JP4826092B2 - Patterning substrate and cell culture substrate - Google Patents

Description

  The present invention is used for forming a patterning substrate for cell culture capable of adhering cells in a high-definition pattern, a patterning substrate used for forming the patterning substrate for cell culture, and the patterning substrate. The present invention relates to a patterning substrate coating solution and a cell culture substrate in which cells are adhered in a high-definition pattern.

  Currently, various animal and plant cell cultures are being performed, and new cell culture methods have been developed. Cell culture techniques are used for the purpose of elucidating biochemical phenomena and properties of cells and producing useful substances. In addition, attempts have been made to examine the physiological activity and toxicity of artificially synthesized drugs using cultured cells.

  Some cells, especially many animal cells, have an adhesion dependency that grows by adhering to something, and cannot survive for a long time in a floating state in vitro. In order to culture such cells having adhesion dependency, a carrier for cell adhesion is required, and generally, a plastic culture in which cell adhesion proteins such as collagen and fibronectin are uniformly applied. A dish is used. These cell adhesion proteins are known to act on cultured cells to facilitate cell adhesion and affect cell morphology.

  On the other hand, a technique for adhering and arranging cultured cells only on a minute part on a substrate has been reported. Such a technique makes it possible to apply cultured cells to artificial organs, biosensors, bioreactors and the like. As a method for arranging cultured cells, use a base material that has a pattern on the surface that has a different ease of adhesion to the cells, culture the cells on this surface, and process the cells to adhere A method has been adopted in which cells are arranged by adhering cells only to the surface.

  For example, in Patent Document 1, a charge holding medium in which an electrostatic charge pattern is formed is applied to cell culture for the purpose of growing nerve cells in a circuit form. In Patent Document 2, an attempt is made to arrange cultured cells on a surface obtained by patterning a photo-sensitive hydrophilic polymer having cell adhesion inhibitory or cell adhesion properties by photolithography.

  Furthermore, Patent Document 3 discloses a cell culture substrate patterned with a substance such as collagen that affects the cell adhesion rate and morphology, and a method for producing the substrate by photolithography. By culturing cells on such a substrate, many cells are adhered to the surface on which collagen or the like is patterned, thereby realizing cell patterning.

  However, such patterning of the cell culture site may be required to have high definition depending on the application. When patterning by a photolithography method using a photosensitive material as described above, a high-definition pattern can be obtained, but the cell adhesive material needs to have photosensitivity, for example, a biopolymer or the like In many cases, it is difficult to perform chemical modification for imparting such photosensitivity, and there is a problem that the range of selectivity of the cell adhesive material is extremely narrow. Further, in the photolithography method using a photoresist, it is necessary to use a developer or the like, which may have an adverse effect on cell culture.

  Furthermore, as a method for forming a pattern of a high-definition cell adhesive material, a micro contact printing method has been proposed by George M. Whitesides of Harvard University (for example, Patent Document 4, Patent) Document 5, Patent Document 6, Patent Document 7, etc.). However, there is a problem that it is difficult to produce a cell culture substrate having a pattern of a cell adhesive material industrially using this method.

JP-A-2-245181 Japanese Patent Laid-Open No. 3-7576 Japanese Patent Laid-Open No. 5-176653 US Pat. No. 5,512,131 US Patent No. 5,900,160 JP-A-9-240125 Japanese Patent Laid-Open No. 10-12545

  Therefore, it is desired to provide a cell culture patterning substrate used for bonding and culturing cells on a substrate in a high-definition pattern, a cell culture substrate in which cells are bonded in a high-definition pattern, and the like. ing.

  The present invention comprises a base material, a cell adhesive material formed on the base material, which has adhesiveness with cells and is decomposed or modified by the action of a photocatalyst accompanying energy irradiation, and a cell adhesive layer containing a binder. A patterning substrate is provided.

  According to the present invention, since the cell adhesion layer contains the cell adhesion material, the cell adhesion layer is irradiated with energy in a pattern using, for example, a photocatalyst-containing layer containing a photocatalyst. A patterning substrate capable of forming a region where the adhesive material is decomposed or denatured and has no adhesion to cells and a region where the cell adhesion material remains and has good adhesion to cells is provided. it can.

  In the said invention, the light shielding part shall be formed on the said base material. Thereby, for example, the cell adhesion layer and the photocatalyst-containing layer containing the photocatalyst are opposed to each other and irradiated with energy from the base material side, so that only the region where the light-shielding part is not formed is irradiated with energy. This is because the adhesive material can be decomposed or modified.

  In the present invention, the cell adhesion layer of the patterning substrate comprises a cell adhesion inhibition part in which the cell adhesion material is decomposed or denatured in a pattern, and a cell adhesion part that is an area other than the cell adhesion inhibition part. The present invention provides a patterning substrate for cell culture characterized by comprising:

  According to the present invention, a cell adhesion inhibitor having low adhesion to cells due to degradation or denaturation of the cell adhesion material, and cells having good adhesion to cells without degradation or denaturation of the cell adhesion material A cell culture patterning substrate capable of adhering cells with high precision only to the cell adhesion part without using a complicated process or a treatment solution that adversely affects the cell. can do.

  The present invention also provides a cell culture substrate characterized in that cells adhere to the cell adhesion portion of the cell culture patterning substrate.

  According to the present invention, since the cell culture patterning substrate is formed with a cell adhesion portion having good adhesion to cells and a cell adhesion inhibition portion having low adhesion to cells, Cells can be adhered only on the cell adhesion part, and a cell culture substrate in which cells are adhered in a high-definition pattern can be obtained. At this time, since the photocatalyst does not need to be contained in the cell culture substrate, there is an advantage that the photocatalyst can have no possibility of affecting the cells over time.

  The present invention also provides a cell adhesion material that has adhesiveness to cells and is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation, and cell adhesion inhibition that inhibits adhesion to cells after at least energy irradiation. A coating liquid for a patterning substrate, comprising: a material comprising:

  According to the present invention, by applying the patterning substrate coating liquid onto a substrate or the like to form a layer, the cell adhesive material can be used to form a layer having good adhesion to cells. In addition, the cell adhesion material can be decomposed or denatured by irradiating the layer with energy using, for example, a photocatalyst-containing layer containing a photocatalyst, and further, the cell adhesion inhibiting material is contained. , The adhesiveness with cells can be lowered. Therefore, it can be set as the coating liquid for patterning substrates which can form easily the area | region with favorable adhesiveness with a cell, and the area | region with low adhesiveness with a cell.

Furthermore, the present invention provides a cell adhesion layer that forms a cell adhesion layer containing a binder, and a cell adhesion material that has adhesiveness to cells and is decomposed or modified by the action of a photocatalyst associated with energy irradiation on a substrate. Forming process;
After the cell adhesion layer, the photocatalyst containing layer containing the photocatalyst and the photocatalyst containing layer side substrate having the substrate are arranged so that the cell adhesion layer and the photocatalyst containing layer face each other, energy irradiation is performed from a predetermined direction. And an energy irradiation step for forming a pattern consisting of a cell adhesion inhibiting part in which the cell adhesion material contained in the cell adhesion layer is decomposed or denatured and a cell adhesion part other than the cell adhesion inhibiting part. A method for producing a patterning substrate for cell culture is provided.

  According to the present invention, since a cell adhesion material is contained in the cell adhesion layer formed by the cell adhesion layer formation step, in the energy irradiation step, energy is formed in a pattern using the photocatalyst-containing layer side substrate. Patterning substrate for cell culture in which a cell adhesion inhibiting part in which a cell adhesion material is easily decomposed or denatured by irradiation and a cell adhesion part having good adhesion to cells not irradiated with energy are formed It can be. Further, according to the present invention, since the photocatalyst-containing layer side substrate is used, it is not necessary to contain a photocatalyst in the cell culture patterning substrate. For example, even when cells adhere to the cell adhesion portion, Thus, it is possible to obtain a high-quality cell culture patterning substrate in which the photocatalyst does not affect the cells over time.

  Further, the present invention provides a cell culture substrate comprising a cell adhesion step for adhering cells on the cell adhesion portion of the cell culture patterning substrate produced by the method for producing a cell culture patterning substrate. A manufacturing method is provided.

  According to the present invention, on the cell culture patterning substrate, a cell adhesion portion having good adhesion to cells and a cell adhesion inhibition portion having low adhesion to cells are formed. However, it is possible to easily manufacture a cell culture substrate that is adhered only on the cell adhesion portion with high precision.

Furthermore, the present invention provides a cell adhesion layer comprising a base material, a cell adhesive material formed on the base material, having adhesiveness with cells and decomposed or modified by the action of a photocatalyst accompanying energy irradiation, and a binder. And cells formed in a pattern on the cell adhesion layer, the cell adhesion layer comprising: a cell adhesion inhibition part in which the cell adhesion material is decomposed or denatured, and cells other than the cell adhesion inhibition part. A method for producing a cell culture substrate having an adhesive part, wherein the cells are formed on the cell adhesive part,
After performing the cell adhesion step for adhering cells on the cell adhesion part, the cell adhesion inhibition part is provided with a photocatalyst containing layer and a photocatalyst containing layer side substrate having a substrate, the cell adhesion layer and the photocatalyst. A method for producing a cell culture substrate, comprising a cell maintaining step of irradiating energy from a predetermined direction and maintaining a pattern of cells adhered on the cell adhesion part after being disposed so as to face the containing layer I will provide a.

  According to the present invention, after the cells are adhered on the cell adhesion portion by the cell adhesion step, energy is applied to the cell adhesion inhibition portion of the cell culture substrate using the photocatalyst-containing layer side substrate in the cell maintenance step. By irradiating, it becomes possible to remove cells and the like adhered to the cell adhesion-inhibiting portion, and a cell culture substrate in which cells are adhered in a high-definition pattern only to the cell adhesion portion can be obtained.

  According to the present invention, since the cell adhesion layer containing the cell adhesion material is included, the cell adhesion layer is irradiated with energy in a pattern using, for example, a photocatalyst containing layer containing a photocatalyst, thereby A patterning substrate capable of forming a region where the adhesive material is decomposed or denatured and has no adhesiveness with cells and a region with a good adhesiveness with cells where the cell adhesive material remains can be formed. , Has the effect.

  The present invention is used for forming a patterning substrate for cell culture capable of adhering cells in a high-definition pattern, a patterning substrate used for forming the patterning substrate for cell culture, and the patterning substrate. The present invention relates to a patterning substrate coating solution, a cell culture substrate in which cells are adhered in a high-definition pattern, and the like. Each will be described separately below.

A. Patterning Substrate Coating Solution First, the patterning substrate coating solution of the present invention will be described. The patterning substrate coating solution of the present invention has a cell adhesion material that has adhesiveness to cells and is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation, and adheres to cells after at least energy irradiation. It contains a cell adhesion inhibiting material having a cell adhesion inhibiting property to inhibit.

Since the patterning substrate coating solution of the present invention contains the cell adhesion material, when the layer is formed by applying the patterning substrate coating solution on a substrate or the like, for example, Adhesiveness can be good. In addition, when the layer is irradiated with energy such as a photocatalyst-containing layer containing a photocatalyst, the cell adhesion material is decomposed or denatured by the action of the photocatalyst accompanying the energy irradiation, and the adhesion to the cell is increased. Can be low. At this time, since the cell adhesion inhibitory material is contained in the coating liquid for patterning substrate, the cell adhesion inhibitory property of the cell adhesion inhibitory material makes the adhesion to cells lower. Can do. Therefore, according to the present invention, it is possible to allow cells to adhere to a high-definition pattern only in the region where the energy is not irradiated in the layer to which the coating liquid for patterning substrate is applied. .
Hereinafter, each structure used for the coating liquid for patterning substrates of the present invention will be described.

1. Cell Adhesive Material First, the cell adhesive material used for the patterning substrate coating solution of the present invention will be described. The cell adhesion material used in the coating solution for a patterning substrate of the present invention is not particularly limited as long as it has adhesiveness with cells and is decomposed or modified by the action of a photocatalyst accompanying energy irradiation. It is not something. Here, having adhesiveness with a cell means that it adheres favorably to a cell, and means that it adheres favorably to a target cell when the adhesiveness with the cell differs depending on the type of the cell. .

  The cell adhesion material used in the present invention has adhesion with such cells, and is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation, and has no adhesion with cells, What changes into what has cell adhesion inhibitory property which inhibits adhesion with a cell, etc. are used.

  Here, there are two types of materials having adhesiveness to cells as described above: materials having adhesiveness to cells based on physicochemical characteristics, and materials having adhesiveness to cells based on biochemical characteristics.

  Examples of the physicochemical factors that determine the adhesion to cells of a material having adhesion to cells based on physicochemical characteristics include surface free energy and electrostatic interaction. For example, when the adhesion 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 adhesion between the cell and the material will be good. The adhesion between the material and the material will be reduced. Examples of such changes in cell adhesion due to surface free energy include the leading edge of the biomaterials published by CMC publication Yoshito Tsuji (supervised) p. Experimental results as shown in the lower part of 109 are known. Examples of the material having adhesiveness to cells due to such factors include hydrophilic polystyrene and poly (N-isopropylacrylamide). When such a material is used, the surface free energy changes due to the action of the photocatalyst associated with energy irradiation, for example, due to substitution or decomposition of the functional group on the surface of the material, and adhesion to the cell. It may be one having no sex or one having cell adhesion inhibitory properties.

  Further, when the adhesion between the cell and the material is determined by electrostatic interaction or the like, the adhesion with the cell is determined by, for example, the amount of positive charge of the material. Examples of the material having adhesiveness to cells by such electrostatic interaction include basic polymers such as polylysine, aminopropyltriethoxysilane, N- (2-aminoethyl) -3-aminopropyltrimethoxysilane, and the like. And basic compounds such as these and condensates containing them. When such a material is used, the above-mentioned material is decomposed or modified by the action of the photocatalyst associated with energy irradiation, so that, for example, the amount of positive charges existing on the surface can be changed, and adhesion to cells can be improved. What does not have or what has cell adhesion inhibition property can be used.

  In addition, examples of materials having adhesiveness to cells due to biological characteristics include those having good adhesion to specific cells, or those having good adhesion to many cells, and specifically, fibronectin. , Laminin, tenascin, vitronectin, RGD (arginine-glycine-aspartic acid) sequence-containing peptide, YIGSR (tyrosine-isoleucine-glycine-serine-arginine) sequence-containing peptide, collagen, atelocollagen, gelatin and the like. When such materials are used, those that do not have adhesiveness to cells by, for example, destroying a part of the structure of the material or destroying the main chain by the action of the photocatalyst accompanying energy irradiation, Or it can have a cell adhesion inhibitory property.

  Such a cell adhesion material is appropriately optimized depending on the cell adhesion ability of the material, and is usually 0.0001% by weight to 30% by weight in the coating liquid for patterning substrate, and more preferably 0.001% by weight to The content is preferably 10% by weight. This is because the region containing the cell adhesive material can be made a region having good adhesion to cells.

2. Cell Adhesion Inhibiting Material Next, the cell adhesion inhibiting material used in the patterning substrate coating solution of the present invention will be described. The cell adhesion-inhibiting material used for the patterning substrate coating solution of the present invention has a cell adhesion-inhibiting property that inhibits adhesion to cells after at least energy irradiation. Here, the cell adhesion inhibitory property refers to a property that inhibits cells from adhering to a region irradiated with energy when a patterning substrate coating solution is applied to form a layer. . Moreover, when adhesiveness with a cell changes with cell types, it has the property which inhibits adhesion with the target cell.

  In the present invention, the material is not particularly limited as long as it has such a cell adhesion inhibitory property after being irradiated with energy at least, and for example, the cell adhesion inhibitory property before being irradiated with energy. It does not have cell adhesion inhibitory properties before it is irradiated with energy, and its adhesion to cells is lowered by the action of the photocatalyst accompanying energy irradiation, and exhibits cell adhesion inhibitory properties. The thing etc. may be sufficient.

  In the present invention, it is preferable to use a cell adhesion-inhibiting material that has cell adhesion-inhibiting property, particularly due to the action of a photocatalyst accompanying energy irradiation. Thereby, in the region before the energy irradiation, the adhesion of the cell adhesive material to the cell is not inhibited, and only the region irradiated with the energy has a low adhesiveness to the cell. Because it can.

  As a material used as such a cell adhesion inhibiting material, for example, the main skeleton has a high binding energy that is not decomposed by photoexcitation of the photocatalyst, and has an organic substituent that is decomposed by the action of the photocatalyst. For example, (1) organopolysiloxane that exhibits high strength by hydrolyzing and polycondensing chloro or alkoxysilane by sol-gel reaction or the like, and (2) reactive silicone excellent in water repellency and oil repellency. Can be mentioned.

In the case of (1) above, the general formula:
Y _n SiX _(4-n)
(Here, Y represents an alkyl group, vinyl group, amino group, phenyl group or epoxy group, X represents an alkoxy group, acetyl group or halogen. N is an integer from 0 to 3.)
It is preferable that it is the organopolysiloxane which is a 1 type, or 2 or more types of hydrolysis condensate or cohydrolysis condensate of the silicon compound shown by these. Here, the number of carbon atoms of the group represented by Y is preferably in the range of 1 to 20, and the alkoxy group represented by X is a methoxy group, an ethoxy group, a propoxy group, or a butoxy group. preferable.

  Examples of the reactive silicone (2) include compounds having a skeleton represented by the following general formula.

However, n is an integer of 2 or more, R ^1, R ² are each a substituted or unsubstituted alkyl having 1 to 20 carbon atoms, alkenyl, aryl or cyanoalkyl group, the total molar ratio of 40% or less Vinyl, phenyl and phenyl halide. Further, those in which R ¹ and R ² are methyl groups are preferable because the surface energy becomes the smallest, and the methyl groups are preferably 60% or more by molar ratio. In addition, the chain end or side chain has at least one reactive group such as a hydroxyl group in the molecular chain. By using such a material, the surface of the region irradiated with energy can have high hydrophilicity due to the action of the photocatalyst accompanying energy irradiation. This is because adhesion with the cells is inhibited, and the cells can be prevented from adhering to the region irradiated with energy.

  When using the said material as a cell adhesion inhibiting material, it is preferable that the contact angle with water before energy irradiation is in the range of 15 ° to 120 °, particularly 20 ° to 100 °. This is because it is possible to prevent the cell adhesion material from adhering to the cells.

Moreover, when energy is irradiated to this cell adhesion inhibiting material, it is preferable that the contact angle with water is 10 ° or less. It is because it can have high hydrophilicity by setting it as the said range, and can make adhesiveness with a cell low.
In addition, the contact angle with water here is measured using a contact angle measuring instrument (CA-Z type manufactured by Kyowa Interface Science Co., Ltd.) with water or a liquid having an equivalent contact angle (micro type). 30 seconds after dropping a droplet from the syringe), and the result was obtained or a graph was obtained.

  In addition to the above organopolysiloxane, a stable organosilicon compound that does not undergo a crosslinking reaction, such as dimethylpolysiloxane, may be mixed in the binder.

  Further, in the present invention, the cell adhesion inhibition may be caused by a degradation substance or the like that lowers the adhesiveness to the cells or assists such a change by causing a change in wettability of the region irradiated with energy. You may use with material.

  Examples of such degrading substances include surfactants that are degraded by the action of a photocatalyst associated with energy irradiation and become hydrophilic, thereby reducing adhesion to cells. Specifically, hydrocarbons such as NIKKOL BL, BC, BO, BB series manufactured by Nikko Chemicals Co., Ltd., ZONYL FSN, FSO manufactured by DuPont, Surflon S-141, 145 manufactured by Asahi Glass Co., Ltd., Dainippon Megafac F-141, 144 manufactured by Ink Chemical Industry Co., Ltd., Footgent F-200, F251 manufactured by Neos Co., Ltd., Unidyne DS-401, 402 manufactured by Daikin Industries, Ltd., Fluorard FC-170 manufactured by 3M Co., Ltd. 176 or the like, and nonionic surfactants such as cationic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants can also be used.

  In addition to surfactants, polyvinyl alcohol, polyethylene glycol, zwitterionic materials having a betaine structure, phospholipid-containing materials such as poly (2-methacryloyloxyethyl) phosphorylcholine, poly (2-methoxyethyl) acrylate , Unsaturated polyester, acrylic resin, polyethylene, diallyl phthalate, ethylene propylene diene monomer, epoxy resin, phenol resin, polyurethane, melamine resin, polycarbonate, polyvinyl chloride, polyamide, polyimide, styrene butadiene rubber, chloroprene rubber, polypropylene, polybutylene, Polystyrene, polyvinyl acetate, nylon, polyester, polybutadiene, polybenzimidazole, polyacrylonitrile, epichlorohydrin, polysulfur It can be mentioned id, polyisoprene and the like, a polymer or the like.

  In the present invention, such a cell adhesion-inhibiting material is contained in the coating liquid for patterning substrate in an amount of 0.001% to 60% by weight, particularly 0.01% to 40% by weight, particularly 0.1% by weight to It is preferably contained within the range of 20% by weight.

3. Next, the patterning substrate coating solution of the present invention will be described. The coating solution for a patterning substrate of the present invention is not particularly limited as long as it contains the cell adhesion material and the cell adhesion-inhibiting material, and appropriately contains components such as a binder as necessary. It may be what you are doing. By including a binder, the coating liquid for patterning substrate can be easily applied when applied onto, for example, a base material, or imparts strength and resistance to the formed layer, etc. This is because various characteristics can be imparted. Such a binder is appropriately selected according to the purpose for which the patterning substrate coating liquid is required. In the present invention, it is preferable that the cell adhesion-inhibiting material fulfills the role as the binder.

  The patterning substrate coating liquid of the present invention preferably contains no photocatalyst. This is because, for example, when the cells are adhered onto the layer to which the patterning substrate coating solution is applied, it is possible that the photocatalyst has no possibility of affecting the cells over time.

B. Next, the patterning substrate of the present invention will be described. The patterning substrate of the present invention contains a base material, a cell adhesive material formed on the base material, having adhesiveness with cells and decomposed or denatured by the action of a photocatalyst accompanying energy irradiation, and a binder. It has a cell adhesion layer.

  In the patterning substrate of the present invention, for example, as shown in FIG. 1, a cell adhesion layer 2 containing the cell adhesion material and a binder is formed on a base material 1. According to the present invention, since the cell adhesion material is contained in the cell adhesion layer, for example, the photocatalyst-containing layer containing a photocatalyst is opposed to the cell adhesion layer and irradiated with energy. The cell adhesion material can be decomposed or denatured by the action of the photocatalyst accompanying the energy irradiation, and can have no adhesion to cells. Further, since the cell adhesive material remains in the region where energy is not irradiated, the cell adhesive material can be adhered to the cell by the adhesiveness with the cell. Therefore, by irradiating energy using the above-mentioned photocatalyst-containing layer or the like in a pattern without requiring special equipment or complicated processes, the adhesion between the cells and the adhesion between the cells is improved. Thus, a cell culture patterning substrate capable of forming a low region can be formed.

Here, the patterning substrate of the present invention may be one in which a light shielding portion 3 is formed on a base material 1 as shown in FIG. When such a light shielding portion is formed, the light shielding portion is formed by irradiating the entire surface with energy from the base material side, for example, with the photocatalyst-containing layer containing the photocatalyst facing the cell adhesion layer. Only the cell adhesion material contained in the cell adhesion layer on the untreated region will be degraded or denatured. Thereby, for example, energy can be irradiated in a pattern without using a photomask or the like, and it is possible to easily form a region where cells adhere and a region where cells do not adhere.
Hereinafter, each configuration of the patterning substrate of the present invention will be described.

1. Cell Adhesion Layer First, the cell adhesion layer used in the present invention will be described. The cell adhesion layer used in the present invention comprises a cell adhesion material that is formed on a substrate to be described later, has cell adhesiveness, and is decomposed or modified by the action of a photocatalyst accompanying energy irradiation, and a binder. If it is, it will not specifically limit.

  As such a cell adhesion layer, a layer is formed using, for example, a coating solution containing a cell adhesion material and a binder that has adhesiveness with cells and is decomposed or modified by the action of a photocatalyst accompanying energy irradiation. Or the like. Specifically, a layer is formed by using a patterning substrate coating solution containing a cell adhesion material and a cell adhesion inhibiting material as a binder as described in the section “A. Patterning substrate coating solution”. It is preferable that As a result, when the cell adhesion layer is irradiated with energy, the adhesiveness with the cells in the region irradiated with energy can be made lower, and only the region where the energy is not irradiated has high definition. This is because cells can be adhered in a pattern.

  As a method of forming such a cell adhesion layer, it can be formed by applying the above-mentioned patterning substrate coating solution or the like by a general application method, and the specific application method is a spin coating method. Spray coating method, dip coating method, roll coating method, bead coating method and the like can be used. Moreover, an adsorption method can also be used suitably.

  In addition, for example, when a recess is formed in the base material to be described later, a casting method in which a coating solution for a patterning substrate is dropped into the recess of the base material and dried to form a cell adhesion layer, or the base material An adsorption method or the like in which a patterning substrate coating solution or the like is dropped into the recesses and washed after a predetermined time can be used.

Since the cell adhesion material and the cell adhesion inhibiting material used for the cell adhesion layer in the present invention are the same as those described in the above-mentioned section “A. Coating liquid for patterning substrate”, the explanation here is as follows. Omitted.
The film thickness of the cell adhesion layer is appropriately selected depending on the type of the patterning substrate and the like, but is usually about 0.001 to 1 μm, and more preferably about 0.005 to 0.1 μm.

  The cell adhesion layer in the present invention preferably contains no photocatalyst. Thereby, for example, when cells are adhered on the cell adhesion layer, the photocatalyst can be prevented from affecting the cells over time, and the quality can be improved. In addition, as a method of decomposing | disassembling or denature | denaturing the said cell adhesion material of such a cell adhesion layer, it can carry out by making a photocatalyst containing layer containing a photocatalyst face, for example, and irradiating energy.

2. Next, the substrate used in the present invention will be described. The substrate used in the present invention is not particularly limited as long as it is a layer capable of forming the cell adhesion layer, and examples thereof include inorganic materials such as metal, glass and silicon, and organic materials represented by plastics. Can be used.

  Here, the flexibility and the like of the base material are appropriately selected depending on the type and use of the patterning substrate. The transparency of the base material is appropriately selected depending on the type of the patterning substrate, the irradiation direction of the energy applied to decompose or denature the cell adhesive material, and the base material has, for example, a light shielding part. And when the irradiation of the energy is performed from the substrate side, the substrate has transparency.

  In addition, in this invention, although a base material may be flat, the recessed part may be formed in the base material. As such a base material, for example, as shown in FIG. 13 (a), one concave portion may be formed, and for example, as shown in FIG. 13 (b), a plurality of concave portions may be formed. It may be formed.

  In this case, a treatment may be performed so that the cell adhesion layer is not formed on the side wall of the base material having the concave portion. As such a process, for example, a method of attaching a liquid-repellent material only to the side wall by a CVD method using a mask or the like, or a liquid-repellent material is attached to the entire surface of the recess, and then cylindrical For example, there may be mentioned a method in which only the bottom surface of the concave portion is made lyophilic by performing ultraviolet treatment, plasma treatment or the like using the above mask.

  Here, in the present invention, the base material may be subjected to chemical cleaning such as alkali cleaning, or may be subjected to dry cleaning such as oxygen plasma treatment or ultraviolet treatment. In this case, the wettability of the patterning substrate coating liquid and the like is improved. Furthermore, since a reactive functional group is arranged on the surface, there is an advantage that the adhesion of the cell adhesion layer is improved.

  Here, as described above, the light shielding portion may be formed on the base material of the present invention. The light-shielding part that can be used in the present invention is not particularly limited as long as it can block the energy applied to the patterning substrate. For example, by a sputtering method, a vacuum deposition method, or the like. It may be formed by forming a metal thin film such as chromium having a thickness of about 1000 to 2000 mm and patterning the thin film. As this patterning method, a normal patterning method such as sputtering can be used.

  Alternatively, a method may be used in which a layer containing light-shielding particles such as carbon fine particles, metal oxides, inorganic pigments, and organic pigments in a resin binder is formed in a pattern. As the resin binder to be used, polyimide resin, acrylic resin, epoxy resin, polyacrylamide, polyvinyl alcohol, gelatin, casein, cellulose, or a mixture of one or more kinds, photosensitive resin, or O / A W emulsion type resin composition, for example, an emulsion of a reactive silicone can be used. The thickness of such a resin light-shielding portion can be set within a range of 0.5 to 10 μm. As a method for patterning such a resin light shielding portion, a generally used method such as a photolithography method or a printing method can be used.

  The light shielding portion may be formed on the surface of the base material on which the cell adhesion layer is formed or may be formed on the opposite surface. As shown in FIG. 2, it is preferable that the light shielding part 3 is formed on the surface of the base material 1 on the side where the cell adhesion layer 2 is formed, as shown in FIG.

3. Next, the patterning substrate of the present invention will be described. The patterning substrate of the present invention is not particularly limited as long as the cell adhesion layer is formed on the above-described base material. For example, the patterning substrate may be one in which other layers are laminated as necessary. May be.

C. Next, the cell culture patterning substrate of the present invention will be described. In the cell culture patterning substrate of the present invention, the cell adhesion layer of the patterning substrate is a region other than the cell adhesion inhibition portion in which the cell adhesion material is decomposed or modified in a pattern and the cell adhesion inhibition portion. It has a cell adhesion part.

  The cell culture patterning substrate of the present invention has a substrate 1 and a cell adhesion layer formed on the substrate 1 as shown in FIG. 3, for example, and the cell adhesion contained in the cell adhesion layer. The material is decomposed or denatured, and has a cell adhesion inhibiting part 4 that does not have adhesion to cells and a cell adhesion part 2 'that has good adhesion to cells other than the cell adhesion inhibiting part.

  According to the present invention, since the cell adhesion portion and the cell adhesion inhibition portion are provided, the cells can be easily adhered only on the cell adhesion portion, for example, when the cells are applied to the entire surface of the cell adhesion layer. However, it becomes possible to adhere cells with high precision only on the cell adhesion part.

  Further, according to the present invention, after the cells are adhered on the cell adhesion portion, for example, the cell adhesion layer and the photocatalyst containing layer containing the photocatalyst are arranged to face each other, and the entire surface is irradiated with energy. By doing so, it is possible to decompose or denature the cell adhesion material of the cell adhesion part to which the cells adhere, and to make the adhesion to the cells low. Thereby, the cells adhered on the cell adhesion part can be peeled off, and the cells formed in a pattern can be obtained. The energy irradiated at this time is set to an energy that does not affect the cells.

  Here, in the present invention, for example, as shown in FIG. 4, the light shielding part 3 may be formed on the base material 1 in the same pattern as the cell adhesion part 2 ′ of the cell adhesion layer 2. By forming such a light-shielding portion, for example, the patterning substrate described in “B. Patterning substrate” described above is formed, and the photocatalyst-containing layer containing the photocatalyst is disposed facing the cell adhesion layer. Then, by irradiating energy from the substrate side, the cell adhesion material only on the region where the light shielding portion is not formed can be decomposed or denatured, and the cell adhesion inhibiting portion is easily formed. Because it can.

  Further, after adhering cells on the cell adhesion portion of the cell culture patterning substrate of the present invention, the cell adhesion inhibiting portion is irradiated with energy from the substrate side with the photocatalyst-containing layer facing the cell adhesion layer. Only energy can be irradiated. As a result, even when cells adhere to the cell adhesion inhibiting portion, cells on the cell adhesion inhibiting portion can be removed by this energy irradiation, and a high-definition pattern can be maintained. .

  Here, the cell adhesion part is an area containing a cell adhesion material that has adhesiveness to cells and is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation, and has an adhesion property with a target cell. It is a good area. Here, having adhesion with cells means having physicochemical cell adhesion and biological cell adhesion as described in the above-mentioned section “A. Patterning substrate coating solution”. .

  On the other hand, the cell adhesion-inhibiting part is an area where the cell adhesion material is decomposed or denatured and is an area having low adhesion to cells. The cell adhesion material is decomposed or denatured means that the cell adhesion material is not contained, or the cell adhesion material is contained in a small amount compared to the amount of the cell adhesion material contained in the cell adhesion part. It means being done. For example, when the 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 a degradation product of the cell adhesion material, etc. Or the cell adhesive material layer is completely decomposed and removed to contain only the binder. When the cell adhesion material is denatured by the action of a photocatalyst accompanying energy irradiation, the denatured product or the like is contained in the cell adhesion inhibition portion. In the present invention, it is preferable that the cell adhesion-inhibiting portion contains a cell adhesion-inhibiting material having a cell adhesion-inhibiting property that inhibits adhesion to cells. Thereby, the adhesiveness with the cells of the cell adhesion-inhibiting part can be made lower, and the cells can be adhered with high definition only to the cell adhesion part.

  Here, the base material and the cell adhesive layer used in the cell culture patterning substrate of the present invention are the same as those described in the above-mentioned patterning substrate section, and thus the description thereof is omitted. A method for forming a cell adhesion layer having the cell adhesion part and the cell adhesion inhibition part will be described.

  First, using the patterning substrate coating solution described in “A. Patterning substrate coating solution” described above, for example, as shown in FIG. 5, the cell adhesion layer 2 containing at least a cell adhesion material and a binder is formed. It forms on the base material 1 (FIG.5 (a)). Next, a substrate 11 and a photocatalyst containing layer side substrate 13 having a photocatalyst containing layer 12 containing at least a photocatalyst formed on the substrate 11 are prepared, and the photocatalyst containing layer 12 and the cell adhesion layer 2 are opposed to each other. (Fig. 5B). Subsequently, the energy 6 is irradiated using a photomask 5 or the like, for example, in a pattern that forms the cell adhesion inhibition portion (FIG. 5B). Thereby, the cell adhesion material contained in the cell adhesion layer 2 is decomposed or denatured by the action of the photocatalyst contained in the photocatalyst containing layer 12, and the cell adhesion inhibitor 4 having low adhesion to cells, Since energy is not irradiated and the cell adhesion material is not decomposed, a cell adhesion part 2 ′ having good adhesion to cells is formed (FIG. 5C). The method for forming the cell adhesion layer is the same as the method described in “B. Patterning Substrate”, and therefore, the description thereof is omitted and used for the formation of such a cell adhesion inhibiting portion. The photocatalyst containing layer side substrate having the photocatalyst containing layer, energy, and the like will be described below.

(Photocatalyst containing layer side substrate)
First, a photocatalyst containing layer side substrate having a photocatalyst containing layer containing a photocatalyst will be described. The photocatalyst-containing layer side substrate used in the present invention usually has a photocatalyst-containing layer containing a photocatalyst, and usually a substrate and a photocatalyst-containing layer formed on the substrate. This photocatalyst containing layer side substrate may have a photocatalyst containing layer side light shielding part, a primer layer, etc. formed in the shape of a pattern, for example. Hereinafter, each structure of the photocatalyst containing layer side board | substrate used for this process is demonstrated.

a. Photocatalyst containing layer First, the photocatalyst containing layer used for a photocatalyst containing layer side board | substrate is demonstrated. The photocatalyst containing layer used in the present invention 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. It may be composed of a binder, or may be formed of a photocatalyst alone. Further, the surface characteristics may be particularly lyophilic or lyophobic.

  The photocatalyst-containing layer used in the present invention may be formed on the entire surface of the substrate 11 as shown in FIG. 5B, for example. However, as shown in FIG. The photocatalyst containing layer 12 may be formed on the pattern.

  By forming the photocatalyst-containing layer in a pattern in this way, when irradiating energy to form the cell adhesion inhibiting portion, it is not necessary to irradiate the pattern using a photomask or the like, and by irradiating the entire surface. The cell adhesion material contained in the cell adhesion layer can form a decomposed or denatured pattern.

  The method for patterning the photocatalyst-containing layer is not particularly limited, but can be performed by, for example, a photolithography method.

  In addition, since the cell adhesion material is actually decomposed or modified only in the part on the cell adhesion layer facing the photocatalyst containing layer, the direction of energy irradiation faces the photocatalyst containing layer and the cell adhesion layer. As long as the portion is irradiated with energy, it may be irradiated from any direction. Further, the irradiated energy is not particularly limited to parallel light such as parallel light.

Examples of the photocatalyst used in the present invention include titanium dioxide (TiO ₂ ), zinc oxide (ZnO), tin oxide (SnO ₂ ), strontium titanate (SrTiO ₃ ), tungsten oxide (WO ₃ ), which are known as photo semiconductors. Bismuth oxide (Bi ₂ O ₃ ) and iron oxide (Fe ₂ O ₃ ) can be mentioned, and one or a mixture of two or more selected from these can be used.

  In the present invention, titanium dioxide is particularly preferably used because it has a high band gap energy, is chemically stable, has no toxicity, and is easily available. Titanium dioxide includes an anatase type and a rutile type, and both can be used in the present invention, but anatase type titanium dioxide is preferred. Anatase type titanium dioxide has an excitation wavelength of 380 nm or less.

Examples of such anatase type titanium dioxide include hydrochloric acid peptizer type anatase type titania sol (STS-02 manufactured by Ishihara Sangyo Co., Ltd. (average particle size 7 nm), ST-K01 manufactured by Ishihara Sangyo Co., Ltd.), nitric acid solution An anatase type titania sol (TA-15 manufactured by Nissan Chemical Co., Ltd. (average particle size 12 nm)) and the like can be mentioned.
The smaller the particle size of the photocatalyst, the more effective the photocatalytic reaction occurs. The average particle size is preferably 50 nm or less, and the photocatalyst of 20 nm or less is particularly preferable.

  The photocatalyst-containing layer in the present invention may be formed by a photocatalyst alone as described above, or may be formed by mixing with a binder.

  In the case of a photocatalyst-containing layer consisting of only a photocatalyst, the efficiency of decomposition or denaturation of the cell adhesion material in the cell adhesion layer is improved, which is advantageous in terms of cost such as shortening of the treatment time. On the other hand, in the case of a photocatalyst-containing layer comprising a photocatalyst and a binder, there is an advantage that the formation of the photocatalyst-containing layer is easy.

  Examples of a method for forming a photocatalyst-containing layer composed only of a photocatalyst include a method using a vacuum film forming method such as a sputtering method, a CVD method, or a vacuum deposition method. By forming the photocatalyst-containing layer by a vacuum film-forming method, it is possible to obtain a photocatalyst-containing layer that is a uniform film and contains only the photocatalyst, thereby decomposing or denaturing the cell adhesion material in the cell adhesion layer. Since it can be performed uniformly and consists of only a photocatalyst, the cell adhesion material can be efficiently decomposed or modified as compared with the case of using a binder.

  In addition, as another example of a method for forming a photocatalyst-containing layer composed only of a photocatalyst, for example, when the photocatalyst is titanium dioxide, amorphous titania is formed on a substrate, and then the phase is changed to crystalline titania by firing. Is mentioned. As the amorphous titania used here, for example, hydrolysis, dehydration condensation, tetraethoxytitanium, tetraisopropoxytitanium, tetra-n-propoxytitanium, tetrabutoxytitanium, titanium inorganic salts such as titanium tetrachloride and titanium sulfate, An organic titanium compound such as tetramethoxytitanium can be obtained by hydrolysis and dehydration condensation in the presence of an acid. Next, it can be modified to anatase titania by baking at 400 ° C. to 500 ° C. and modified to rutile type titania by baking at 600 ° C. to 700 ° C.

  Moreover, when using a binder, what has the high bond energy that the main frame | skeleton of a binder is not decomposed | disassembled by photoexcitation of said photocatalyst is preferable, for example, organopolysiloxane etc. can be mentioned.

  When organopolysiloxane is used as a binder in this way, the photocatalyst-containing layer is prepared by dispersing the photocatalyst and the binder organopolysiloxane in a solvent together with other additives as necessary. The coating solution can be formed by coating on a substrate. As 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 or bead coating. When an ultraviolet curable component is contained as a binder, the photocatalyst-containing layer can be formed by irradiating with ultraviolet rays and performing a curing treatment.

  Moreover, an amorphous silica precursor, surfactant, etc. can be used as a binder. As such a material, the same materials as those described in JP 2000-249821 A can be used.

b. Substrate Next, the substrate used for the photocatalyst-containing layer side substrate will be described. In the present invention, as shown in FIG. 5 (b), the photocatalyst-containing layer side substrate has at least a base 11 and a photocatalyst-containing layer 12 formed on the base 11. At this time, the material constituting the substrate to be used is appropriately selected depending on the energy irradiation direction to be described later and whether the pattern forming body to be obtained requires transparency.

The substrate used in the present invention may be a flexible substrate such as a resin film, or may be a non-flexible substrate such as a glass substrate. This is appropriately selected depending on the energy irradiation method.
In order to improve the adhesion between the substrate surface and the photocatalyst containing layer, an anchor layer may be formed on the substrate. Examples of such an anchor layer include silane-based and titanium-based coupling agents.

c. Photocatalyst containing layer side light-shielding part As the photocatalyst containing layer side light shielding part used for this invention, you may use what the photocatalyst containing layer side light shielding part formed in pattern shape was formed. Thus, by using the photocatalyst containing layer side substrate having the photocatalyst containing layer side light-shielding portion, it is not necessary to use a photomask or perform drawing irradiation with laser light when irradiating energy. Therefore, since alignment between the photocatalyst-containing layer side substrate and the photomask is not necessary, it is possible to use a simple process, and an expensive apparatus necessary for drawing irradiation is also unnecessary, so that the cost is reduced. Has the advantage of being advantageous.

The photocatalyst-containing layer side substrate having such a photocatalyst-containing layer side light-shielding part can have the following two modes depending on the formation position of the photocatalyst-containing layer side light-shielding part.
For example, as shown in FIG. 7, for example, a photocatalyst containing layer side light shielding part 14 is formed on a substrate 11, and a photocatalyst containing layer 12 is formed on the photocatalyst containing layer side light shielding part 14 to form a photocatalyst containing layer side substrate. It is an aspect to make. For example, as shown in FIG. 8, a photocatalyst containing layer 12 is formed on a substrate 11, and a photocatalyst containing layer side light shielding portion 14 is formed thereon to form a photocatalyst containing layer side substrate.

  In any aspect, compared to the case where a photomask is used, the photocatalyst containing layer side light shielding portion is arranged in the vicinity of the arrangement portion of the photocatalyst containing layer and the cell adhesion layer. Since the influence of energy scattering can be reduced, energy pattern irradiation can be performed very accurately.

  Here, in the present invention, when the photocatalyst containing layer side light shielding portion 14 is formed on the photocatalyst containing layer 12 as shown in FIG. 8, the photocatalyst containing layer and the cell adhesion layer are placed at predetermined positions. When arranging, the photocatalyst containing layer side light shielding part is used as a spacer for keeping the gap constant by making the film thickness of the photocatalyst containing layer side light shielding part coincide with the width of the gap. Has the advantage of being able to.

  That is, when the photocatalyst-containing layer and the cell adhesion layer are arranged to face each other with a predetermined gap, the photocatalyst-containing layer side light-shielding portion and the cell adhesion layer are arranged in close contact with each other. It is possible to make the predetermined gap accurate, and by irradiating energy in this state, the cell adhesion layer in the portion where the cell adhesion layer and the light shielding portion are in contact with each other is decomposed or Since it is not denatured, it becomes possible to accurately form the cell adhesion inhibiting portion.

  The method for forming such a photocatalyst-containing layer side light-shielding part is not particularly limited, and is appropriately selected according to the characteristics of the formation surface of the photocatalyst-containing layer side light-shielding part, the shielding property against the required energy, and the like. Since it can be the same as the light-shielding portion provided on the base material described in “B. Patterning Substrate”, detailed description thereof is omitted here.

  In the above description, the two positions of the photocatalyst containing layer side light shielding portion between the substrate and the photocatalyst containing layer and the surface of the photocatalyst containing layer have been described as the formation position of the photocatalyst containing layer side. It is also possible to adopt a mode in which the photocatalyst-containing layer side light shielding portion is formed on the surface that is not provided. In this embodiment, for example, a case where a photomask is attached to the surface to such an extent that it can be attached and detached is conceivable, and it can be suitably used when the pattern of the cell adhesion inhibiting portion is changed in a small lot.

d. Next, the primer layer used for the photocatalyst containing layer side substrate of the present invention will be described. In the present invention, when the photocatalyst-containing layer side light-shielding portion is formed in a pattern on the substrate as described above, and the photocatalyst-containing layer is formed on the photocatalyst-containing layer side substrate, the photocatalyst-containing layer side substrate is used. A primer layer may be formed between the side light shielding part and the photocatalyst containing layer.

  Although the action and function of this primer layer are not necessarily clear, by forming a primer layer between the photocatalyst-containing layer side light-shielding part and the photocatalyst-containing layer, the primer layer can decompose the cell adhesion material by the action of the photocatalyst. Or impurities from the opening present between the photocatalyst-containing layer side light-shielding part and the photocatalyst-containing layer side light-shielding part, which are factors that inhibit modification, in particular, residues generated when patterning the photocatalyst-containing layer side light-shielding part, metal, It is considered that it has a function of preventing diffusion of impurities such as metal ions. Therefore, by forming the primer layer, degradation or denaturation treatment of the cell adhesion material proceeds with high sensitivity, and as a result, a cell adhesion inhibition portion formed with high definition can be obtained.

In the present invention, the primer layer prevents impurities existing in not only the photocatalyst containing layer side light shielding part but also the opening formed between the photocatalyst containing layer side light shielding parts from affecting the action of the photocatalyst. The primer layer is preferably formed over the entire surface of the photocatalyst containing layer side light shielding portion including the opening.
The primer layer in the present invention 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 is not in contact with 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 the photocatalyst is preferable. Specific examples include amorphous silica. When such amorphous silica is used, the precursor of the amorphous silica is represented by the general formula SiX ₄ and X is a silicon compound such as halogen, methoxy group, ethoxy group, or acetyl group, Silanol which is a hydrolyzate thereof or polysiloxane having an average molecular weight of 3000 or less is preferable.
The thickness of the primer layer is preferably in the range of 0.001 μm to 1 μm, particularly preferably in the range of 0.001 μm to 0.1 μm.

(Energy irradiation)
Next, energy irradiation at the time of forming the cell adhesion inhibition part will be described. The energy irradiation can be performed by arranging the cell adhesion layer and the photocatalyst containing layer in the photocatalyst containing layer side substrate with a predetermined gap and irradiating energy from a predetermined direction.

  The above arrangement refers to a state where the photocatalyst acts substantially on the surface of the cell adhesion layer. In addition to a state where the photocatalyst actually touches, a predetermined interval is provided. Thus, the photocatalyst-containing layer and the cell adhesion layer are arranged. This gap is preferably 200 μm or less.

  In the present invention, the gap has a very good pattern accuracy and a high photocatalyst sensitivity. Therefore, in consideration of the efficiency of decomposition or denaturation of the cell adhesion material in the cell adhesion layer, it is particularly 0.2 μm to 10 μm. It is preferable to be within the range of 1 μm to 5 μm. Such a gap range is particularly effective for a cell adhesion layer having a small area that can control the gap with high accuracy.

  On the other hand, when the treatment is performed on a cell adhesion layer having a large area of, for example, 300 mm × 300 mm or more, a fine gap as described above is formed between the photocatalyst-containing layer side substrate and the cell adhesion layer without contact. It is extremely difficult to form. Therefore, when the cell adhesion layer has a relatively large area, the gap is preferably in the range of 10 to 100 μm, particularly in the range of 50 to 75 μm. By setting the gap within such a range, problems such as a decrease in pattern accuracy such as blurring of the pattern and a problem that the sensitivity of the photocatalyst deteriorates and the efficiency of decomposing or denaturing the cell adhesion material may occur. This is because there is an effect that unevenness does not occur in the decomposition or denaturation of the cell adhesive material.

  Thus, when irradiating energy to a cell adhesion layer having a relatively large area, the setting of the gap in the positioning device between the photocatalyst containing layer side substrate and the cell adhesion layer in the energy irradiation device is within the range of 10 μm to 200 μm, In particular, it is preferable to set within a range of 25 μm to 75 μm. By setting the set value within such a range, the pattern accuracy and the photocatalyst sensitivity are not significantly degraded, and the photocatalyst-containing layer side substrate and the cell adhesion layer are not in contact with each other. Because it becomes possible.

  Thus, by disposing the photocatalyst-containing layer and the cell adhesion layer surface at a predetermined interval, oxygen, water, and active oxygen species generated by the photocatalytic action are easily desorbed. That is, when the interval between the photocatalyst-containing layer and the cell adhesion layer is narrower than the above range, the desorption of the active oxygen species becomes difficult, and as a result, the rate of decomposing or denaturing the cell adhesion material can be reduced. It is not preferable because of its properties. In addition, when it is arranged at a distance from the above range, the generated active oxygen species are difficult to reach the cell adhesion layer, and in this case as well, there is a possibility of slowing down the decomposition or denaturation of the cell adhesion material. Is not preferable.

  An example of a method for uniformly forming such an extremely narrow gap and arranging the photocatalyst-containing layer and the cell adhesion layer is a method using a spacer. By using the spacer in this way, a uniform gap can be formed, and the portion in contact with the spacer does not reach the surface of the cell adhesion layer due to the action of the photocatalyst. By having the same pattern as the adhesion part, the cell adhesion material of only the part where the spacer is not formed can be decomposed or denatured, and the cell adhesion inhibition part can be formed with high definition. . In addition, by using such a spacer, the active oxygen species generated by the action of the photocatalyst reaches the cell adhesion layer surface at a high concentration without diffusing, thereby efficiently forming a high-definition cell adhesion inhibitor. can do.

  In the present invention, such an arrangement state of the photocatalyst-containing layer side substrate only needs to be maintained at least during the energy irradiation.

Here, the energy irradiation (exposure) as used in the present invention is a concept including irradiation of any energy rays that can decompose or denature the cell adhesion material by the action of the photocatalyst accompanying the energy irradiation. It is not limited to irradiation.
Usually, the wavelength of light used for such energy irradiation is set in the range of 400 nm or less, preferably in the range of 380 nm or less. This is because, as described above, the preferred photocatalyst used as the photocatalyst is titanium dioxide, and as the energy for activating the photocatalytic action by the titanium dioxide, light having the above-described wavelength is preferable.
Examples of light sources that can be used for such energy irradiation include mercury lamps, metal halide lamps, xenon lamps, excimer lamps, and various other light sources.

  In addition to the method of performing pattern irradiation through a photomask using the light source as described above, it is also possible to use a method of drawing and irradiating in a pattern using a laser such as excimer or YAG. Further, as described above, when the substrate has the light shielding portion in the same pattern as the cell adhesion portion, it can be performed by irradiating the entire surface with energy from the substrate side. Moreover, when it has a light-shielding part in the same pattern as a cell adhesion part on a photocatalyst content layer, it can carry out by irradiating the whole surface from any direction. In these cases, there is an advantage that a photomask or the like is not necessary and a step such as alignment is not necessary.

Here, as described above, when the substrate has a recess and the cell adhesion layer is formed in the recess, the entire surface may be irradiated with energy by the method described above. For example, when a plurality of the concave portions are formed, for example, the exposure may be performed in different patterns for each concave portion. In this way, as a method of performing exposure for each concave portion, for example, a different mask is arranged in each concave portion, and a method of irradiating energy using the photocatalyst-containing layer side substrate, for example, the tip of an optical fiber, Examples include a method in which a chromium mask, a stencil mask, and the like and the photocatalyst-containing layer side substrate are arranged and energy is irradiated.
Note that, for example, a cylindrical mask or the like may be used to expose only the bottom surface of the recess so that the sidewalls of the recess are not irradiated with energy.

In addition, the energy irradiation amount 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 the photocatalyst.
In this case, it is preferable in that the energy can be increased while heating the photocatalyst-containing layer, so that the sensitivity can be increased and the cell adhesion material can be efficiently decomposed or denatured. Specifically, it is preferable to heat within a range of 30 ° C to 80 ° C.

  Moreover, as for the direction of energy irradiation performed through the photomask in the present invention, when the above-described base material is transparent, energy irradiation may be performed from any direction of the base material side and the photocatalyst containing layer side substrate. On the other hand, when the substrate is opaque, it is necessary to irradiate energy from the photocatalyst containing layer side substrate side.

3. Next, the cell culture patterning substrate of the present invention will be described. The cell culture patterning substrate of the present invention is not particularly limited as long as the cell adhesion layer having the cell adhesion part and the cell adhesion inhibition part is formed on the base material. Depending on the situation, other layers may be appropriately formed.
Further, in the present invention, after forming the cell adhesion inhibiting part, a part of the cell culture patterning substrate is cut out and a part of the cell culture patterning substrate is attached to the bottom of a concave base material or the like Or the like may be used as a cell culture patterning substrate.

D. Cell Culture Substrate Next, the cell culture substrate of the present invention will be described. The cell culture substrate of the present invention is obtained by adhering cells on the cell adhesion portion in the above-described cell culture patterning substrate.
In the cell culture substrate of the present invention, for example, as shown in FIG. 9, cells 7 adhere only on the cell adhesion part 2 ′ of the cell adhesion layer 2, and cells 7 adhere on the cell adhesion inhibition part 4. It is not.

  According to the present invention, on the cell culture patterning substrate, a cell adhesion portion having good adhesion to cells and a cell adhesion inhibition portion having no adhesion to cells are formed. For example, even when the cells are applied to the entire surface of the cell culture patterning substrate, the cells on the cell adhesion inhibiting portion can be easily removed by adhering the cells only to the cell adhesion portion. Thereby, it can be easily formed without using a complicated process or a treatment solution that adversely affects cells. In addition, since it is not necessary for the cell culture patterning substrate to contain a photocatalyst, it is possible that the photocatalyst has no possibility of affecting the cells over time, and a high-quality cell culture substrate can be obtained. It can be.

  Here, according to the present invention, for example, the photocatalyst-containing layer of the photocatalyst-containing layer side substrate is disposed so as to face the cell adhesion layer, and the cell adheres by irradiating the entire surface of the cell culture substrate with energy. The cell adhesion material in the cell adhesion part can be decomposed or denatured, and the adhesion to cells can be lowered. Thereby, the cell etc. which adhere | attached on the cell adhesion part can be peeled easily, and only the cell formed in the pattern shape can be obtained. The energy irradiated at this time is set to an energy that does not affect the cells.

In addition, as described above, when the light-shielding part is formed on the base material in the same pattern as the cell adhesion part, for example, it is opposed to the photocatalyst-containing layer side substrate, and if necessary, the entire surface from the base material side. By irradiating the cell with energy, it becomes possible to remove cells adhering to the cell adhesion-inhibiting portion, and the pattern in which the cells adhere to the cell adhesion portion can be maintained with high definition.
When the light shielding part is not formed, a photomask or the like in which openings are formed in the same pattern as the cell adhesion inhibiting part with the photocatalyst containing layer side substrate and the cell adhesive layer facing each other is used. By irradiating energy, a high-definition pattern can be maintained.
Hereinafter, the cells used for the cell culture substrate of the present invention will be described. Here, the cell culture patterning substrate is the same as that described in “C. Patterning substrate for cell culture”, and a description thereof will be omitted.

(cell)
The cells used for the cell culture substrate of the present invention are not particularly limited as long as they adhere to the cell adhesion part of the cell culture patterning substrate and do not adhere to the cell adhesion inhibition part. Absent.

  Examples of cells used in the present invention include any tissue existing in a living body and cells derived from it other than non-adherent cells such as nerve tissue, liver, kidney, pancreas, blood vessel, brain, cartilage, and blood cells. be able to. In general, for non-adherent cells, in recent years, a technique for modifying a cell membrane has been devised for adhesion fixation, and it can be used in the present invention by using such a technique as necessary. It is.

  Here, since each tissue as described above is formed by cells having various functions, it is necessary to select and use desired cells. For example, in the case of the liver, it is formed from epithelial cells, endothelial cells, Kupffer cells, fibroblasts, fat intake cells and the like in addition to liver parenchymal cells. In this case, since the adhesiveness to the cell adhesion material differs depending on the cell type, it is necessary to select the cell adhesion material used in the cell adhesion part and the composition ratio depending on the cell type.

  In addition, as a method for adhering cells to the cell adhesion part, it is possible to adhere cells only to the cell adhesion part of the cell culture patterning substrate having the cell adhesion part and the cell adhesion inhibition part. There is no particular limitation. For example, a method of adhering cells with an ink jet printer or a manipulator may be used, but after seeding the cell suspension and adhering the cells to the cell adhesion part, the cell adhesion inhibition part no longer needed A method of washing cells with a phosphate buffer and removing the cells is generally used. Such methods include, for example, the reference “Spatial distribution of mammalian cells dedicated by material surface chemistry”, Kevin E. Healy et al., Biotech. Bioeng. (1994) The method described in p.792 etc. can be used.

E. Next, the manufacturing method of the cell culture patterning substrate of the present invention will be described. The method for producing a patterning substrate for cell culture according to the present invention comprises a cell adhesion material containing a binder, and a cell adhesion material that has adhesiveness to cells and is decomposed or modified by the action of a photocatalyst associated with energy irradiation. A cell adhesion layer forming step for forming a layer; the cell adhesion layer; a photocatalyst containing layer containing a photocatalyst; and a photocatalyst containing layer side substrate having a substrate so that the cell adhesion layer and the photocatalyst containing layer face each other. After being placed on the cell adhesion layer, it is irradiated with energy from a predetermined direction, and is composed of a cell adhesion inhibiting part in which the cell adhesion material contained in the cell adhesion layer is decomposed or denatured, and a cell adhesion part other than the cell adhesion inhibiting part. And an energy irradiation step for forming a pattern.

  The method for producing a patterning substrate for cell culture according to the present invention, for example, as shown in FIG. 5, forms a cell adhesion layer 2 that forms a cell adhesion layer containing a cell adhesion material and a binder on a substrate 1. A photocatalyst containing layer side substrate 13 having a layer forming step (FIG. 5 (a)) and a base 11 and a photocatalyst containing layer 12 containing a photocatalyst is prepared, and the cell adhesion layer 2 and the photocatalyst containing layer 12 are opposed to each other. Cells that are disposed and irradiated with energy 6 using, for example, a photomask 5 (FIG. 5B), and the cell adhesion material contained in the cell adhesion layer 2 in the region irradiated with the energy is decomposed or denatured It has an energy irradiation step (FIG. 5C) that forms an adhesion inhibiting portion 4 and a cell adhesion portion 2 ′ that is not irradiated with energy and has good adhesion to cells.

According to the present invention, by irradiating energy using the photocatalyst-containing layer side substrate, the cell adhesion material contained in the cell adhesion layer can be easily decomposed or denatured into a high-definition pattern. It is possible to form a cell adhesion part and a cell adhesion inhibition part. Moreover, since it is not necessary to contain a photocatalyst in the cell adhesion layer, a high-quality cell culture patterning substrate that does not affect the photocatalyst over time can be obtained.
Hereinafter, each process of the present invention will be described.

1. Cell Adhesion Layer Formation Step First, the cell adhesion layer formation step of the present invention will be described. In the cell adhesion layer forming step of the present invention, a cell adhesion layer containing a binder and a cell adhesion material that has adhesiveness to cells and is decomposed or modified by the action of a photocatalyst accompanying energy irradiation is formed on a substrate. It is a process to do. In the present invention, the coating can be carried out on a substrate using the above-mentioned cell adhesion material and a coating solution containing a binder, and the coating method and the material thereof are particularly limited. However, it is preferable that a cell adhesion-inhibiting material having a cell adhesion inhibitory property that inhibits adhesion to cells after at least energy irradiation is used as a binder. Thereby, in the energy irradiation process to be described later, the adhesiveness with the cells of the cell adhesion-inhibiting portion that is the region irradiated with energy can be made low, and the cells can be adhered in a high-definition pattern. This is because it can be used as a cell culture patterning substrate.

  The cell adhesion material, binder, cell adhesion inhibitory material, and base material used in this step, and the method for forming the cell adhesion layer are the same as those described in the above section “B. Patterning substrate”. Since there is, explanation here is omitted.

2. Energy Irradiation Step Next, the energy irradiation step in the present invention will be described. In the energy irradiation step of the present invention, the cell adhesion layer, the photocatalyst-containing layer containing the photocatalyst, and the photocatalyst-containing layer side substrate having the substrate are arranged so that the cell adhesion layer and the photocatalyst-containing layer face each other. After that, a pattern consisting of a cell adhesion inhibiting part in which the cell adhesion material contained in the cell adhesion layer is decomposed or denatured and a cell adhesion part other than the cell adhesion inhibiting part is irradiated with energy from a predetermined direction. It is a process of forming.

In this step, the cell adhesion layer formed by the cell adhesion layer formation step described above and the photocatalyst containing layer of the photocatalyst containing layer side substrate are opposed to each other to irradiate energy in a pattern, and the cell adhesion in the cell adhesion layer The method is not particularly limited as long as the material is decomposed or modified to form a cell adhesion-inhibiting part having low adhesion to cells.
Here, as the photocatalyst-containing layer side substrate used in this step and the energy irradiation method, those described in the above-mentioned section “C. Patterning substrate for cell culture” can be used. Detailed explanation here is omitted.

F. Next, a method for producing a cell culture substrate of the present invention will be described. There are two embodiments of the method for producing a cell culture substrate of the present invention. Hereinafter, each embodiment will be described.

1. First Embodiment First, a first embodiment of the cell culture substrate of the present invention will be described. The cell culture substrate manufacturing method in this embodiment is a cell adhesion part of the cell culture patterning substrate manufactured by the cell culture patterning substrate manufacturing method described in “E. Cell culture patterning substrate manufacturing method” described above. It has a cell adhesion process for adhering cells.

  According to this embodiment, the cell culture patterning substrate includes a cell adhesion portion having adhesion to cells and a cell adhesion inhibition portion having no adhesion to cells formed on the cell adhesion layer. Therefore, by applying cells or the like on the cell adhesion layer, it is possible to easily produce a cell culture substrate having cells adhered only on the cell adhesion portion. In addition, since the photocatalyst does not need to be contained in the cell culture patterning substrate, the photocatalyst can be prevented from affecting the cells over time, and a high-quality cell culture substrate and It can be done.

  Here, the cell adhesion step in the present embodiment is not particularly limited as long as it is a method capable of attaching cells onto the cell adhesion portion, and the above-mentioned item “D. Cell culture substrate”. Therefore, the detailed description is omitted here.

2. Second Embodiment Next, a second embodiment of the method for producing a cell culture substrate of the present invention will be described. The method for producing a cell culture substrate according to the present embodiment includes a base material, a cell adhesive material formed on the base material, having adhesiveness with cells and decomposed or modified by the action of a photocatalyst accompanying energy irradiation, and a binder A cell adhesion layer containing a cell formed in a pattern on the cell adhesion layer, the cell adhesion layer comprising: a cell adhesion inhibition part in which the cell adhesion material is decomposed or denatured; and the cell A cell culture substrate having a cell adhesion part other than an adhesion inhibition part, wherein the cells are formed on the cell adhesion part,

  After performing the cell adhesion step of adhering cells on the cell adhesion part, the cell adhesion inhibition part is provided with a photocatalyst-containing layer containing a photocatalyst and a substrate containing a photocatalyst-containing layer side substrate. After arrange | positioning so that a photocatalyst content layer may oppose, it has a cell maintenance process of irradiating energy from a predetermined direction and maintaining the pattern of the cell adhere | attached on the said cell adhesion part.

For example, as shown in FIG. 10, the method for producing a cell culture substrate according to this embodiment includes a base material 1 and a cell adhesion layer formed on the base material 1 and having a cell adhesion part 2 ′ and a cell adhesion inhibition part 4. 2 and a method for producing a cell culture substrate having cells 7 formed on the cell adhesion part 2. In this embodiment, after performing the cell adhesion step of adhering the cells 7 on the cell adhesion part 2 ′, the substrate 11 and the photocatalyst containing layer side substrate 13 in which the photocatalyst containing layer 12 is formed on the substrate 11. The photocatalyst-containing layer 12 and the cell adhesion layer 2 are arranged to face each other, and for example, a cell maintenance step of irradiating only the cell adhesion inhibition part 4 with energy 6 using a photomask 5 or the like is provided. . Thereby, even when cells adhere to the cell adhesion-inhibiting part, proteins, organic substances, cells, etc. on the cell adhesion-inhibiting part can be removed by the action of the photocatalyst contained in the photocatalyst-containing layer. Thus, a cell culture substrate in which cells are adhered with high precision only on the cell adhesion portion can be obtained.
Hereafter, the cell maintenance process in the manufacturing method of the cell culture substrate of this embodiment is demonstrated.

(Cell maintenance process)
The cell maintenance step in the method for producing a cell culture substrate of the present embodiment includes performing a cell adhesion step for adhering cells on the cell adhesion portion, and then using the photocatalyst-containing layer side substrate for the cell adhesion inhibition portion. If it is a step of maintaining the pattern of cells adhered on the cell adhesion part by performing irradiation, and if it is possible to maintain the cell pattern on the cell adhesion part with high definition, the energy irradiation method Etc. are not particularly limited.

  In the present embodiment, for example, as shown in FIG. 10, after the photocatalyst containing layer 12 and the cell adhesion layer 2 in the photocatalyst containing layer side substrate 13 are arranged to face each other, the openings are formed in the pattern of the cell adhesion inhibiting portion. For example, a method of irradiating energy 6 using the photomask 5 or the like that is included. Further, for example, as shown in FIG. 11, the light-catalyst portion 14 is provided on the photocatalyst-containing layer side substrate 13, and the photocatalyst-containing layer 12 and the cell adhesion layer 2 on the photocatalyst-containing layer side substrate 13 are arranged to face each other. Thereafter, a method of irradiating the entire surface with energy 6 may be used. For example, as shown in FIG. 12, when the light-shielding part 3 is formed on the substrate 1 in the same pattern as the cell adhesion part 2 ′, the cell adhesion layer 2 and the photocatalyst containing layer 12 are opposed to each other. After that, by irradiating the entire surface with the energy 6 from the substrate 1 side, a method of irradiating the energy 6 only to the cell adhesion inhibiting portion 4 can be obtained.

  At this time, the energy to be irradiated is not particularly limited as long as it is an energy capable of removing cells adhered on the cell adhesion inhibiting portion by the action of the photocatalyst accompanying energy irradiation, specifically, It can be the same as that described as the method for forming the cell adhesion layer of “C. Patterning substrate for cell culture”. Further, the photocatalyst-containing layer side substrate, the arrangement position of the photocatalyst-containing layer side substrate, and the like may be the same as those described as the method for forming the cell adhesion layer of “C. Patterning substrate for cell culture”. Since it can, detailed explanation here is omitted.

In addition, the time when this step is performed may be performed immediately after the cell adhesion step in which cells are adhered on the cell adhesion portion, and when the cells on the cell adhesion portion are cultured for a predetermined period, It may be performed appropriately according to the type and state of the cell so that there is no problem such as a cell adhering to the adhesion-inhibiting part and a thick pattern. Moreover, this process may be performed repeatedly.
In addition, since it can be made to be the same as that of the cell adhesion process of the 1st embodiment mentioned above about the cell adhesion process which adheres a cell on the said cell adhesion part, description here is abbreviate | omitted.

(Other)
In the method for producing a cell culture substrate of this embodiment, in addition to the energy irradiation step and the cell adhesion step, for example, a cell adhesion layer is formed by applying a coating solution for a patterning substrate containing a photocatalyst and a cell adhesion material on the substrate. And the step of forming a cell adhesion layer having a cell adhesion part and a cell adhesion inhibition part by irradiating the cell adhesion layer with energy using a photocatalyst-containing layer side substrate. You may have.

  The cell adhesion material, base material, and photocatalyst-containing layer side substrate used in the method for producing a cell culture substrate of this embodiment, the method for forming the cell adhesion layer, and the like are described in “E. Patterning for cell culture”. Since it is the same as that described in “Manufacturing Method of Substrate”, description thereof is omitted here.

  The present invention is not limited to the above embodiment. The above-described embodiment is an exemplification, and the present invention has substantially the same configuration as the technical idea described in the claims of the present invention, and any device that exhibits the same function and effect is the present invention. It is included in the technical scope of the invention.

  The following examples illustrate the present invention more specifically.

[Example 1]
(Formation of cell adhesion layer)
3 g of isopropyl alcohol, 0.4 g of organosilane TSL8114 (GE Toshiba Silicone) and 0.4 g of aminopropyltriethoxysilane were mixed and heated at 100 ° C. for 20 minutes with stirring. This solution is applied to a 0.7 mm thick glass substrate that has been previously alkali-treated by spin coating, and the substrate is dried at a temperature of 150 ° C. for 10 minutes to allow hydrolysis and polycondensation reactions to proceed. An organopolysiloxane layer containing an amino group having a thickness of about 80 nm was formed on the substrate to obtain a patterning substrate.

(Patterning of patterning substrate)
The photomask on which the photocatalyst layer is formed is allowed to stand so that the photocatalyst layer and the cell adhesion layer of the patterning substrate face each other, and UV irradiation of 15 J / cm ² (wavelength 254 nm) is performed through the photomask with a mercury lamp. Thus, a cell culture patterning substrate having a cell adhesive surface in which the unexposed portion was cell adhesive and the exposed portion was patterned to inhibit cell adhesion was obtained.

(Cell adhesion process)
The details of the experimental procedure for culturing cells derived from various tissues are described in, for example, Togori Weave Culture Technology 3rd Edition, Basic Edition, Japanese Tissue Culture Society, Asakura Shoten, etc.
In this application, the substrate was evaluated using rat hepatocytes.
First, the liver extracted from the rat was transferred to a petri dish and subdivided into 5 mm pieces with a scalpel, 20 ml of DMEM medium was added, and the mixture was lightly suspended with a pipette and then filtered with a cell filter. The obtained coarsely dispersed cell suspension was centrifuged at 500 to 600 rpm for 90 seconds, and the supernatant was aspirated and removed. DMEM medium was newly added to the remaining cells and centrifuged again. By repeating this operation three times, almost uniform hepatocytes were obtained. To the obtained hepatocytes, 20 ml of DMEM medium was added and suspended to prepare a hepatocyte suspension.
Next, 900 ml of distilled water was added to 14.12 g of Waymouth MB752 / 1 medium (containing L-glutamine, not containing NaHCO ₃ ) (Gibco). To this, 2.24 g of NaHCO ₃ , 10 ml of amphotericin B solution (ICN) and 10 ml of penicillin streptomycin solution (Gibco) were added and stirred. After adjusting this to pH 7.4, the total volume was 1000 ml, and sterilized by filtration through a 0.22 μm membrane filter was used as the Waymouth MB752 / 1 medium solution.
The previously prepared liver parenchymal cell suspension was suspended in the same prepared Waymouth MB752 / 1 medium solution and then seeded on the above-mentioned cell culture patterning substrate having a cell adhesion part and a cell adhesion inhibition part. . The substrate was allowed to stand for 24 hours in an incubator with 37 ° C. and 5% CO _2, and hepatocytes were adhered 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 fresh medium. Cell culture was continued for 48 hours while exchanging the medium solution, and the cells were observed with an optical microscope. As a result, it was confirmed that the cells were adhered along the cell adhesion part on the cell culture patterning substrate.

[Example 2]
A hole with a diameter of 14 mm was made in the center of the bottom surface of a commercially available 35-mm diameter plastic dish (Corning). Subsequently, a cell culture patterning substrate was formed using a glass substrate having a thickness of about 0.1 mm in the same manner as in Example 1, and the cell culture patterning substrate was cut into 21 mm squares. Thereafter, the cut glass substrate of the cell culture patterning substrate was attached to the plastic dish using an adhesive KE45T (manufactured by Shin-Etsu Silicone).
(Cell adhesion)
The plastic dish was sterilized with 70% ethanol, washed with PBS, and then washed with DMEM medium. Then, when cells were cultured in the same manner as in Example 1, it was confirmed that the cells were adhered along the cell adhesion portion in the plastic dish.

It is a schematic sectional drawing which shows an example of the board | substrate for patterning of this invention. It is a schematic sectional drawing which shows the other example of the patterning board | substrate of this invention. It is a schematic sectional drawing which shows an example of the patterning board | substrate for cell cultures of this invention. It is a schematic sectional drawing which shows the other example of the patterning board | substrate for cell cultures of this invention. It is process drawing which shows an example of the formation method of the cell adhesion inhibition part in the patterning board | substrate for cell cultures of this invention. It is a schematic sectional drawing which shows an example of the photocatalyst containing layer side board | substrate used for this invention. It is a schematic sectional drawing which shows the other example of the photocatalyst containing layer side board | substrate used for this invention. It is a schematic sectional drawing which shows the other example of the photocatalyst containing layer side board | substrate used for this invention. It is a schematic sectional drawing which shows an example of the cell culture substrate of this invention. It is a schematic sectional drawing which shows an example of the energy irradiation process in the manufacturing method of the cell culture substrate of this invention. It is a schematic sectional drawing which shows the other example of the energy irradiation process in the manufacturing method of the cell culture substrate of this invention. It is a schematic sectional drawing which shows the other example of the energy irradiation process in the manufacturing method of the cell culture substrate of this invention. It is a schematic sectional drawing for demonstrating the base material used for the patterning board | substrate for cell cultures of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Board | substrate 2 ... Cell adhesion layer 2 '... Cell adhesion part 3 ... Light-shielding part 4 ... Cell adhesion inhibition part 5 ... Photomask 6 ... Energy 11 ... Base | substrate 12 ... Photocatalyst containing layer 13 ... Photocatalyst containing layer side board | substrate

Claims (6)

A base material, a cell adhesive material formed on the base material, having adhesiveness with cells and decomposed or modified by the action of a photocatalyst accompanying energy irradiation, and a cell adhesive layer containing a binder,
A light shielding portion is formed on the substrate;
All SANYO that have a high binding energy, such as the binder is the main backbone not degraded by photoexcitation of the photocatalyst,
A patterning substrate, wherein the cell adhesion material is a material having adhesion to cells by electrostatic interaction or a material having adhesion to cells by biochemical characteristics . The cell adhesion layer of the substrate for patterning according to claim 1 comprises a cell adhesion inhibition part in which the cell adhesion material is decomposed or denatured in a pattern, and a cell adhesion part that is an area other than the cell adhesion inhibition part. A patterning substrate for cell culture, comprising: The cell culture substrate according to claim 2, wherein cells are adhered on the cell adhesion part of the patterning substrate for cell culture. A cell adhesion layer forming step for forming a cell adhesion layer containing a binder, and a cell adhesion material that has adhesiveness to cells and is decomposed or modified by the action of a photocatalyst accompanying energy irradiation;
After the cell adhesion layer, the photocatalyst containing layer containing the photocatalyst and the photocatalyst containing layer side substrate having the substrate are arranged so that the cell adhesion layer and the photocatalyst containing layer face each other, energy irradiation is performed from a predetermined direction. And an energy irradiation step of forming a pattern consisting of a cell adhesion inhibiting part in which the cell adhesion material contained in the cell adhesion layer is decomposed or denatured, and a cell adhesion part other than the cell adhesion inhibiting part. ,
A light shielding portion is formed on the substrate;
All SANYO that have a high binding energy, such as the binder is the main backbone not degraded by photoexcitation of the photocatalyst,
A method for producing a patterning substrate for cell culture, wherein the cell adhesive material is a material having adhesiveness to cells by electrostatic interaction or a material having adhesiveness to cells by biochemical characteristics . A cell culture substrate production method comprising a cell adhesion step for adhering cells on the cell adhesion portion of the cell culture patterning substrate produced by the method for producing a cell culture patterning substrate according to claim 4. Method. A cell adhesion layer containing a base material, a cell adhesion material formed on the base material, having adhesiveness with cells and decomposed or modified by the action of a photocatalyst accompanying energy irradiation, and a binder, and the cell adhesion layer Cells formed in a pattern on the cell adhesion layer, the cell adhesion layer has a cell adhesion inhibition part in which the cell adhesion material is decomposed or denatured, and a cell adhesion part other than the cell adhesion inhibition part And a method for producing a cell culture substrate in which the cells are formed on the cell adhesion part,
After performing the cell adhesion step of adhering cells on the cell adhesion part, the photocatalyst-containing layer side substrate having a photocatalyst-containing layer and a substrate containing a photocatalyst is formed on the cell adhesion inhibition part and the photocatalyst. A cell maintaining step of maintaining the pattern of cells adhered on the cell adhesion part by irradiating energy from a predetermined direction after being arranged so as to face the containing layer;
A light shielding portion is formed on the substrate;
All SANYO that have a high binding energy, such as the binder is the main backbone not degraded by photoexcitation of the photocatalyst,
A method for producing a cell culture substrate, wherein the cell adhesion material is a material having adhesion to cells by electrostatic interaction or a material having adhesion to cells by biochemical characteristics .

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