WO2005103227A1 - 細胞培養用パターニング基板およびその製造方法 - Google Patents
細胞培養用パターニング基板およびその製造方法 Download PDFInfo
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- WO2005103227A1 WO2005103227A1 PCT/JP2005/007775 JP2005007775W WO2005103227A1 WO 2005103227 A1 WO2005103227 A1 WO 2005103227A1 JP 2005007775 W JP2005007775 W JP 2005007775W WO 2005103227 A1 WO2005103227 A1 WO 2005103227A1
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- cell adhesion
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0068—General culture methods using substrates
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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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/20—Material Coatings
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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
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/06—Plates; Walls; Drawers; Multilayer plates
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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
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2535/00—Supports or coatings for cell culture characterised by topography
- C12N2535/10—Patterned coating
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2539/00—Supports and/or coatings for cell culture characterised by properties
- C12N2539/10—Coating allowing for selective detachment of cells, e.g. thermoreactive coating
Definitions
- the present invention relates to a method for producing a cell culture patterning substrate capable of adhering cells in a high-definition pattern.
- Some cells particularly many animal cells, have an adhesion dependency of growing by adhering to something, and cannot survive for a long period of time in a floating state outside a living body. Cultivation of cells having such adhesion dependence requires a carrier for the cells to adhere to the cells. Generally, a plastic-made cell on which a cell adhesion protein such as collagen fibronectin is uniformly applied is generally used. A culture dish is used. These cell adhesion proteins are known to act on cultured cells, facilitating cell adhesion and affecting cell morphology.
- a technique for arranging cultured cells is to use a substrate having a patterned surface with different ease of adhesion to the cells, cultivate the cells on this surface, and allow the cells to adhere.
- a method is used in which cells are arranged by adhering the cells only to the processed surface.
- Patent Document 1 a charge holding medium having an electrostatic charge pattern formed thereon is applied to cell culture for the purpose of, for example, growing nerve cells in a circuit form. Further, Patent Document 2 attempts to arrange cultured cells on a surface obtained by patterning a non-cell-adhesive or cell-adhesive photosensitive hydrophilic polymer by a photolithography method.
- Patent Document 3 describes a cell culture substrate on which a substance such as collagen which affects cell adhesion rate and morphology is patterned, and a method for producing the substrate by photolithography. Has been disclosed. By culturing the cells on such a base material, more cells can be adhered to the surface on which collagen or the like is put on, thereby realizing the cell patterning.
- Patent Document 1 JP-A-2-245181
- Patent Document 2 JP-A-3-7576
- Patent Document 3 JP-A-5-176753
- the present invention has a substrate having a convex portion, and a cell culture region for culturing cells formed on the surface of the substrate, wherein the cell culture region has a convex portion.
- the present invention provides a pattern culturing substrate for cell culturing, characterized in that the substrate is partitioned by the above-mentioned projections.
- the height and width of the projections can prevent the cells attached to the adjacent cell culture area from binding, and can be used to culture cells in a desired pattern. It can be.
- the region having the convex portion is a cell adhesion inhibiting portion having an adhesion inhibitory property to a cell
- the cell culture region is a cell adhesive portion having an adhesive property to a cell.
- Such a putter substrate for cell culture can have the following six embodiments.
- a photocatalyst and a photocatalyst-containing cell adhesion layer containing a cell adhesion material having an adhesive property to cells and being decomposed or denatured by the action of a photocatalyst accompanying energy irradiation are provided on the substrate.
- the cell adhesion inhibitor is formed, and the cell adhesion material is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation.
- the substrate has at least a photocatalyst and a cell adhesion-inhibiting property of inhibiting adhesion to cells, and is degraded or denatured by the action of a photocatalyst accompanying energy irradiation.
- a photocatalyst-containing cell adhesion-inhibiting layer containing a cell adhesion-inhibiting material is formed, and the cell-adhesion-inhibiting material is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation. .
- the photocatalyst-containing cell adhesion layer containing the photocatalyst or the photocatalyst-containing cell adhesion inhibition layer is irradiated with energy, whereby the photocatalyst contained in these layers themselves is irradiated.
- the cell adhesion material or the cell adhesion inhibitor can be decomposed or denatured, and the cell adhesion part and the cell adhesion inhibition part can be easily formed. Therefore, it is easy to form a layer without the necessity of separately forming another layer and the like, and it is possible to make the surface power such as manufacturing efficiency preferable.
- a photocatalyst-containing layer containing at least a photocatalyst and a cell that is adhesive to cells and is decomposed or denatured by the action of the photocatalyst accompanying energy irradiation are provided on the base material.
- a cell adhesive layer containing an adhesive material is formed, and the cell adhesion inhibitor is obtained by decomposing or denaturing the cell adhesive material by the action of a photocatalyst accompanying energy irradiation.
- a photocatalyst-containing layer containing at least a photocatalyst on the base material and an action of the photocatalyst having cell adhesion inhibitory property of inhibiting adhesion to cells and accompanying energy irradiation
- a cell adhesion-inhibiting layer containing a cell adhesion-inhibiting material that is degraded or denatured by the above is formed, and the cell adhesion portion is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation. Is what it is.
- the photocatalyst-containing cell adhesion layer or the photocatalyst-containing cell adhesion inhibition layer is irradiated with energy, so that the adjacent photocatalyst-containing cell adhesion layer is irradiated with energy.
- the photocatalyst in the layer is excited, the cell adhesion material or the cell adhesion inhibiting material can be decomposed or denatured, and the cell adhesion portion or the cell adhesion inhibiting portion can be easily formed.
- the photocatalyst is unlikely to be exposed on the surface, so that the cells may be affected by the photocatalyst over time when the cells are cultured on the cell culture area. It also has the advantage that the performance can be reduced.
- a cell adhesion layer containing a cell adhesion material that has cell adhesion to at least adhere to cells and that is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation is formed.
- the cell adhesion material is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation.
- the present invention relates to a cell adhesion on the substrate, which has at least cell adhesion to prevent adhesion to cells and is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation.
- a cell adhesion-inhibiting layer containing an inhibitory material is formed, and the cell-adhesion portion is formed by decomposing or denaturing the cell-adhesion-inhibiting material by the action of a photocatalyst accompanying energy irradiation.
- a photocatalyst-containing layer-side substrate having at least a photocatalyst-containing layer containing a photocatalyst is used as the cell adhesion layer or the cell adhesion-inhibiting layer, for example.
- the photocatalyst contained in the photocatalyst-containing layer can easily decompose or denature the cell adhesion material or the cell adhesion inhibition material by the action of the photocatalyst. Can be formed.
- it is not necessary to include a photocatalyst in the cell adhesion layer or the cell adhesion inhibition layer! Therefore, the cells can be prevented from being affected by the photocatalyst over time, and the cells can be adhered.
- a high-quality cell culture substrate can be obtained.
- the present invention includes a substrate having a convex portion and a cell adhesive layer containing a cell adhesive material having adhesiveness to cells and being decomposed or denatured by the action of a photocatalyst accompanying energy irradiation.
- the patterning region is formed in the region having the convex portion.
- Irradiate energy from a predetermined direction Energy for forming a pattern consisting of a cell adhesion inhibitor in which the cell adhesion material contained in the cell adhesion layer is decomposed or denatured, and a cell adhesion portion which has not been irradiated with energy and has adhesive properties to cells.
- a method for producing a cell culture buttering substrate which comprises an irradiation step.
- the cell adhesion material in the region having the convex portion is decomposed or denatured, and the cell adhesion layer is decomposed.
- the present invention provides a substrate having a convex portion, and a cell-adhesive material having a cell adhesion-inhibiting property of inhibiting adhesion to cells and being decomposed or denatured by the action of a photocatalyst accompanying energy irradiation.
- a photocatalyst-containing layer side substrate having a photocatalyst-containing layer and a substrate containing a photocatalyst were arranged such that the cell adhesion layer and the photocatalyst-containing layer faced each other.
- the cell culture region on the cell adhesion-inhibiting layer is irradiated with energy using the photocatalyst-containing layer-side substrate, whereby the cell-adhesion-inhibiting material is decomposed or degraded, and the cell adhesion region is exposed to energy.
- a cell adhesive portion having good adhesiveness can be obtained.
- the region where the projections are formed when the energy is not applied remains as the cell adhesion inhibiting portion because the cell adhesion inhibiting material remains. Therefore, cells are adhered to the cell adhesion portion of the putter substrate for cell culture manufactured according to the present invention, that is, the cell culture region.
- the height and width of the protrusions and the cell adhesion-inhibiting property of the cell adhesion-inhibiting layer can prevent the cells attached to the adjacent cell culture region from being bonded, thereby achieving high-definition. This makes it possible to culture cells in a pattern.
- the cell culture region is partitioned by the convex portion, it is possible to prevent the cells attached to the adjacent cell culture region from binding due to the height of the convex portion.
- a cell culture patterning substrate capable of culturing cells in a desired pattern can be obtained.
- FIG. 1 is a schematic cross-sectional view showing one example of a patterning substrate for cell culture of the present invention.
- FIG. 2 is a process chart showing an example of a method for forming a cell adhesion portion and a cell adhesion inhibition portion of the cell culture patterning substrate of the present invention.
- FIG. 3 is a schematic sectional view showing an example of a photocatalyst-containing layer-side substrate used in the present invention.
- FIG. 4 is a schematic cross-sectional view showing another example of the photocatalyst-containing layer side substrate used in the present invention.
- FIG. 5 is a schematic sectional view showing another example of the photocatalyst-containing layer-side substrate used in the present invention.
- FIG. 6 is an explanatory view showing one example of a method for forming a cell adhesion portion and a cell adhesion inhibition portion of the patterning substrate for cell culture of the present invention.
- FIG. 7 is a process chart showing an example of a method for producing a puttering substrate for cell culture of the present invention. Explanation of symbols
- the present invention relates to a cell culture patterning substrate used for adhering and culturing cells on a substrate in a high-definition pattern, and a method for producing the same. Each of these is described separately below.
- the patterning substrate for cell culture of the present invention has a substrate having a convex portion, and a cell culture region which is a region for culturing cells formed on the surface of the substrate, wherein the cell culture region comprises: It is characterized in that the base material having the convex portions is partitioned by the convex portions.
- a cell culture region 3 which is a region for culturing cells, is partitioned by a convex portion 2 of the base material 1. Things.
- the protrusions are formed between the adjacent cell culture regions, the cells attached to the adjacent cell culture regions adhere to each other depending on the height or width of the protrusions. Can be prevented. As a result, it is possible to obtain a cell-cultured patterning substrate capable of culturing cells in a desired pattern.
- a cell-cultured patterning substrate capable of culturing cells in a desired pattern.
- the substrate having a convex portion used in the present invention is not particularly limited as long as it can prevent cells attached to adjacent cell culture regions from binding to each other by the convex portion.
- inorganic materials such as metal, glass, and silicon, and organic materials typified by plastics can be used.
- a method for forming the projections a method for forming a base material or the like can be generally used. For example, a method of molding a material into a shape having a convex portion, a method of bonding a member having a convex portion on a flat member, a method of printing a convex portion on a flat member, and a method of forming a flat member.
- a method in which a photoresist is applied and a convex portion is formed by one photolithography method a method in which a flat member is shaved to form a convex portion, a coating layer is provided on a flat member, and a convex portion is formed by etching the coating layer.
- Examples thereof include a method, a method of molding a resin by hot pressing using a mold having an uneven shape, and a method of pouring a curable resin into a mold and curing the resin.
- the flat member and the member to be bonded may be made of materials having different material strengths.
- the same member may be used.
- the flat member has a cell adhesive property
- a member to be bonded as a convex portion is made of a material V that does not have an adhesive property to cells, so that a region where a convex portion is formed and a negative region is formed.
- a cell culture region having adhesiveness to cells and a region where a convex portion is formed as a cell adhesion inhibiting region having no adhesiveness to cells are also possible.
- the convex portion when using a method of printing a convex portion on a flat member, even when the convex portion is printed using a material made of the same material as the material of the flat member, etc. It may be a material printed on a flat member having cell adhesion and having no adhesiveness to cells and printed using a material.
- the shape of the convex portion is not particularly limited as long as it is possible to prevent cells attached to the adjacent cell culture region from binding.
- the cross-sectional shape is rectangular.
- the shape may be circular or semi-circular, or may be triangular.
- the height of the convex portion is appropriately selected depending on the size of the cells to be cultured and the like, and is usually 0.1 ⁇ m to 1000 ⁇ m, and particularly 1 ⁇ m to 500 ⁇ m. In particular, it is preferably about 10 ⁇ m to 200 ⁇ m.
- the width of the convex portion is preferably 10 ⁇ m to 1000 ⁇ m, particularly preferably 50 ⁇ m to 800 ⁇ m, and particularly preferably about 100 ⁇ m to 500 ⁇ m.
- the width refers to the width of the bottom of the formed protrusion. By forming the protrusions having such a width or height, cells adhering to the adjacent cell culture area can be bound. Is prevented.
- the width of the convex portion may be wider or narrower than the width between adjacent convex portions, that is, the width of the cell culture region.
- the cell culture region in the putter substrate for cell culture of the present invention is a region used for culturing cells, and is a region partitioned by the above-mentioned convex portions, and the shape of the cell culture region is determined according to the type and purpose of the cells. Selected as appropriate.
- the term “partitioned by the protrusion” may mean that the protrusion is formed on the entire surface around the cell culture region, or the protrusion is formed only in a part of the periphery of the cell culture region. Once formed, the cell culture area may be partitioned!
- the cell culture region is not particularly limited as long as it is formed so as to have good adhesion to cells. It may be one having adhesiveness or one having adhesiveness to cells due to physical properties. In addition, when the adhesiveness to cells differs depending on the type of cells, etc., any adhesive can be used as long as it adheres well to target cells.
- Such a cell culture region may be, for example, a cell adhesive layer formed on a substrate using a cell adhesive material having an adhesive property to cells, or the like.
- a cell culture area may be formed by using one having adhesive properties to cells.
- materials having adhesiveness to cells and also used as a base material include silicone resin, styrene resin, acrylic resin, metharyl resin, polyester, polyethylene, polydalicholic acid, polylactic acid, Examples include quartz glass, soda glass, apatites, and aluminas.
- a cell adhesive material used for the cell adhesive layer a cell adhesive material used for a general cell culture substrate or the like can be used.
- basic polymers such as hydrophilized polystyrene, poly (N-isopropylacrylamide) and polylysine
- basic polymers such as aminopropyltriethoxysilane and N- (2aminoethyl) 3aminopropyltrimethoxysilane
- conjugation products and condensates containing them include conjugation products and condensates containing them.
- Cell adhesion materials include fibronectin, laminin, tenascin, vitronectin, RGD (arginine glycine-aspartate) sequence-containing peptide, YIGSR (tyrosine isoleucine-glycine-serine-arginine) sequence-containing peptide, collagen, atherocollagen, Gelatin and the like can be mentioned.
- the pattern culturing substrate for cell culture of the present invention is not particularly limited as long as it has the above-mentioned cell culture region on the above-mentioned substrate, but in the present invention, in particular, the above-mentioned convex portion is formed. It is preferable that the region in which the cells are cultured is a cell adhesion inhibitor having cell adhesion inhibitory property for inhibiting adhesion to cells, and the cell culture region for culturing cells is a cell adhesion region having adhesion to cells. As a result, it is possible to prevent cells in adjacent cell culture areas from adhering to each other even by the cell adhesion inhibiting properties of the protrusions, which are determined only by the heights and widths of the protrusions.
- the region where the above-mentioned projections are formed has the cell adhesion-inhibiting property only when the above-mentioned projections themselves have cell-cell-adhesion inhibiting properties. Is formed to have a cell adhesion inhibitory property.
- a substrate made of a material having cell and adhesion inhibition properties By forming a cell adhesive layer or the like having an adhesive property to cells, the convex portion has a cell adhesive inhibitory property, and the cell adhesive region may be formed to have the cell adhesive property. It may be formed by forming a cell adhesion-inhibiting layer or the like having cell-adhesion-inhibiting properties on the projections of the cell-adhesive substrate, and furthermore, cell culture of a substrate having a projection. A cell adhesion layer may be formed in a region, and a cell adhesion inhibition layer may be formed on each of the projections.
- the cell adhesion portion and the cell adhesion inhibition portion are formed by using a layer whose adhesiveness to cells changes by the action of a photocatalyst accompanying energy irradiation. It is preferred that it is. This makes it possible to easily perform the pattern junging of the cell adhesion part and the cell adhesion inhibition part.
- a photocatalyst containing at least a photocatalyst and a cell adhesive material that has adhesiveness to cells and is decomposed or modified by the action of the photocatalyst accompanying energy irradiation is provided on a substrate having a convex portion.
- the cell adhesion-containing layer is formed and the cell adhesion inhibitor is decomposed or degraded by the action of a photocatalyst accompanying energy irradiation.
- the photocatalyst-containing cell adhesion layer contains the photocatalyst and the above-mentioned cell adhesion material
- energy is applied to the photocatalyst-containing cell adhesion layer on the region where the convex portion is formed!
- the cell adhesion material is decomposed or denatured by the action of the photocatalyst, and can be used as a cell adhesion inhibitor to which cells do not adhere.
- the cell adhesive material in the area where the energy is not irradiated, since the cell adhesive material remains, it is possible to form a cell adhesive portion having good adhesiveness to cells.
- the cell cultivation area which is a cell adhesion part, does not have cell-adhesive properties. This makes it possible to easily form the adhesion inhibiting portion.
- the cell adhesion inhibition portion formed with the convex portion is formed.
- the cell adhesion inhibitor can be removed by the action of the photocatalyst. This also has the advantage that cells cultured in a high-definition pattern can be maintained.
- the photocatalyst-containing cell adhesion layer used in the present embodiment contains at least a photocatalyst and the above-mentioned cell adhesive material. This is a layer that is decomposed or denatured to have low adhesion to cells.
- photocatalyst-containing cell adhesive layer-forming coating solution containing a photocatalyst-containing cell adhesive material and a photocatalyst-containing photocatalyst-containing cell adhesive material to be decomposed or modified by the action of a photocatalyst accompanying energy irradiation. It can be performed by, for example, coating on a base material having a portion.
- the application of the coating solution for forming a photocatalyst-containing cell adhesive layer can be performed using a general coating method, for example, a spin coating method, a spray coating method, a dip coating method, a roll coating method, a bead coating method, or the like. Can be used.
- the film thickness of the photocatalyst-containing cell adhesion layer is a force which is appropriately selected depending on the type of the cell culture patterning substrate and the like, usually about 0.01 m to 1.0 O / zm, especially
- the force S can be set to about 0.1 ⁇ m to 0.3 ⁇ m.
- the type of the cell adhesive material contained in the photocatalyst-containing cell adhesive layer of this embodiment is not particularly limited as long as it has adhesiveness to cells and is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation. Not something.
- having an adhesive property with a cell means that the cell adheres well to the cell, and when the adhesive property with the cell varies depending on the type of the cell, etc., it adheres well to a target cell. That means.
- the cell adhesive material used in the present embodiment has such adhesion to cells, and is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation, and has adhesion to cells. Those that disappear or those that change to those having cell adhesion inhibitory properties that inhibit adhesion to cells are used.
- the above-mentioned materials having adhesive properties to cells include a material having adhesive properties to cells due to physical properties and a material having adhesive properties to cells due to biochemical properties. There are two types; ⁇ .
- Physically determinable factors that determine the adhesiveness between cells and a material having adhesiveness to cells based on physicochemical properties include surface free energy and electrostatic interaction. .
- surface free energy For example, when the adhesiveness to cells is determined by the surface free energy of the material, if the material has a surface free energy within a predetermined range, the adhesiveness between the cells and the material becomes good, and if the material falls outside the range, the material has good surface free energy. Adhesion between the cells and the material will be reduced.
- the change in cell adhesiveness due to such surface free energy for example, the experimental results shown in the lower part of Yoshinobu Raft, supervised by CMC Publishing Noo Materials, p. 109, are known.
- Materials having adhesiveness to cells due to such factors include, for example, hydrophilized polystyrene, poly (N-isopropylacrylamide), and the like.
- the surface free energy changes due to, for example, substitution or decomposition of a functional group on the surface of the material due to the action of a photocatalyst accompanying energy irradiation, and adhesion to cells. It may be one having no property, or one having cell adhesion inhibitory property.
- the adhesiveness between cells and a material is determined by electrostatic interaction or the like
- the adhesiveness to cells is determined by, for example, the amount of positive charge of the material.
- the material having an adhesive property to cells by such electrostatic interaction include a basic polymer such as polylysine, aminopropyltriethoxysilane, N- (2aminoethyl) 3aminopropyltrimethoxysilane and the like. Basic conjugates and condensates containing them, and the like.
- the above-mentioned material is decomposed or denatured by the action of a photocatalyst accompanying the energy irradiation, for example, the amount of positive charges present on the surface can be changed, and the adhesion to cells can be changed. It may be one having no property, or one having cell adhesion inhibitory property.
- Examples of the material having adhesive properties to cells due to its biological properties include those having good adhesive properties with specific cells or those having good adhesive properties with many cells. Examples include fibronectin, laminin, tenascin, vitronectin, RGD (arginine-glycine-aspartate) sequence-containing peptide, YIGSR (tyrosine isoleucine-glycine-serine arginine) sequence-containing peptide, collagen, atelocollagen, gelatin and the like. When such a material is used, it does not have adhesiveness to cells by the action of a photocatalyst accompanying energy irradiation, for example, by destroying a part of the structure of the material or by destroying the main chain. Or have cell adhesion inhibitory properties Wear.
- Such a cell adhesive material varies depending on the kind of the above-mentioned material and the like, but is usually 0.01% to 95% by weight, particularly 1% to 10% by weight in the photocatalyst-containing cell adhesive layer. % Is preferably contained. Thereby, the region containing the cell adhesive material can be a region having good adhesion to cells.
- the photocatalyst used in the present embodiment is not particularly limited as long as it can decompose or modify the above-mentioned cell adhesive material by the action of the photocatalyst accompanying energy irradiation.
- the action mechanism of a photocatalyst typified by titanium oxide as described later is not necessarily clear, but it is possible to directly react with a compound near the carrier force generated by light irradiation, or It is thought that the reactive oxygen species generated in the presence of oxygen, water, and water change the chemical structure of organic matter. In this embodiment, it is considered that this carrier has an effect on the above-mentioned cell adhesion material.
- titanium dioxide TiO 2
- zinc oxide ZnO
- tin oxide SnO 2
- strontium titanate S
- titanium dioxide is particularly preferably used because it has a high band gap energy, is chemically stable, is toxic, and is easily available. Titanium dioxide has an anatase type and a rutile type, and any of them can be used in the present embodiment. However, an anatase type titanium dioxide is preferred.
- anatase-type titanium dioxide examples include, for example, anatase-type titania sol of peptic hydrochloride type (STS-02 (average particle size: 7 nm) manufactured by Ishihara Sangyo Co., Ltd., ST-K01 manufactured by Ishihara Sangyo Co., Ltd.) ), Nitrate peptized anatase titania sol (TA-15 (Nissan Chemical Industries, Ltd. Particle size 12 nm))).
- STS-02 average particle size: 7 nm
- ST-K01 manufactured by Ishihara Sangyo Co., Ltd.
- TA-15 Nitrate peptized anatase titania sol
- a photocatalyst having a preferred average particle diameter of 50 nm or less, preferably 20 nm or less, because the smaller the particle diameter of the photocatalyst, the more effectively the photocatalytic reaction occurs.
- the content of the photocatalyst in the photocatalyst-containing cell adhesion layer of this embodiment can be set in the range of 5 to 95% by weight, preferably 10 to 60% by weight, and more preferably 20 to 40% by weight. Wear.
- the photocatalyst used in the present embodiment preferably has low adhesiveness to cells, for example, by having high hydrophilicity. This makes it possible to use the region where the photocatalyst is exposed due to the decomposition of the cell adhesive material or the like as the region having low adhesion to cells.
- the photocatalyst-containing cell adhesive layer may contain, for example, a binder or the like that improves the strength, resistance, or the like, if necessary, in addition to the cell adhesive material and the photocatalyst alone.
- a material having a cell adhesion-inhibiting property of inhibiting adhesion to cells at least after energy irradiation is used as the binder.
- a material having the above-described cell adhesion inhibitory property before the energy irradiation may be made to have the cell adhesion inhibitory property by the action of the photocatalyst accompanying the energy irradiation. You may.
- the binder a material that has cell adhesion inhibitory property particularly by the action of a photocatalyst accompanying energy irradiation.
- a material that has cell adhesion inhibitory property particularly by the action of a photocatalyst accompanying energy irradiation.
- the main skeleton has the above-mentioned photocatalyst. Preference is given to those having a high binding energy such that they are not decomposed by photoexcitation and have an organic substituent that can be decomposed by the action of a photocatalyst. And (2) organopolysiloxane obtained by crosslinking a reactive silicone excellent in water repellency and oil repellency, and the like.
- Y is an alkyl group, a fluoroalkyl group, a butyl group, an amino group, a phenyl group or an epoxy group, or an organic group containing these, and X represents an alkoxyl group, an acetyl group or a halogen.
- ⁇ is an integer from 0 to 3.
- the organopolysiloxane is one or more hydrolytic condensates or cohydrolytic condensates of the silicon compound represented by
- the carbon number of the organic group represented by ⁇ is preferably in the range of 1 to 20.
- Alkoxy group represented by X is a methoxy group, an ethoxy group, a propoxy group, or a butoxy group. Preferably, there is.
- Examples of the reactive silicone of the above (2) include compounds having a skeleton represented by the following general formula.
- n is an integer of 2 or more
- R 1 and R 2 are each a substituted or unsubstituted alkyl, alkaryl, aryl or cyanoalkyl group having 1 to 20 carbon atoms, and the molar ratio is 40% of the whole.
- the following are the bullets, fillers and halogenated fillers.
- the surface energy is lowest when R 2 is a methyl group, the methyl group is preferably at least 60% in a preferred molar ratio.
- at least one chain end or side chain It has a reactive group such as the above hydroxyl group.
- the surface of the region irradiated with the energy can be made highly hydrophilic by the action of the photocatalyst accompanying the energy irradiation. Thereby, the adhesion to the cells is inhibited, and the cells do not adhere to the energy-irradiated region.
- a stable organosilicon conjugate which does not undergo a crosslinking reaction like dimethylpolysiloxane, may be mixed with the above-mentioned organopolysiloxane in a binder.
- the contact angle with water before irradiation with energy is preferably in the range of 15 ° to 120 °, particularly preferably in the range of 20 ° to 100 °.
- the present invention is a glass which does not inhibit the adhesiveness of the above-mentioned cell adhesive material to cells.
- the material When the cell adhesion inhibiting material is irradiated with energy, the material preferably has a contact angle force with water of S 10 ° or less.
- the content By setting the content in the above range, it is possible to have high hydrophilicity and low adhesiveness to cells.
- the contact angle with water here is determined by measuring the contact angle with water or a liquid having an equivalent contact angle using a contact angle measuring device (CA-Z type manufactured by Kyowa Interface Science Co., Ltd.). The measurement (micro-syringe force was also performed 30 seconds after dropping the droplet) was obtained from the results or as a graph.
- a contact angle measuring device CA-Z type manufactured by Kyowa Interface Science Co., Ltd.
- the adhesiveness to cells is reduced or the decomposition is assisted by causing a change in the wettability of an area irradiated with energy. It may contain substances and the like.
- Examples of such a decomposed substance include, for example, a surfactant that is decomposed by the action of a photocatalyst accompanying energy irradiation, becomes hydrophilic, and thereby reduces adhesion to cells. it can.
- a surfactant that is decomposed by the action of a photocatalyst accompanying energy irradiation, becomes hydrophilic, and thereby reduces adhesion to cells. it can.
- hydrocarbons such as NIKKOL BL, BC, BO, and BB series from Nikko Chemicals, ZONYL FSN and FSO from DuPont, Surflon S-141, 145 from Asahi Glass, and Dainippon Japan.
- polyvinyl alcohol unsaturated polyester, acrylic resin, polyethylene, diaryl phthalate, ethylene propylene diene monomer, epoxy resin, phenol resin, polyurethane, melamine resin , Polycarbonate, polychlorinated vinyl, polyamide, polyimide, styrene butadiene rubber, chloroprene rubber, polypropylene, polybutylene, polystyrene, polyvinyl acetate, nylon, polyester, polybutadiene, polybenzimidazole, polyacryl-tolyl, epi Examples thereof include oligomers and polymers such as chlorhydrin, polysulfide, and polyisoprene.
- such a binder is contained in the photocatalyst-containing cell adhesion layer in an amount of 5 wt% to 95 wt%, particularly 40 wt% to 90 wt%, particularly 60 wt% to 80 wt%. It is preferable to be contained within the range.
- the substrate used in the present embodiment is not particularly limited as long as it has the above-mentioned convex portion.
- a base for example, inorganic materials such as metal, glass, and silicon, and organic materials typified by plastics can be used.
- the flexibility and the like of the base material are appropriately selected depending on the type and use of the cell culturing puttering substrate. Further, the transparency of the base material is appropriately selected depending on the type of the pattern culturing substrate for cell culture, the irradiation direction of energy applied for decomposing or denaturing the cell adhesive material, and the like.
- the substrate has a light-shielding portion and the like, and when the irradiation of the energy is performed from the substrate side, the substrate is made to have transparency.
- the substrate has permeability to the irradiated energy
- the substrate and the photocatalyst-containing cell adhesion are adhered so that the cell culture region is not irradiated with the energy.
- a light-shielding portion is formed between the layers and at a position corresponding to the cell culture area. Is also good.
- irradiating energy to the region where the convex portion is formed to form the cell adhesion inhibiting portion by irradiating the entire surface of the substrate rear surface with energy without using a photomask or the like, It is also a force capable of decomposing or denaturing the cell adhesion material in the photocatalyst-containing cell adhesion layer formed on the projection.
- any light-shielding portion that can block the energy applied to the cell culture patterning substrate when forming the cell adhesion inhibitory portion can be used.
- a metal thin film of chromium or the like having a thickness of about 1000 to 2000 A may be formed by a sputtering method, a vacuum evaporation method, or the like, and the thin film may be formed by patterning.
- a normal patterning method such as sputtering can be used.
- a method in which a layer containing light-shielding particles such as carbon fine particles, metal oxides, inorganic pigments, and organic pigments in a resin binder may be formed in a pattern.
- the resin binder used include one or a mixture of two or more resins such as polyimide resin, acrylic resin, epoxy resin, polyacrylamide, polybutyl alcohol, gelatin, casein, and cellulose.
- a reactive resin, or an OZW emulsion type resin composition for example, an emulsion obtained by emulsifying a reactive silicone can be used.
- the thickness of such a resin light-shielding portion can be set in the range of 0.5 to 10 / ⁇ .
- a method of patterning the resin light-shielding portion a commonly used method such as a photolithography method and a printing method can be used.
- a cell adhesion inhibiting portion is formed on the photocatalyst-containing cell adhesive layer 4 formed on the base material 1 having a convex portion by using, for example, a photomask 5 or the like.
- the cell adhesive material in the photocatalyst-containing cell adhesive layer 4 in the energy irradiated area is irradiated.
- the cell adhesion inhibitor contains a photocatalyst, a decomposed product or a modified product of the cell bonding material, and the like.
- energy irradiation (exposure) also includes irradiation with a single energy beam capable of decomposing or denaturing a cell adhesive material by the action of a photocatalyst accompanying the energy irradiation. This is a concept and is not limited to light irradiation.
- the wavelength of light used for such energy irradiation is generally set to a range of 400 nm or less, preferably 380 nm or less. This is because, as described above, a preferred photocatalyst used as a photocatalyst is titanium dioxide, and light having the above-mentioned wavelength is preferred as energy for activating photocatalysis by the titanium dioxide. is there.
- Examples of the light source that can be used for such energy irradiation include a mercury lamp, a metal halide lamp, a xenon lamp, an excimer lamp, and various other light sources.
- a method of performing pattern drawing irradiation using a laser such as excimer or YAG can also be used.
- the substrate-side power can also be applied by irradiating the entire surface with energy. In this case, there is an advantage that a step such as alignment that requires a photomask or the like is not required.
- the amount of energy irradiation at the time of energy irradiation is an irradiation amount necessary for the cell adhesion material to be decomposed or denatured by the action of a photocatalyst.
- the sensitivity can be increased, and the cell adhesion material can be efficiently decomposed or denatured, which is preferable. .
- heating within the range of 30 ° C to 80 ° C is preferred! / ⁇
- the direction of the energy irradiation performed through the photomask is such that, when the above-described base material is transparent, the energy irradiation is performed from the direction of deviation between the base material side and the photocatalyst-containing cell adhesive layer side. Is also good.
- the substrate when the substrate is opaque, it is necessary to perform energy irradiation from the photocatalyst-containing cell-adhesive layer side.
- the photocatalyst and the A photocatalyst-containing cell-adhesion-inhibiting layer containing a cell-adhesion-inhibiting material that has cell-adhesion-inhibiting properties that inhibits adhesion to cells and that is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation are one in which the cell adhesion inhibiting material is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation.
- the photocatalyst-containing cell adhesion-inhibiting layer contains the photocatalyst and the cell adhesion-inhibiting material
- energy is applied to regions other than the convex portions of the photocatalyst-containing cell adhesion-inhibiting layer.
- the energy-irradiated region becomes a cell-adhesion portion having adhesiveness to cells, that is, a cell culture region. It can be done.
- the cell adhesion-inhibiting material remains, and the cell adhesion-inhibiting property can be obtained.
- the photocatalyst-containing cell adhesion inhibiting layer used in this embodiment is a layer containing the photocatalyst and the above-mentioned cell adhesion-inhibiting material. This is a layer having adhesiveness.
- Such formation of the photocatalyst-containing cell adhesion inhibitory layer is performed by using a cell adhesive material that is decomposed or modified by the action of the photocatalyst accompanying energy irradiation and a coating solution for forming a photocatalyst-containing cell adhesion inhibitory layer that contains the photocatalyst. It can be carried out by, for example, applying the composition on the base material having the convex portions.
- the coating solution for forming the photocatalyst-containing cell adhesion inhibiting layer can be applied by a general coating method, for example, a spin coating method, a spray coating method, a dip coating method, a roll coating method, a bead coating method, or the like. Can be used.
- the thickness of the photocatalyst-containing cell adhesion-inhibiting layer is a force which is appropriately selected depending on the type of the cell culture patterning substrate and the like, and is usually 0.01 m to 1.0 m. About 0.1 ⁇ m to 0.3 ⁇ m.
- photocatalyst-containing cell adhesion-inhibiting layer materials used for the photocatalyst-containing cell adhesion-inhibiting layer will be described.
- the photocatalyst used in the present embodiment can be the same as the photocatalyst used in the above-described first embodiment, and a detailed description thereof will be omitted.
- the cell adhesion-inhibiting material used in the present embodiment has a cell adhesion-inhibiting property of inhibiting adhesion to cells and is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation.
- the type and the like are not particularly limited.
- having the cell adhesion inhibitory property means that the cell has the property of inhibiting the cell from adhering to the cell adhesion inhibitory material, that is, when the adhesiveness to the cell differs depending on the type of the cell. And the like means having the property of inhibiting adhesion to a target cell.
- the cell adhesion-inhibiting material used in this embodiment has such cell adhesion-inhibiting properties, and is degraded or denatured by the action of a photocatalyst associated with energy irradiation, and has no cell adhesion-inhibiting properties. And those having good adhesion to cells are used.
- a material having a high hydration ability can be used as such a cell adhesion-inhibiting material.
- a material with high hydration ability forms a hydration layer in which water molecules are gathered around it.
- a substance with such high hydration ability has a higher adhesiveness to water molecules than an adhesiveness to cells. Therefore, the cells cannot adhere to the material having a high hydration ability and have low adhesion to the cells.
- the above-mentioned hydration ability means a property of hydration with water molecules, and a high hydration ability means that it is easy to hydrate with water molecules.
- Examples of the material having a high hydration ability and used as a cell adhesion inhibiting material include polyethylene glycol, a zwitterionic material having a betaine structure and the like, and a phospholipid-containing material.
- the cell adhesion-inhibiting material is decomposed or deteriorated by the action of a photocatalyst when irradiated with energy in an energy irradiation step described below, and the surface of the material is damaged. By leaving the hydrated layer, it is possible to have no such cell adhesion inhibitory property.
- a surfactant having a water-repellent or oil-repellent organic substituent which can be decomposed by the action of a photocatalyst can also be used as the cell adhesion-inhibiting material.
- a surfactant include hydrocarbons such as NIKKOL BL, BC, BO, and BB series manufactured by Nikko Chemicals Co., Ltd., ZONYL FSN and FS0 manufactured by DuPont, and Surflon S manufactured by Asahi Glass Co., Ltd.
- the cell adhesion-inhibiting material is unevenly distributed on the surface.
- the water repellency and oil repellency of the surface can be increased, and the interaction with the cell, which has a small interaction with the cell, can be reduced.
- the layer is irradiated with energy in the energy irradiation step, the layer is easily decomposed by the action of the photocatalyst to expose the photocatalyst and not have the cell adhesion inhibitory property. You can.
- the present embodiment it is particularly preferable to use, as the above-mentioned cell adhesion-inhibiting material, a material having good adhesion to cells by the action of a photocatalyst accompanying energy irradiation.
- a material having good adhesion to cells by the action of a photocatalyst accompanying energy irradiation examples thereof include materials having oil repellency and water repellency.
- the water-repellent or oil-repellent properties of the cell-adhesion-inhibiting material for example, a hydrophobic property between the cell and the cell adhesion-inhibiting material. Adhesion with cells with small interaction such as sexual interaction can be reduced.
- Such a water-repellent or oil-repellent material is, for example, a material having a high binding energy such that the skeleton is not decomposed by the action of a photocatalyst and which is decomposed by the action of a photocatalyst.
- Those having a water-repellent or oil-repellent organic substituent may be mentioned.
- Examples of those having a high binding energy such that the skeleton is not decomposed by the action of a photocatalyst and having a water-repellent or oil-repellent organic substituent which is decomposed by the action of a photocatalyst include those described above.
- Organopolysiloxane which exerts high strength by hydrolyzing and polycondensing the dye or alkoxysilane by sol-gel reaction, etc., and (2) Cross-linking reactive silicone And the like organopolysiloxanes.
- the side chain of the organopolysiloxane or the like is decomposed or denatured at a high rate by the action of a photocatalyst accompanying energy irradiation.
- the side chains of the organopolysiloxane and the like are completely decomposed by the action of the photocatalyst accompanying the energy irradiation.
- the region irradiated with the energy can be made to have adhesiveness to cells.
- a stable organosilicon compound that does not undergo a crosslinking reaction like dimethylpolysiloxane may be separately mixed with the above-mentioned organopolysiloxane and the like.
- the material having a contact angle with water of 80 ° or more, particularly in the range of 100 ° to 130 ° is usually used as the cell adhesion-inhibiting material. It is preferable to use them. This is because the photocatalyst-containing cell adhesion-inhibiting layer before energy irradiation can have low adhesiveness to cells.
- the upper limit of the angle is the upper limit of the contact angle of the cell adhesion-inhibiting material with water on a flat substrate, for example, the water of the cell adhesion-inhibiting material on a substrate having irregularities.
- the contact angle with water is preferably 10 ° to 40 °, more preferably 15 ° to 30 °. It is preferable to irradiate the energy so as to be within. This makes it possible to enhance the adhesiveness of the photocatalyst-containing cell adhesion-inhibiting layer to cells after energy irradiation. It is.
- the contact angle with water is obtained by the above-described method.
- Such a cell adhesion-inhibiting material is preferably contained in the photocatalyst-containing cell adhesion-inhibiting layer in a range of 0.01% to 95% by weight, particularly preferably 1% to 10% by weight. This is a force that enables the region containing the cell adhesion inhibiting material to be a region having low adhesion to cells.
- the above-mentioned cell adhesion inhibiting material preferably has surface activity.
- a coating solution for forming a photocatalyst-containing cell adhesion inhibitor layer containing the above-mentioned cell adhesion inhibitor material is applied and then dried, the rate of uneven distribution on the surface of the coating film increases, resulting in good cell adhesion. This is because inhibitory properties can be obtained.
- the photocatalyst-containing cell adhesion-inhibiting layer of the present embodiment contains a binder or the like in accordance with required properties such as coatability when forming the layer, strength and resistance when forming the layer, and the like. May be. Further, the cell adhesion-inhibiting material may function as the binder.
- a binder having a high binding energy such that the main skeleton is not decomposed by the action of the photocatalyst can be used.
- polysiloxane having no organic substituent or having a small amount of organic substituent that does not affect the adhesion can be mentioned, such as tetramethoxysilane and tetraethoxysilane. Can be obtained by hydrolysis and polycondensation.
- such a binder is contained in the photocatalyst-containing cell adhesion-inhibiting layer in an amount of 5% by weight to 95% by weight, particularly 40% by weight to 90% by weight, particularly 60% by weight to 80% by weight. It is preferable to be contained within the range. This makes it possible to exhibit properties such as facilitating formation of the photocatalyst-containing cell adhesion inhibition layer and imparting strength to the photocatalyst-containing cell adhesion inhibition layer.
- the photocatalyst-containing cell adhesion-inhibiting layer contains a cell-adhesive material having an adhesive property to cells at least after being irradiated with energy.
- the photocatalyst-containing cell adhesion-inhibiting layer improves the adhesion to the cells in the cell-adhesion area, which is the area irradiated with energy, that is, the cells in the cell culture area. It is the power that can be done.
- a cell adhesive material may be a material used as the above-mentioned binder, or may be a material used separately from the binder.
- the precursor may be one having good adhesiveness to the cells due to the energy irradiation, or one having good adhesiveness to the cells by the action of the photocatalyst accompanying the energy irradiation.
- having the adhesiveness with the cells means that the cells adhere well to the cells. If the adhesiveness with the cells differs depending on the type of the cells, etc., it should adhere well to the target cells. ⁇ ⁇ .
- adhesion to cells may be, for example, hydrophobic interaction or electrostatic. It may be good due to physical interaction such as interaction, hydrogen bonding, van der Waals force, etc. .
- such a cell adhesion material is contained in the photocatalyst-containing cell adhesion inhibition layer.
- the photocatalyst-containing cell adhesion-inhibiting layer is also capable of improving the adhesiveness of the cell adhesion portion, which is the region irradiated with energy, to cells.
- the cell adhesion inhibition in a region not irradiated with the energy, that is, a region serving as the cell adhesion inhibitor. It is preferably contained to such an extent that the cell adhesion inhibiting property of the material is not inhibited.
- the substrate used in the present embodiment is not particularly limited as long as it has the above-mentioned convex portion, and may be the substrate described in the first embodiment.
- a light-shielding portion is formed in a region where the convex portion is formed on the base material. It may be.
- the photocatalyst-containing cell adhesion inhibition layer on the cell culture region is irradiated with energy to form a cell adhesion portion, it is not necessary to use a photomask or the like from the back side of the substrate. By irradiating the entire surface with energy, it is also a force that can easily form a cell adhesion portion.
- a light-shielding portion may be separately formed in addition to the convex portion of the base material, but the light-shielding portion may be formed as a convex portion of the base material. This is a force that enables the formation of a putter substrate for cell culture with high production efficiency.
- the type of the base material used in the present embodiment the method of forming the light-shielding portion, the type, and the like are the same as those described in the first embodiment, and a detailed description thereof will be omitted. You.
- the photocatalyst-containing cell adhesion inhibition layer on the cell culture region is irradiated with energy using, for example, a photomask.
- the cell adhesion inhibitor in the region irradiated with energy can be decomposed or denatured, and a cell adhesion portion (cell culture region) having adhesiveness to cells can be obtained.
- the cell adhesion portion contains a photocatalyst, a decomposition product or a denatured product of the cell adhesion inhibitor, and the like.
- the cell adhesion-inhibiting material remains, and the cell adhesion-inhibiting portion having no adhesiveness to cells can be formed.
- energy irradiation refers to irradiation of a line of energy capable of decomposing or denaturing a cell adhesion-inhibiting material by the action of a photocatalyst accompanying the energy irradiation. And is not limited to light irradiation.
- a photocatalyst-containing layer containing at least a photocatalyst and a cell that has adhesiveness to cells and is degraded or denatured by the action of the photocatalyst accompanying energy irradiation are provided on the base material.
- a cell adhesive layer containing an adhesive material is formed, and the cell adhesion inhibitor is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation.
- the cell adhesion layer is formed on the photocatalyst-containing layer, by irradiating energy to the region where the projections are formed, the cell adhesion layer in the cell adhesion layer is irradiated.
- the material is decomposed or denatured by the action of the photocatalyst in the adjacent photocatalyst-containing layer, so that it is possible to form a cell-adhesion-inhibited portion having reduced adhesion to cells in that region.
- the cell adhesion inhibitor contains a small amount of the cell adhesion material or
- the photocatalyst-containing layer is exposed due to the presence of a decomposition product or the like, or the cell adhesion layer is completely decomposed and removed.
- the denatured product or the like is contained in the cell adhesion inhibitor.
- the cell adhesion inhibiting portion formed with the projections is formed.
- the cells attached to the cell adhesion inhibitor can be removed by the action of the photocatalyst-containing layer. This has the advantage that cells cultured in a high-definition pattern can be maintained.
- the substrate used in the present embodiment and the method for forming the cell adhesion inhibitor in the present embodiment are the same as those in the first embodiment described above, and thus description thereof will be omitted.
- the cell adhesive layer used in this embodiment is a layer having at least a cell adhesive material having an adhesive property to cells, and a layer generally used as a layer having an adhesive property to cells can be used.
- the cell adhesion layer of the present embodiment also contains the material having the cell adhesion inhibitory property described in the photocatalyst-containing cell adhesion layer described in the first embodiment. Thereby, the adhesiveness with the cells of the cell adhesion inhibitor, which is the area irradiated with energy, can be reduced.
- the formation of such a cell adhesion layer can be performed by, for example, applying a coating solution for forming a cell adhesion layer containing the above cell adhesion material by a general application method.
- the method can be the same as the method for forming the photocatalyst-containing cell adhesive layer of the embodiment, the description thereof is omitted here.
- the adsorption method may be applied to the formation of the cell adhesive layer.
- the thickness of such a cell adhesion layer is appropriately selected depending on the type of the cell culturing pattern substrate, etc. Normally about 0.001 / ⁇ to 1.O / zm. Especially, it can be about 0.01 ⁇ m to 0.3 ⁇ m.
- the photocatalyst-containing layer used in this embodiment is not particularly limited as long as it is a layer containing at least a photocatalyst.
- the photocatalyst-containing layer may be a layer having only the power of the photocatalyst, or may be a layer containing other components such as a binder. It may be.
- the photocatalyst used in this embodiment can be the same as that used for the photocatalyst-containing cell adhesion layer in the first embodiment, and in this embodiment, titanium oxide is particularly used. Is preferred.
- Examples of a method for forming a photocatalyst-containing layer in which only the photocatalyst is effective include a method using a vacuum film-forming method such as a sputtering method, a CVD method, and a vacuum evaporation method.
- a vacuum film-forming method such as a sputtering method, a CVD method, and a vacuum evaporation method.
- amorphous titania is formed on a substrate, and then fired. And a method of changing the phase to crystalline titania.
- the amorphous titanium used herein include, for example, hydrolysis, dehydration condensation of inorganic salts of titanium such as titanium tetrachloride and titanium sulfate, tetraethoxytitanium, tetraisopropoxytitanium, tetra-n-propoxytitanium, and tetrabutoxytitanium.
- an organic titanium conjugate such as tetramethoxytitanium can be obtained by hydrolysis and dehydration condensation in the presence of an acid. Then, it can be modified to anatase type titania by baking at 400 ° C to 500 ° C, and can be modified to rutile type titania by baking at 600 ° C to 700 ° C.
- a binder When a binder is used, those having a high binding energy such that the main skeleton of the binder is not decomposed by the photoexcitation of the photocatalyst are preferable.
- a binder includes the cell adhesion layer described above.
- the organopolysiloxane used in the above section is preferable.
- the photocatalyst-containing layer is formed by dispersing the organocatalyst, which is a photocatalyst, and a binder in a solvent together with other additives as necessary. It can be formed by preparing a coating solution and applying the coating solution onto a substrate.
- the solvent to be used alcohol-based organic solvents such as ethanol and isopropanol are preferable.
- the coating can be performed by a known coating method such as spin coating, spray coating, dip coating, roll coating, and bead coating.
- the photocatalyst-containing layer can be formed by performing a curing treatment by irradiating ultraviolet rays.
- an amorphous silica precursor can be used as a binder.
- This amorphous silica precursor is represented by the general formula SiX, where X is a halogen, methoxy, ethoxy, or acetyl group.
- silicon compounds such as hydroxyl groups, silanols which are hydrolysates thereof, and polysiloxanes having an average molecular weight of 3000 or less!
- Specific examples include tetraethoxysilane, tetraisopropoxysilane, tetra-n-propoxysilane, tetrabutoxysilane, tetramethoxysilane and the like.
- the precursor of the amorphous silica and the particles of the photocatalyst are uniformly dispersed in a non-aqueous solvent, and the transparent substrate is hydrolyzed with moisture in the air to form silanol.
- the photocatalyst-containing layer can be formed by dehydration-condensation polymerization at room temperature.
- the content of the photocatalyst in the photocatalyst-containing layer can be set in the range of 5 to 60% by weight, preferably 20 to 40% by weight.
- the thickness of the photocatalyst-containing layer is preferably in the range of 0.05 to: LO / zm.
- the photocatalyst-containing layer may also contain a surfactant or the like used in the cell adhesion layer described above.
- the surface of the photocatalyst-containing layer preferably has low adhesiveness to cells, for example, a low hydrophilicity due to the hydrophilic surface.
- the region can be a region having low adhesion to cells.
- a light-shielding portion may be formed on the photocatalyst-containing layer.
- the photocatalyst on the region where the light-shielding portion is formed is not excited, and is contained in the cell adhesive layer other than the region where the light-shielding portion is formed.
- a force that can degrade or denature cell adhesion materials is not particularly limited.
- a photocatalyst-containing layer containing at least a photocatalyst on the above-mentioned base material, and a photocatalytic action having cell adhesion inhibitory property of inhibiting adhesion to cells and accompanying energy irradiation
- a cell adhesion-inhibiting layer containing a cell adhesion-inhibiting material that is decomposed or denatured by the reaction is formed, and the cell adhesion portion is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation. Is what it is.
- the cell adhesion inhibiting layer is formed on the photocatalyst-containing layer, the cell adhesion inhibiting layer formed in a region other than the region in which the convex portion is formed has a different shape.
- the photocatalyst contained in the photocatalyst containing layer is excited,
- the cell adhesion-inhibiting material in the cell adhesion-inhibiting layer can be decomposed or denatured, and a cell adhesion portion (cell culture region) can be formed. Further, at this time, a region where the energy is not irradiated and the convex portion where the cell adhesion inhibiting material remains is formed, can be a cell adhesion inhibiting portion.
- the cell adhesion-inhibiting material is decomposed or denatured!” Means that the cell adhesion-inhibiting material is contained, or that the cell adhesion-inhibiting material is contained in the cell adhesion-inhibiting portion. That the cell adhesion-inhibiting material is contained in a smaller amount than the amount of the cell adhesion inhibitor.
- the cell adhesion portion when the cell adhesion inhibiting material is decomposed by the action of a photocatalyst accompanying energy irradiation, the cell adhesion portion contains a small amount of the cell adhesion inhibiting material, or the cell adhesion inhibiting material
- the photocatalyst-containing layer is exposed due to the presence of decomposed products and the like, and the cell adhesion-inhibiting material is completely decomposed to expose the photocatalyst-containing layer.
- the above-mentioned cell adhesion-inhibiting material is modified by the action of a photocatalyst accompanying energy irradiation, the cell adhesion portion contains the denatured product or the like.
- the cell adhesive portion contains a cell adhesive substance having an adhesive property to cells after at least energy irradiation.
- the adhesiveness between the cell adhesion portion and the cell can be further enhanced, and the cell can be adhered to only the cell adhesion portion with high definition.
- the photocatalyst-containing layer used in the present embodiment can be the same as that described in the third embodiment described above, and the substrate and the cell adhesion used in the present embodiment can be used.
- the method of forming the portion can be the same as in the above-described second embodiment, and a description thereof is omitted here.
- the cell adhesion inhibitory layer used in this embodiment is formed on the photocatalyst-containing layer, has a cell adhesion inhibitory property of inhibiting adhesion to cells, and is decomposed or degraded by the action of a photocatalyst accompanying energy irradiation. It is not particularly limited as long as it contains a cell adhesion inhibiting material to be denatured.
- the formation method and the like are particularly limited.
- it can be formed by applying a coating solution for forming a cell adhesion-inhibiting layer containing the cell adhesion-inhibiting material to the photocatalyst-containing layer by a general application method. it can.
- the thickness of such a cell adhesion-inhibiting layer is appropriately selected depending on the type of the cell culture patterning substrate and the like.
- the force can be set to about 0 ⁇ m, especially about 0.1 ⁇ m to 0.3 ⁇ m.
- the material is used for the photocatalyst-containing cell adhesion-inhibiting layer described in the first embodiment. Since the same material as the cell adhesion inhibiting material can be used, detailed description is omitted here. Further, it is preferable that the cell adhesion-inhibiting layer of the present embodiment also contains the material having the cell adhesion described in the photocatalyst-containing cell adhesion-inhibiting layer described in the first embodiment. Thereby, the adhesiveness of the cell adhesion portion (cell culture region), which is the region irradiated with energy, to the cells can be increased.
- a cell adhesive layer containing a cell adhesive material having at least adhesive properties to cells and being decomposed or denatured by the action of a photocatalyst accompanying energy irradiation is formed.
- the inhibition portion is one in which the cell adhesion material is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation.
- the cell adhesion layer and the photocatalyst-containing layer are arranged so as to face each other, and energy is applied to a pattern forming a cell adhesion inhibition portion, that is, a region where the projections are formed.
- a cell adhesion inhibition portion that is, a region where the projections are formed.
- the cell adhesion inhibition portion formed with the convex portion is formed.
- the photocatalyst-containing layer-side substrate By irradiating energy using the photocatalyst-containing layer-side substrate, cells adhering to the cell adhesion inhibitor can be removed by the action of a photocatalyst, and cells cultured in a high-definition pattern can be removed. Can be maintained, t also have advantages.
- the photocatalyst-containing layer-side substrate used in the present embodiment and the photocatalyst-containing layer-side substrate will be used. Then, a method of forming the cell adhesion inhibitor will be described. Note that the cell adhesive layer used in this embodiment is the same as that used in the above-described third embodiment, and a description thereof will be omitted.
- the photocatalyst-containing layer-side substrate used in the present embodiment usually has a photocatalyst-containing layer containing a photocatalyst, and usually has a substrate and a photocatalyst-containing layer formed on the substrate.
- the photocatalyst-containing layer-side substrate may include, for example, a photocatalyst-containing layer-side light-shielding portion or a primer layer formed in a pattern.
- the photocatalyst containing layer used for the photocatalyst containing layer side substrate will be described.
- the photocatalyst-containing layer used in the present embodiment is not particularly limited as long as the photocatalyst in the photocatalyst-containing layer decomposes or denatures the cell adhesion material in the adjacent cell adhesion layer.
- the film may be composed of a binder and a photocatalyst alone.
- the characteristics of the surface may be lyophilic or lyophobic.
- the photocatalyst-containing layer used in the present embodiment may be formed on the entire surface of the substrate, but for example, as shown in Fig. 3, a photocatalyst-containing layer 12 is formed on the substrate 11 on a pattern. It may have been done.
- the photocatalyst-containing layer in a pattern as described above, when irradiating energy to form the cell adhesion inhibition portion, the entire surface where irradiation with a pattern using a photomask or the like is not necessary is required. By irradiation, the cell adhesion material contained in the cell adhesion layer can be decomposed or denatured to form a cell adhesion inhibitor.
- the method for patterning the photocatalyst-containing layer is not particularly limited, but can be performed by, for example, one photolithography method or the like.
- the energy irradiation direction is the same as that of the photocatalyst containing layer. Irradiation may be performed from any direction as long as the energy is applied to the portion where the layer and the cell adhesion layer face each other, and the energy to be applied is not particularly limited to parallel light such as parallel light. This has the following advantages.
- the photocatalyst-containing layer used in this embodiment can be the same as the photocatalyst-containing layer described in the above-described third embodiment, and a detailed description thereof will be omitted. .
- the photocatalyst-containing layer-side substrate has at least a substrate and a photocatalyst-containing layer formed on the substrate.
- the material constituting the base to be used is appropriately selected depending on the direction of energy irradiation described later, whether the obtained pattern formed body needs transparency, and the like.
- the substrate used in the present embodiment may be a flexible substrate, for example, a resin film, or a non-flexible substrate, for example, a glass substrate. This is appropriately selected depending on the energy irradiation method.
- an anchor layer may be formed on the substrate.
- an anchor layer include silane-based and titanium-based coupling agents.
- the photocatalyst-containing layer-side substrate used in the present embodiment may be one having a photocatalyst-containing layer-side light-shielding portion formed in a pattern.
- the photocatalyst-containing layer-side substrate having such a photocatalyst-containing layer-side light-shielding portion can be in the following two modes depending on the position where the photocatalyst-containing layer-side light-shielding portion is formed.
- One is to form a photocatalyst-containing layer-side light-shielding portion 14 on a substrate 11 as shown in FIG. 4, for example.
- the photocatalyst-containing layer 12 is formed on the photocatalyst-containing layer-side light-shielding portion 14 to form a photocatalyst-containing layer-side substrate.
- the other is a mode in which a photocatalyst-containing layer 12 is formed on a base 11 and a photocatalyst-containing layer-side light-shielding portion 14 is formed thereon to form a photocatalyst-containing layer-side substrate, as shown in FIG. 5, for example.
- the photocatalyst-containing layer-side light-shielding portion is disposed in the vicinity of the portion where the photocatalyst-containing layer and the cell adhesive layer are disposed, as compared with the case where a photomask is used. Since the influence of energy scattering in the body or the like can be reduced, it is possible to perform energy pattern irradiation extremely accurately.
- the photocatalyst-containing layer-side light-shielding portion 14 is formed on the photocatalyst-containing layer 12 as shown in FIG.
- the photocatalyst-containing layer-side light-shielding portion is arranged at the position shown in FIG. If it can be used as a spacer, it has the following advantages.
- the photocatalyst-containing layer and the cell adhesive layer are arranged so as to face each other with a predetermined gap, the photocatalyst-containing layer-side light-shielding portion and the cell adhesive layer are in close contact with each other.
- the predetermined gap can be made accurate, and by irradiating energy in this state, the portion of the cell adhesive layer where the cell adhesive layer and the light shielding portion are in contact with each other is Since the adhesive material is not decomposed or denatured, the cell adhesion inhibitor can be formed with high accuracy.
- the method of forming the light-blocking portion on the photocatalyst-containing layer side is not particularly limited, and may be appropriately determined according to the characteristics of the surface on which the light-blocking portion on the photocatalyst-containing layer side is formed, the shielding property against required energy, and the like. Since it can be selected and used and can be the same as the light-shielding portion provided on the base material described in the first embodiment, detailed description is omitted here.
- the photocatalyst-containing layer-side light-shielding portion is formed at two positions, that is, between the substrate and the photocatalyst-containing layer and on the surface of the photocatalyst-containing layer. It is also possible to adopt a mode in which a photocatalyst containing layer side light-shielding portion is formed on the surface on the side where the containing layer is not formed. In this embodiment, for example, a case may be considered in which a photomask is brought into close contact with the surface to such an extent that the photomask can be detached. It can be suitably used in a case where a no-turn is changed in a small lot.
- the primer layer used in the photocatalyst-containing layer-side substrate of the present embodiment will be described.
- the photocatalyst-containing layer-side substrate is formed by forming the photocatalyst-containing layer-side light-shielding portion on the substrate in a pattern and forming the photocatalyst-containing layer thereon.
- a primer layer may be formed between the light-shielding portion on the containing layer side and the photocatalyst containing layer.
- the function and function of the primer layer are not necessarily clear, but by forming the primer layer between the light-shielding portion and the photocatalyst-containing layer on the photocatalyst-containing layer side, the primer layer becomes a cell by the action of the photocatalyst. Impurities from the photocatalyst-containing layer-side light-shielding portion and the openings existing between the photocatalyst-containing layer-side light-shielding portions that cause degradation or denaturation of the adhesive material, particularly residues generated when patterning the photocatalyst-containing layer-side light-shielding portion It is considered to have a function of preventing diffusion of impurities such as metal and metal ions. Therefore, by forming the primer layer, the process of decomposing or denaturing the cell adhesion material proceeds with high sensitivity, and as a result, it is possible to obtain a cell adhesion inhibition portion formed with high definition. You.
- the primer layer prevents impurities present not only in the light-shielding portion on the photocatalyst-containing layer side but also in the opening formed between the light-shielding portions on the photocatalyst-containing layer from affecting the action of the photocatalyst. Therefore, the primer layer is preferably formed over the entire light-shielding portion on the photocatalyst-containing layer side including the opening.
- the primer layer in this embodiment is not particularly limited as long as the primer layer is formed so that the photocatalyst-containing layer-side light-shielding portion of the photocatalyst-containing layer-side substrate does not contact the photocatalyst-containing layer.
- the material constituting the primer layer is not particularly limited, but an inorganic material that is not easily decomposed by the action of a photocatalyst is preferable.
- Specific examples include amorphous silica.
- the precursor of the amorphous silica is represented by the general formula SiX, wherein X is a halogen, a methoxy group, an ethoxy group, or an acetyl group.
- the thickness of the primer layer is preferably in the range of 0.001 ⁇ m to 1 ⁇ m, particularly preferably in the range of 0.001 ⁇ m to 0.1 ⁇ m.
- the cell adhesion layer 8 formed on the substrate 1 and the photocatalyst containing layer 12 of the photocatalyst containing layer side substrate 13 are arranged at a predetermined gap. Then, for example, using a photomask 5 or the like, a region where the above-mentioned convex portion is formed is irradiated with energy 6 in a predetermined direction (FIG. 6A). As a result, the cell adhesive material in the energy-irradiated area is degraded or denatured, and has no adhesion to the cells!
- a cell adhesion inhibitor 7 is formed in the cell adhesive 9 (FIG. 6). (b)).
- the cell adhesion inhibitor is, for example, when the above-mentioned cell adhesion material is decomposed by the action of a photocatalyst accompanying energy irradiation.
- the cell adhesion inhibitor contains a small amount of the cell adhesion material, or contains a decomposition product of the cell adhesion material, or the cell adhesion layer is completely decomposed and removed to expose the substrate. And so on. Further, when the cell adhesive material is modified by the action of a photocatalyst accompanying energy irradiation, the modified substance or the like is contained in the cell adhesion inhibitor.
- the above-mentioned arrangement refers to a state in which the action of the photocatalyst substantially extends to the surface of the cell adhesive layer.
- the photocatalyst-containing layer and the cell adhesion layer are arranged at an interval. This gap is preferably 200 ⁇ m or less.
- the gap is particularly excellent in consideration of the fact that the pattern accuracy is extremely good, the sensitivity of the photocatalyst is high, and the efficiency of decomposition or denaturation of the cell adhesive material in the cell adhesive layer is good. It is preferably in the range of 2 ⁇ m to 10 ⁇ m, preferably in the range of 1 ⁇ m to 5 ⁇ m. Such a range of the gap is particularly effective for a small-area cell adhesion layer capable of controlling the gap with high accuracy.
- the gap is preferably in the range of 10 to: LOO / zm, particularly preferably in the range of 50 to 75 m.
- the gap in the positioning device between the photocatalyst-containing layer-side substrate and the cell adhesive layer in the energy irradiation device is set to 10 ⁇ m to 10 ⁇ m. It is preferable to set within the range of 200 ⁇ m, especially within the range of 25 ⁇ m to 75 ⁇ m. By setting the set value within such a range, it is possible to prevent the cell-adhering layer from being in contact with the photocatalyst-containing layer-side substrate without causing a significant decrease in pattern accuracy or a significant deterioration in photocatalytic sensitivity. This is because it is possible to dispose them.
- An example of a method of forming such an extremely narrow gap uniformly and arranging the photocatalyst-containing layer and the cell adhesion layer includes a method using a spacer.
- a spacer By using the spacer in this manner, a uniform gap can be formed, and the portion where the spacer comes into contact is not affected by the action of the photocatalyst on the surface of the cell adhesive layer.
- the spacer By making the spacer have a pattern similar to that of the above-mentioned cell-adhesive portion, the cell-adhesive material only in a portion where the spacer is not formed can be decomposed or denatured, and the cell can be highly defined.
- the adhesion inhibition part can be formed.
- such an arrangement state of the photocatalyst-containing layer-side substrate should be maintained only at least during one energy irradiation.
- energy irradiation also includes irradiation with a single energy beam capable of decomposing or denaturing a cell adhesive material by the action of a photocatalyst accompanying the energy irradiation. This is a concept and is not limited to light irradiation.
- the direction of the energy irradiation performed through the photomask is such that when the above-described base material is transparent, the energy irradiation is performed from the direction of displacement between the base material side and the photocatalyst containing layer side substrate. May be.
- the substrate when the substrate is opaque, it is necessary to perform energy irradiation from the photocatalyst-containing layer side substrate side.
- a cell adhesion inhibitory layer that at least inhibits adhesion to cells is provided on the substrate, and is degraded or denatured by the action of a photocatalyst accompanying energy irradiation.
- a cell adhesion-inhibiting layer containing an inhibitory material is formed, and the above-mentioned cell-adhesion inhibitor is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation.
- the cell adhesion-inhibiting layer contains a cell adhesion-inhibiting material that is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation
- the cell adhesion-inhibiting layer The photocatalyst-containing layer and the photocatalyst-containing layer are arranged to face each other, and energy is irradiated in a pattern in the cell-adhesion culture area. Alternatively, it can be denatured to form a cell culture region (cell adhesion region) having adhesive properties to cells.
- the cell adhesion-inhibiting material remains in the region that has not been irradiated with energy, it can be assumed that the region has no adhesiveness to cells and can be used as a cell adhesion-inhibiting portion. Because
- the cell adhesion-inhibiting material is degraded or denatured! Means that the cell adhesion-inhibiting material is contained, or that the cell adhesion-inhibiting material is contained in the cell adhesion-inhibiting portion. That the cell adhesion-inhibiting material is contained in a smaller amount than the amount of the cell adhesion inhibitor.
- the cell adhesion portion contains a small amount of the cell adhesion inhibiting material, or the cell adhesion inhibiting material A degradation product or the like is contained, or the cell adhesion-inhibiting material is completely decomposed to expose the substrate.
- the above-mentioned cell adhesion-inhibiting material is modified by the action of a photocatalyst accompanying energy irradiation,
- the cell adhesive portion contains a cell adhesive substance having an adhesive property to cells after at least energy irradiation. Thereby, the adhesiveness between the cell adhesion portion and the cell can be further enhanced, and the cell can be adhered to only the cell culture region with high definition.
- the cell adhesion inhibiting layer used in the present embodiment is the same as the cell adhesion inhibiting layer described in the fourth embodiment, and the substrate on the photocatalyst containing layer side, the arrangement thereof, the method of irradiating energy, and the like are described. Since this is the same as that described in the fifth embodiment, a detailed description thereof will be omitted.
- the above-described pattern culturing pattern substrate may be used for culturing vascular cells.
- a blood vessel can be formed by, for example, culturing vascular cells forming a blood vessel in a pattern on the cell culture region, and then adding a growth factor that promotes vascularization of the vascular cells.
- the vascular cells that organize such blood vessels refer to, for example, vascular endothelial cells, pericytes, smooth muscle cells, vascular endothelial progenitor cells, and smooth muscle progenitor cells obtained from various organisms, particularly human ⁇ animals, In particular, vascular endothelial cells and the like can be used.
- co-culture of a plurality of types of cells such as co-culture of vascular endothelial cells and perisite or co-culture of vascular endothelial cells and smooth muscle cells, can be used.
- the medium is A blood vessel can be obtained by adding a growth factor such as bFGF or VEGF that promotes vascularization of vascular cells. It is thought that the stimulus also receiving such a growth factor force causes the vascular cells to stop growing, differentiate and become vascularized.
- a gel medium or a gel medium containing the above growth factors may be used as a medium for vascularizing vascular cells adhered to the confluent on the vascular culture area.
- a culture medium containing a growth factor as described above which is a combination of a medium and a liquid medium, can be used.
- the medium in the form of a gel collagen, fibrin gel, Matrigel (trade name), synthetic peptide mouth gel, and the like can be used.
- a plurality of the cell culture regions are formed in substantially parallel lines.
- parallel used herein means substantially parallel only when completely parallel, that is, two lines intersect in a certain area. It is assumed that a state where the lines do not intersect, such as a line, exists.
- substantially parallel includes, for example, crossing and branching among crossing or branching structures such as a net-like structure, and the like.
- the shape of the cell culture region forming each blood vessel is not particularly limited as long as it is formed in a line shape, and is appropriately selected according to the shape of the target blood vessel.
- the width of the line in the cell culture region is 10 m to 5000 m, especially 20 m to 100 m, particularly about 40 m to 60 m. Line width is less than 10 ⁇ m In such a case, the vascular cells are not easily adhered, which is not preferable.
- the line width exceeds 5000 ⁇ m, almost all of the vascular cells will adhere to the cell culture area in a spread form, so the cultured vascular cells must be shaped into blood vessels. Difficult and undesirable.
- the cell adhesion auxiliary part is a region formed in a fine pattern in the cell culture region and having no adhesion to vascular cells.
- the cell adhesion auxiliary portion does not inhibit the binding of the vascular cells in the cell culture region.
- vascular cells are cultured by attaching vascular cells to a cell culture region to form a tissue
- the vascular cells are gradually arranged from the outside to the inside of the cell culture region.
- tissue it is necessary that individual vascular cells undergo morphological changes and be arranged.
- This morphological change of vascular cells also gradually proceeds from the end to the center of the cell culture region. It is something to be done. Therefore, when the width of the cell culture region is large, tissue with poor alignment of vascular cells at the center of the cell culture region is not formed, or when vascular cells do not adhere to the center of the cell culture region. Etc.
- the morphological change of vascular cells in the central part of the cell culture region may be poor.
- the end force of the cell adhesion assisting portion can also arrange vascular cells and change its shape, thereby causing chipping and poor shape change. It is possible to culture vascular cells that can help. Further, since the above-mentioned cell adhesion auxiliary portion is formed so as not to inhibit the adhesion between vascular cells adjacent to each other with the cell adhesion auxiliary portion interposed therebetween, the width of the finally cultured vascular cells is as follows. The width can be the same as the width of the cell culture region.
- the cell adhesion auxiliary part is preferably formed in a line in the cell culture region.
- the shape of the line is not particularly limited, and may be, for example, a straight line, a curved line, a dotted line, a broken line, a mesh, or the like.
- the line width of the cell adhesion auxiliary part is 0. It is preferably within a range of 5 ⁇ m to 10 ⁇ m, especially 1 ⁇ m to 5 ⁇ m. Wider than the above range! This is because, in this case, it is not preferable because it becomes difficult for vascular cells adjacent to each other across the cell adhesion assisting part to interact on the cell adhesion assisting part. Further, when the width is smaller than the above range, it is difficult to form using the pattern forming technique of the present invention.
- the cell adhesion auxiliary portion may be formed to have an in-plane concavo-convex pattern such as a zigzag shape.
- the term “in-plane” refers to the surface of the substrate or a surface similar thereto.
- the average value of the distance from the concave end to the convex end of the concavo-convex pattern is such that when the vascular cells are adhered to the cell culture region, the vascular cells are oriented in the same direction as the line direction of the cell culture region.
- the alignment distance may be sufficient, but it is particularly preferable that the distance be in the range of 0.5 ⁇ m to 30 ⁇ m.
- the average of the distance between the concave end force and the convex end of the above-mentioned pattern having irregularities was measured by measuring the distance from the bottom to the top of each irregularity in the range of 200 m of the end of the cell adhesion assisting part. Is measured, and the average is taken as the calculated value.
- the method for forming the above-mentioned cell adhesion assisting portion can be the same as the above-described method for forming the cell adhesion inhibiting portion.
- the method for producing a putter substrate for cell culture according to the present invention has two embodiments. Hereinafter, each embodiment will be described.
- the first embodiment of the method for producing a puttering substrate for cell culture according to the present invention includes a substrate having a convex portion, and a cell adhesion which has adhesiveness to cells and is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation.
- the cell adhesion layer and the photocatalyst-containing layer are opposed to each other by a buttering substrate having a cell adhesion layer containing a material and a photocatalyst-containing layer side substrate having a photocatalyst-containing layer and a substrate containing a photocatalyst.
- the area having the convex portion is irradiated with a predetermined directional force energy, and the cell adhesive material contained in the cell adhesive layer is decomposed or denatured.
- One is not irradiated, and has an energy irradiation step of forming a pattern consisting of a cell adhesion portion having adhesiveness to cells.
- the substrate 1 having the convex portion and the cell adhesion formed on the substrate 1 A patterning substrate having a layer 8 is prepared (FIG. 7A).
- a photocatalyst-containing layer-side substrate 13 having a photocatalyst-containing layer 12 and a substrate 11 is prepared, and the cell adhesion layer 8 of the patterning substrate and the photocatalyst-containing layer 12 are opposed to each other, for example, using a photomask 5.
- the cell adhesion inhibitor is decomposed or denatured by the energy irradiation. This is a step of forming a pattern of 7 and a cell adhesion portion 9 that has not been irradiated with energy and has adhesive properties to cells (FIG. 7 (c)).
- the region of the manufactured patterning substrate for cell culture on which the convex portion is formed has a cell adhesion inhibiting property, and the region where the convex portion is not formed, In other words, in the cell culture region, it can be made to have an adhesive property to cells.
- the adjacent cell culture region is defined by the protrusions, and the protrusions are separated only by the height and width of the protrusions.
- the cell adhesion inhibitory property of the area where the cells are formed can also prevent the cells attached to adjacent cell culture areas from binding to each other, allowing cells to be cultured in a high-definition pattern. This makes it possible to provide a novel cell culture patterning substrate.
- the base material having the convex portion and the cell adhesive layer, the photocatalyst containing layer side substrate, the arrangement of the photocatalyst containing layer side substrate and the cell adhesive layer, and irradiation can be the same as that described in the fifth embodiment of “A. Patterning substrate for cell culture” described above.
- the method for producing a puttering substrate for cell culture of the present embodiment may include necessary steps as appropriate in addition to the energy irradiation step.
- the cell culture is performed by attaching cells to a cell culture region on the cell culture puttering substrate.
- the substrate is used as a substrate, the cells adhered to the cell adhesion inhibitor can be removed by irradiating the cell adhesion inhibitor having the projections with energy using the photocatalyst-containing layer-side substrate. This also has the advantage that cells cultured in a high-definition pattern can be maintained.
- the method for producing a puttering substrate for cell culture according to the present embodiment comprises a substrate having a convex portion, a cell adhesion inhibitory property for inhibiting adhesion to cells, and decomposition by the action of a photocatalyst accompanying energy irradiation.
- a pattern consisting of a cell adhesion portion that has adhesiveness to cells and a cell adhesion inhibitory portion that has not been irradiated with energy and has a cell adhesion inhibitory property that inhibits adhesion to cells is formed. Characterized by having an energy irradiation step.
- the cell adhesion-inhibiting material remains because the convex portion of the manufactured cell-patterning substrate is formed, and the cell adhesion-inhibiting material remains over the region.
- the cell adhesion-inhibiting material is degraded or denatured, so that the material has adhesiveness to cells.
- the adjacent cell culture region is defined by the convex portion, and the convex portion is separated only by the height and width of the convex portion.
- the cell adhesion inhibitory property of the area where the part is formed can also prevent the cells attached to the adjacent cell culture area from binding to each other, allowing cells to be cultured in a high-definition pattern. It can be used as a cell culture patterning substrate.
- the arrangement of the photocatalyst-containing layer-side substrate, the arrangement of the photocatalyst-containing layer-side substrate and the cell adhesion layer, the irradiation energy, and the like used in the energy irradiation step of this embodiment are described above. This can be the same as that described in the fifth embodiment of “A. Putter-jung substrate for cell culture” described above. A. It can be the same as that described in the fourth embodiment of “Patterung substrate for cell culture”, and a detailed description thereof will be omitted here.
- the method for producing a notting substrate for cell culture according to the present embodiment may have a necessary step in addition to the above-described energy irradiation step.
- the present invention is not limited to the above embodiment.
- the above embodiment is an exemplification, and has substantially the same configuration as the technical idea described in the claims of the present invention. Included in the technical scope of the invention.
- a resin line-shaped convex part having a width of 300 m and a height of 25 m was formed on the slide glass by a screen printing method so that the pitch of the convex part was 500 m.
- Isopropyl alcohol 3g Organosilane TSL8114 (GE Toshiba Silicone) 0.4g, Fluoroalkylsilane TSL8223 (GE Toshiba Silicone) 0.4g as cell adhesion inhibiting material, and photocatalytic inorganic coating agent ST—K01 (Ishihara Sangyo) 1. 5 g were mixed and heated at 100 ° C. for 20 minutes with stirring.
- This solution is applied to the above-mentioned slide glass with protrusions by a spin coating method, and the substrate is dried at a temperature of 150 ° C. for 10 minutes to allow hydrolysis and polycondensation reactions to proceed.
- the average height difference between the convex portion and the region sandwiched between the adjacent convex portions after the formation of the coating layer was 24.6 ⁇ m.
- a mask having a line-shaped light-shielding portion having a width of 300 m and a pitch of 500 m was placed on the glass on which the photocatalyst-containing cell adhesion inhibition layer was formed so that the projection and the light-shielding portion faced each other. Irradiation was performed at 5.5 Jcm _2 with plain ultraviolet light. As a result, the exposed portion was partially decomposed and removed from the cell adhesion inhibiting material, and changed to a region having cell adhesiveness.
- the liver extracted from the rat was transferred to a petri dish, subdivided into 5 mm-sized pieces using a scalpel, 20 ml of DMEM medium was added, lightly suspended with a pipette, and filtered with a cell strainer. The obtained coarsely dispersed cell suspension was centrifuged at 500 to 600 rpm for 90 seconds, and the supernatant was removed by suction. DMEM medium was newly added to the remaining cells and centrifuged again. By repeating this operation three times, substantially uniform hepatocytes were obtained. To the obtained hepatocytes, 20 ml of DMEM medium was added and suspended to prepare a hepatocyte suspension.
- Wavmouth MB752 / 1 medium (containing L-glutamine and no NaHCO)
- liver parenchymal cell suspension was suspended in the similarly prepared Waymouth MB752 / 1 medium solution, and the above-mentioned cell culture buttering substrate having a cell adhesion part and a cell adhesion inhibition part. Seeded on top. Place this substrate in an incubator with 37 ° C and 5% CO
- hepatocytes were allowed to adhere to the entire surface of the substrate.
- the substrate was washed twice with PBS to remove non-adherent cells and dead cells, and then replaced with a fresh medium.
- This solution is applied onto the polystyrene plate with the convex portions by a spin coating method, and the substrate is dried at a temperature of 150 ° C. for 10 minutes to allow hydrolysis and polycondensation reactions to proceed, and the photocatalyst is converted into a polyorganosiloxane.
- a photocatalyst-containing cell adhesion layer firmly fixed on the substrate was formed on the substrate.
- the average height difference between the region between the adjacent convex portions after the formation of the coating layer and the convex portion was 29.
- a mask having a line-shaped light-shielding portion with a width of 200 m and a pitch of 400 m was placed on a polystyrene plate on which the photocatalyst-containing cell adhesive layer was formed so that the region sandwiched by the protrusions and the light-shielding portion faced each other. arrangement, and an exposure system power ultraviolet and 12Jcm_ 2 irradiation. As a result, the cell adhesion material in the exposed part is almost completely decomposed and removed, and the cell adhesion inhibiting property is extremely high! ⁇ Changed to the area.
- Example 1 Cells were seeded as in Example 1. At this time, as in Example 1, it was confirmed that the cells adhered along the cell adhesion portion sandwiched between the protrusions, that is, along the cell culture region on the cell culture patterning substrate. In addition, it was confirmed that there was no adhesion between cells between the cell adhesion regions (on the convex portions).
- This solution is applied to the above-mentioned slide glass with projections by a spin coating method, and the substrate is dried at a temperature of 150 ° C. for 10 minutes to allow hydrolysis and polycondensation reactions to proceed, whereby the photocatalyst is converted into an organopolysiloxane.
- a photocatalyst-containing layer firmly fixed on the substrate was formed on the substrate.
- the average height difference between the convex portion and the region sandwiched between the adjacent convex portions was 24.
- fibronectin F-4759 Sigma
- 200 ml of pure water 0.2 mg
- this aqueous solution is dropped onto the photocatalyst-containing layer of the above-mentioned slide glass at a ratio of 3001 per lcm 2 of the substrate area.
- the mixture was allowed to stand at 24 ° C for 24 hours. Further, the substrate was washed twice with PBS to obtain a slide glass having a photocatalyst-containing layer and a cell adhesion layer on a substrate having a convex portion.
- a mask having a line-shaped light-shielding portion with a width of 200 / zm and a pitch of 500 m is placed so that the light-shielding portion and the region sandwiched by the convex portions on the slide glass face each other. It was 12Jcm_ 2 irradiation. As a result, the exposed surface of the convex portion was almost completely decomposed and removed by fibronectin, which is a cell adhesive material, and turned into a very hydrophilic region having cell adhesion inhibiting properties.
- Example 1 A cell adhesion experiment was performed in the same manner as in Example 1. At this time, as in Example 1, it was confirmed that the cells adhered along the cell adhesion portion sandwiched between the protrusions, that is, along the cell culture region, on the pattern cultivation substrate substrate. In addition, it was confirmed that the cells did not adhere to each other between the cell adhesion regions (on the convex portions).
- a substrate similar to the substrate used in Example 1 was used.
- fibronectin F-4759 Sigma
- 200 ml of pure water 0.2 mg
- this aqueous solution is dropped on the above-mentioned slide glass at a ratio of 300 1 per lcm 2 of substrate area, and the mixture is added at 4 ° C for 24 hours. Let stand for hours. Further, the substrate was washed twice with PBS to obtain a slide glass having a cell adhesive layer on a substrate having a convex portion.
- a photomask with a 200 m light-shielding part and a 300 ⁇ m opening (pitch 500 ⁇ m) light-shielding part on the photocatalyst-containing layer side was prepared on quartz glass. did.
- the photocatalyst-containing layer-forming coating solution is applied to the surface of the photomask by spin coating, and the substrate is dried at 150 ° C for 10 minutes to allow hydrolysis and polycondensation reactions to proceed.
- the photocatalyst-containing layer-side substrate was arranged so that the region interposed between the protrusions on the slide glass and the light-shielding portion were opposed to each other, and irradiated with ultraviolet light having an exposure power of 12 Jcm_2.
- fibronectin which is a cell adhesive material, was almost completely decomposed and removed from the exposed surface of the convex portion, and the exposed convex surface was changed to a very highly hydrophilic region having cell adhesion inhibiting properties.
- Example 1 A cell adhesion experiment was performed in the same manner as in Example 1. At this time, in the same manner as in Example 1, the cells were attached to the cell culture pattern It was confirmed that they adhered along the culture area. In addition, it was confirmed that the cells did not adhere to each other between the cell adhesion regions (on the convex portions).
- resin line-shaped protrusions having a width of 300 m and a height of 25 m were formed by screen printing so that the pitch of the protrusions was 700 m.
- the silane coupling agent XC98-B2472 (GE Toshiba Silicone) was diluted 10-fold with isopropyl alcohol, and 1,3-butanediol was added to a concentration of 10% and stirred to form a cell adhesion inhibitor layer.
- a coating agent for forming was prepared.
- the slide glass on which the line-shaped convex portions were formed was subjected to UV cleaning for 120 seconds, and immediately thereafter, the coating agent was applied by a spin coating method and dried at 150 ° C. for 15 minutes to form a cell adhesion inhibiting layer.
- a photomask was prepared in which linear light-shielding portions having a width of 300 m and linear opening portions having a width of 400 m were alternately formed. At this time, a linear light-shielding portion having a pitch of 40 m and a width of 5 m was formed along the line direction of the opening within the linear opening having a width of 400 m as a portion corresponding to the cell attachment assisting portion. .
- a photocatalyst-containing layer was formed on this photomask in the same manner as in Example 4 to obtain a transparent photocatalyst-containing layer-side substrate.
- the photocatalyst-containing layer of the photocatalyst-containing layer-side substrate having a pattern corresponding to the cell-adhesion assisting portion and the cell-adhesion-inhibiting layer were arranged to face each other. At this time, the alignment was performed such that the linear light-shielding portion was on the linear convex portion of the base material. Subsequently, ultraviolet rays were irradiated at 6 j / cm 2 from the substrate side of the photocatalyst containing layer side.
- the cell adhesion inhibiting material in a region sandwiched between the adjacent convex portions is removed in a pattern, and the cell adhesion portion having the cell adhesion assisting portion is removed. Been formed.
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Abstract
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
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US11/587,363 US20070218554A1 (en) | 2004-04-26 | 2005-04-25 | Cell Culture Patterning Substrate And Method For Manufacturing The Same |
JP2006512616A JP4699361B2 (ja) | 2004-04-26 | 2005-04-25 | 細胞培養用パターニング基板およびその製造方法 |
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US (1) | US20070218554A1 (ja) |
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JP2007312736A (ja) * | 2006-05-29 | 2007-12-06 | Dainippon Printing Co Ltd | 細胞培養用基板 |
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Also Published As
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JPWO2005103227A1 (ja) | 2008-03-13 |
US20070218554A1 (en) | 2007-09-20 |
JP5077449B2 (ja) | 2012-11-21 |
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