step of forming an adhesion-inhibiting hydrophilic film, and the hydrophilic film is subjected to oxidation treatment and / or decomposition treatment in a pattern so that the cell adhesion region and the cell adhesion inhibition region are adjacent to each other on the conductive region. It can be produced by a method comprising a step of modifying to cell adhesion.
- the hydrophilic film may be modified to have cell adhesion by oxidizing and / or decomposing it into a pattern so that the cell adhesion region and the cell adhesion inhibitory region are adjacent to each other. is there.
- the cells to be seeded on the cell culture substrate may be floating cells such as blood cells and lymphoid cells, or may be adhesive cells, but the present invention is preferably used for cells having adhesiveness. It is also preferably used for cells having the property of migrating. Examples of such cells include liver cancer cells, glioma cells, colon cancer cells, kidney cancer cells, pancreatic cancer cells, prostate cancer cells, colon cancer cells, breast cancer cells, lung cancer cells, and ovarian cancer.
- Cancer cells such as cells, liver cells that are liver parenchymal cells, Kupffer cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, epidermis angle Epidermal cells such as keratinocytes, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells such as corneal epithelial cells, mammary cells, pericytes, muscle cells such as smooth muscle cells and cardiomyocytes, kidney cells, Examples include pancreatic islets of Langerhans, nerve cells such as peripheral nerve cells and optic nerve cells, chondrocytes, and bone cells.
- These cells may be primary cells collected directly from tissues or organs, or may be passaged from one generation to another. Furthermore, these cells are undifferentiated embryonic stem cells, pluripotent stem cells such as pluripotent mesenchymal stem cells, unipotent stem cells such as vascular endothelial progenitor cells that have unipotency, and differentiation is completed. Any of cells may be sufficient. In addition, the cells may be cultivated as a single species, or two or more types of cells may be co-cultured.
- the culture sample containing the target cells is preliminarily subjected to a dispersion process in which the biological tissue is finely dispersed in a liquid, a separation process in which impurities other than the target cells in the biological tissue and other cell debris are removed. It is preferable to go.
- a culture sample containing the target cells Prior to seeding of the cells on the cell culture substrate, it is preferable to preliminarily culture a culture sample containing the target cells by various culture methods to increase the target cells.
- a normal culture method such as monolayer culture, coat dish culture, or on-gel culture can be employed.
- one of the methods of culturing with cells attached to the surface of the support is already known as a so-called monolayer culture method.
- a culture sample and a culture solution in a culture container and maintaining the environment at a certain level, only specific living cells are adhered to the surface of a support such as a culture container. Multiply.
- the apparatus to be used, the treatment conditions, etc. are performed according to the usual monolayer culture method.
- cell adhesion factor which is a chemical substance that favors cell adhesion and proliferation, is also applied to the surface of a support such as glass, plastic sheet and plastic film.
- the culture solution in the culture vessel is removed, so that unnecessary components such as clumps and fibrous impurities that do not adhere to the support surface in the culture sample are removed, and only living cells that adhere to the support surface are removed. Can be recovered. Means such as EDTA-trypsin treatment can be applied to recovering living cells adhered to the support surface.
- Cells preliminarily cultured as described above are seeded on a cell culture substrate in a culture solution.
- the seeding method and seeding amount of the cells are not particularly limited, and for example, the method described in Asakura Shoten, “Tissue Culture Technology of the Japanese Tissue Culture Society (1999)”, pages 266 to 270 can be used. It is preferable to seed the cells so that the cells adhere in a single layer in an amount sufficient to prevent the cells from growing on the cell culture substrate. Usually, it is preferable to seed so that cells are contained in the order of 10 4 to 10 6 per ml of culture medium, and seeding so that cells are contained in the order of 10 4 to 10 6 per 1 cm 2 of the substrate. Is preferred. Specifically, seeding is performed at about 2 ⁇ 10 5 per 400 mm 2 .
- any culture medium can be used without particular limitation as long as it is a cell culture medium usually used in the art.
- a basal medium as described in "Tissue culture technology third edition" edited by the Japanese Society for Tissue Culture, page 581 can be used.
- serum such as fetal bovine serum
- various growth factors, antibiotics, amino acids and the like may be added to the basal medium.
- a commercially available serum-free medium such as Gibco serum-free medium (Invitrogen) can also be used.
- the time for culturing the cells depends on the presence or absence of cell manipulation during culturing, but is usually 6 to 96 hours, preferably 12 to 72 hours.
- the temperature for culturing is usually 37 ° C. It is preferable to culture in a CO 2 concentration atmosphere of about 5% using a CO 2 cell culture device or the like. After culturing, the cell culture substrate is washed to wash away non-adherent cells, and the cells can be adhered only to the cell adhesion region.
- Cell migration test As described above, after seeding and culturing the cells on the cell culture substrate of the present invention, attaching the cells to the cell adhesive region, applying a voltage to the conductive region, the cells on the conductive region Cell migration can be tested by modifying the adhesion-inhibiting region to a cell-adhesive region and observing subsequent cell migration.
- the cell migration test method of the present invention comprises: (I) seeding the cells on the cell culture substrate of the present invention, and attaching the cells to the cell adhesive region; (Ii) a step of changing a cell adhesion-inhibiting region on the conductive region to a cell adhesive region by applying a voltage to the conductive region; (Iii) observing the movement of the cells adhering to the cell adhesive region on the conductive region to the region modified to the cell adhesive region in step (ii).
- step (i) it is preferable to perform cell culture after seeding the cells and adhere the cells to the cell adhesive region, and further wash away the cells that have not adhered by washing the cell culture substrate. It is preferable to attach the cells only to the cell adhesive region.
- the voltage applied to the conductive region is preferably a positive voltage.
- the cell adhesion-inhibiting region can be effectively changed to the cell adhesion region, and in particular, when the conductive region is made of an ITO film, it can be prevented from blackening, and cell migration Can be observed satisfactorily.
- the voltage to be applied can be appropriately determined by those skilled in the art, but is usually 1 to 10 V, preferably 2 to 5 V, and the application time is usually 0.5 to 60 minutes, preferably 1 to 10 minutes. It is.
- the voltage to be applied varies depending on the type of solvent in contact with the electrode, the electrode material, and the electrode shape, but it is usually higher than the voltage at which the cell adhesion-inhibiting region can be changed to the cell adhesive region. It is better to apply a voltage that is low enough not to adversely affect the cells.
- the voltage may be applied between the conductive regions in the substrate plane (for example, between ITO and ITO), or a counter electrode such as Pt is provided in the substrate plane between the conductive region and the counter electrode (for example, Or between ITO and Pt).
- a reference electrode such as Ag / AgCl may be provided in the substrate plane.
- the counter electrode and the reference electrode may not be in the plane of the substrate (the electrode may be immersed in the culture solution).
- Observation of cell movement includes measurement of the speed of cell movement, as well as observation of the migration direction, cell shape during migration, and connections between surrounding cells. Measurement of the speed at which the cells move can be carried out by measuring the area and distance in which the cells infiltrate in a pattern, for example, a linear pattern such as a comb tooth portion.
- the cell adhesion region on the conductive region in the vicinity of the cell adhesion inhibitory region on the conductive region Becomes smaller in area and fewer cells can participate in migration.
- the cell adhesion region and the cell adhesion inhibitory region are formed adjacent to each other on the insulating region, the cell adhesion on the insulating region near the cell adhesion inhibitory region on the conductive region Since cells adhered to the region can also participate in migration, the total number of cells that can participate in migration increases as a result, and migration evaluation can be advantageously performed.
- FIG. 1 (1) to (3) of FIG. 1 show an embodiment of the method for producing a cell culture substrate of the present invention.
- a conductive region (b) and an insulating region (a) are supported on a substrate, and the conductive region supports a plurality of comb teeth (c) and each comb tooth. It forms so that it may be patterned in the comb shape which consists of a base (d).
- a cell adhesion-inhibiting hydrophilic film containing an organic compound having a carbon-oxygen bond is formed on the entire surface of the substrate.
- the hydrophilic film is oxidized and / or decomposed in a pattern (so that the cell adhesion-inhibiting region becomes an elongated (line-shaped) region orthogonal to the comb tooth portion).
- the cell adhesive region (b) and the cell adhesion inhibitory region (b ′) are adjacent to each other on the conductive region, and the cell adhesive region (a) and the cell adhesion inhibitory region on the insulating region.
- a substrate for cell culture in which (a ′) is adjacent is obtained.
- FIG. 1 shows an embodiment of the cell migration test method of the present invention.
- 3 ' cells are seeded on a cell culture substrate, and the cells (e) are adhered to the cell adhesion region.
- a voltage preferably a positive voltage
- the cell adhesion-inhibiting region (b ′) on the conductive region is changed to the cell adhesive region (b) (4 ′).
- the cells adhering to the cell adhesive region on the conductive region move to the region modified to the cell adhesive region (5 ').
- FIGS. Materials and methods for manufacturing a cell culture substrate, shape of the cell culture substrate, cell culture method, cell culture conditions and reagents used, cells to be cultured, applied voltage, voltage application method in this embodiment These conditions are the same as those described for the other embodiments of the present invention unless otherwise specified in this section.
- the cell culture substrate 30 includes at least one linear conductive region 31 on the substrate, and both sides along the length direction of the linear conductive region 31.
- An insulating region 32 is disposed in the area.
- Cell adhesion so as to include at least a part (a part or all) of a part at each position of the linear conductive region at one or two or more positions of the linear conductive region 31 having different lengthwise positions.
- the sex region 33 is formed. 3 to 7, the thick frame indicates the outer edge of the cell adhesion region, and the region outside the frame indicates the cell adhesion inhibitory region. When there are a plurality of cell adhesion regions, they are arranged apart from each other. As shown in FIGS.
- the cell adhesive regions 33, 62 includes the portions of the insulating regions 32, 55, and 61 adjacent to the portion as the remainder.
- the entire cell adhesive region 73 may be formed to be a linear conductive region (wide portion 71) at each position.
- the linear conductive region is formed to have a width corresponding to the width of the cell adhesive region at a position in the length direction corresponding to the cell adhesive region.
- a portion not included in the cell adhesive region 33 is a cell adhesion inhibitory region.
- the length of each cell adhesive region 33 in the length direction of the linear conductive region 31 connected to the cell adhesive region is defined as the length L of the cell adhesive region on the substrate plane with respect to the length direction.
- the dimension in the orthogonal direction is defined as the width W of the cell adhesive region
- the length L and the width W of each cell adhesive region are each independently 1 to 500 ⁇ m.
- the line width of the portion not included in the cell adhesive region (the dimension of the portion in the direction orthogonal to the length direction on the substrate plane) LW is 0.1 to 10 ⁇ m.
- the cell adhesion-inhibiting region of the linear conductive region 31 (the portion of the linear conductive region 31 that is not included in the cell adhesive region 33 in FIG. 3B) is preferably made cell-adhesive by voltage application. It can be modified. That is, when a voltage is applied, as shown in FIG. 3C, the linear conductive region 33 formed in advance and the entire linear conductive region 31 are integrated into a plurality of positions in different longitudinal directions. In addition, a linear cell adhesive region having a wide portion corresponding to the cell adhesive region 33 formed in advance is formed.
- the cell adhesion region 33 can adhere to only 1 to several cells, for example 1 to 3, preferably 1 or 2, and particularly preferably 1 cells to be cultured. It preferably has dimensions, shapes and areas. In order to achieve this object, the size, shape, and area of the cell adhesive region 33 can be appropriately determined according to the type of cells to be cultured.
- the length L and the width W according to the above definition of the cell adhesion region 33 are usually 1 to 500 ⁇ m, preferably 10 to 200 ⁇ m, more preferably 25 to 50 ⁇ m.
- the ratio of the length and width of the cell adhesion region is not particularly limited. Usually, when the length of the cell adhesion region is 1, the width of the cell adhesion region is 0.5 to 2, preferably 0.7.
- Is 1.5, more preferably 0.8 to 1.3, particularly preferably 0.95 to 1.05, and most preferably 1. 3 to 7 exemplify a square as the shape of the cell adhesion region, but it is not particularly limited, and there are many squares, rectangles, rhombuses, parallelograms and other squares, triangles, pentagons, hexagons, or more corners.
- a circular shape such as a square, a perfect circle, an ellipse, and an ellipse can be given.
- the length and width of the cell adhesion region are each 1 to 500 ⁇ m, the area is 1 to 250,000 ⁇ m 2 if the cell adhesion region is square, and about 0.8 to 196,350 ⁇ m 2 if it is circular.
- the area of the cell adhesion region is preferably 50 to 40,000 ⁇ m 2 , more preferably 625 to 2,500 ⁇ m 2 (490 to 1963 ⁇ m 2 in the case of a circle).
- the line width LW of the linear conductive region is preferably 0.1 to 20 ⁇ m, more preferably 0.1 to 10 ⁇ m, and particularly preferably 1 to 5 ⁇ m.
- the line width LW is set to the width W of the cell adhesion region 33 formed in advance. It is formed smaller than.
- the line width LW of the linear conductive region connected to the cell adhesion region is preferably 0.2 or less, more preferably 0.01 to 0.2. It can be.
- the shape of the linear conductive region may be linear or curved. Furthermore, as described later, a plurality of linear conductive regions may be combined to form an arbitrary shape such as a tree shape, a mesh shape, or a lattice shape.
- the portion of the linear conductive region that communicates between the two cell adhesive regions can be appropriately determined according to the type of cells to be cultured, and is usually 10 ⁇ m to 10 mm, preferably 50 to 200 ⁇ m. When used for culturing nerve cells, the length may be set to 100 mm to 30 cm in consideration of high axon extension ability.
- the substrate for cell culture of the present invention forms a linear conductive region 31 and an insulating region 32 on the substrate surface.
- a hydrophilic film or the like is formed on the entire surface of the provided base material to make it cell inhibitory.
- the hydrophilic film is oxidized and / or decomposed in a pattern to form one or a plurality of cell adhesive regions 33.
- the cells 40 are seeded on the cell culture substrate 30 of the present invention, and the cells are adhered to the cell adhesive region 33 (FIG. 4A).
- the cell adhesive region 33 In this case, only 1 to several, for example, 1 to 3, preferably 1 or 2, particularly preferably 1 cell 40 adheres to each cell adhesive region 33.
- FIG. 4B by applying a voltage to the linear conductive region, the cell adhesion-inhibiting region of the linear conductive region is changed to a cell adhesive region.
- FIG. 4C the shape of a part of the cells 40 adhered to the cell adhesive region 33 changes, and along the linear conductive region 31 that is newly cell adhesive.
- the function of the cells can be tested. Observation of cells can be performed from various viewpoints. For example, the speed of the morphological change, the distance, the shape of the cell after the morphological change, etc. can be observed.
- the substrate of the present invention in which the cell adhesive region is formed at two or more positions where the lengthwise position of the linear conductive region is different it is possible to communicate through the linear conductive region. It is also possible to observe the interaction with other cells. According to this method, it is possible to evaluate the cell-cell interaction.
- the above method can also be used to form circuit-like cell constructs.
- the cell 40 is formed in a state where a voltage is applied using the substrate of the present invention in which the cell-adhesive regions are formed at two or more positions where the lengthwise positions of the linear conductive regions are different.
- a part of the cells 40 adhered to the cell adhesive region 33 undergoes a morphological change and extends along the linear conductive region that has newly become cell adhesive.
- the communication part 41 is formed between the other cells communicated via.
- the cell to which this method is applied is not particularly limited. For example, it is possible to form a neural network of a desired shape by using a nerve cell as a cultured cell and inducing axonal extension along a linear conductive region that is newly cell-adhesive. .
- the conductive region can be patterned into a comb shape 52 including a plurality of comb teeth portions 50 and a base portion 51 that supports each comb tooth portion 50. .
- each comb-tooth portion 50 can be used as the linear conductive region.
- a comb-shaped conductive region 53 having a plurality of comb-tooth portions 54 having the same shape can be formed, and the comb-tooth portions 50 and 54 can be arranged alternately.
- FIG. 5A shows an overall view of the apparatus
- FIG. 5B shows an enlarged view in the frame of FIG. 5A.
- the number and arrangement of the linear conductive regions are not particularly limited.
- a plurality of linear conductive regions 60 may be arranged in a lattice pattern.
- the cell adhesive region 62 can be formed so as to include the position of the intersection of the lattice.
- the linear conductive region 60 can be modified to cell adhesiveness by applying a voltage (FIG. 6C).
- the cell adhesive region is formed so as to include a part of the linear conductive region and a part of the insulating region adjacent to the part.
- the entire cell adhesive region may be a conductive region.
- a linear conductive region 70 having a plurality of wide portions 71 is formed at different positions in the length direction.
- the cell adhesive region 73 is formed so as to coincide with the wide portion 71 (FIG. 7B), and the linear conductive region 70 can be altered to cell adhesiveness by applying a voltage (FIG. 7). (C)).
- ITO Indium tin oxide
- a photomask having a titanium oxide photocatalyst applied to the entire surface was prepared.
- a photomask having a square pattern in which openings of 250 ⁇ m square were formed at a pitch of 500 ⁇ m and having a size of 5 inches having openings of about 1.5 cm in width around the photomask was used.
- the illuminance of the exposure machine was measured in advance at a wavelength of 350 nm and used as a guide for setting the exposure time. The illuminance was 25 mW / cm 2 .
- the ITO substrate on which the hydrophilic thin film is formed and the quartz plate with the catalyst are arranged such that the hydrophilic thin film and the photocatalyst layer of the photomask face each other, and the comb tooth portion of the comb-shaped conductive region of the ITO substrate and the square of the photomask It was installed so that the patterns were orthogonal, and installed in the exposure machine so that light was irradiated from the back side of the photomask. The film was exposed for 120 seconds to be oxidized. Then, it cut
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Abstract
L'invention concerne des moyens d'examen de la migration cellulaire an amenant des cellules adhérentes sur un substrat à migrer à l'intérieur d'une région prédéterminée. La présente invention est un substrat pour la culture cellulaire comprenant : une matière de base qui a une région conductrice et une région isolante ; et une région d'adhésion cellulaire et une région inhibitrice d'adhésion cellulaire qui sont respectivement formées sur la région conductrice et la région isolante. La présente invention concerne le substrat mentionné ci-dessus pour la culture cellulaire dans laquelle, dans la région conductrice, la région d'adhésion cellulaire et la région inhibitrice d'adhésion cellulaire sont adjacentes l'une de l'autre, et, dans la région isolante, la région d'adhésion cellulaire et la région inhibitrice d'adhésion cellulaire sont adjacentes l'une de l'autre.
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WO2015068759A1 (fr) * | 2013-11-06 | 2015-05-14 | 独立行政法人科学技術振興機構 | Substrat pour contrôler une direction du mouvement de cellules animales, et procédé d'identification de cellules et procédé de séparation de cellules utilisant ledit substrat |
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Cited By (2)
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WO2015068759A1 (fr) * | 2013-11-06 | 2015-05-14 | 独立行政法人科学技術振興機構 | Substrat pour contrôler une direction du mouvement de cellules animales, et procédé d'identification de cellules et procédé de séparation de cellules utilisant ledit substrat |
US9958429B2 (en) | 2013-11-06 | 2018-05-01 | Japan Science And Technology Agency | Substrate for controlling movement direction of animal cells, and cell identification method and cell separation method using the same |
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