JP4862261B2 - 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 is a cell comprising a substrate and a cell adhesion inhibitor formed on the substrate and having a cell adhesion inhibitory property that inhibits adhesion to cells and denatured by the action of a photocatalyst associated with energy irradiation. There is provided a patterning substrate comprising an adhesion inhibiting layer.

  According to the present invention, since the cell adhesion inhibition layer contains the cell adhesion inhibition material, the adhesion of the cell adhesion inhibition layer to the cells can be lowered. Further, when this cell adhesion inhibiting layer is irradiated with energy using, for example, a photocatalyst containing layer containing a photocatalyst, the cell adhesion inhibiting material is denatured and can have adhesiveness with cells. Therefore, by performing energy irradiation in a pattern using the photocatalyst-containing layer or the like, it is possible to easily form a region having adhesiveness with cells and a region having low adhesiveness with cells. It can be a substrate.

  The present invention also provides a substrate and a cell adhesion inhibitor that is formed on the substrate and has a cell adhesion inhibitory property that inhibits adhesion to cells and is decomposed or denatured by the action of a photocatalyst associated with energy irradiation. A patterning substrate having a material and a cell adhesion-inhibiting layer containing a binder is provided.

  According to the present invention, since the cell adhesion inhibition layer contains the cell adhesion inhibition material, the adhesion of the cell adhesion inhibition layer to the cells can be lowered. In addition, when the cell adhesion inhibition layer is irradiated with energy using, for example, a photocatalyst-containing layer containing a photocatalyst, the cell adhesion inhibition material is decomposed or denatured, and a binder having adhesiveness with cells is exposed. A region or a region containing a degradation product of a cell adhesion inhibiting material and a binder can be used. Therefore, by performing energy irradiation in a pattern using the photocatalyst-containing layer or the like, it is possible to easily form a region having adhesiveness with cells and a region having low adhesiveness with cells. It can be a substrate.

  In the said invention, it is preferable that the said cell adhesion inhibition layer contains the cell adhesion material which has adhesiveness with a cell at least after energy irradiation. This is because a patterning substrate with better adhesion to cells in the region irradiated with energy can be obtained.

  Moreover, in the said invention, it is preferable that the light shielding part is formed on the said base material. Thereby, for example, the cell adhesion inhibiting layer and the photocatalyst containing layer containing the photocatalyst are arranged to face each other, and by irradiating the entire surface from the base material side, cells only in the region where the light shielding part is not formed This is because a patterning substrate capable of decomposing or modifying the adhesion-inhibiting material can be obtained.

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

  According to the present invention, the cell adhesion inhibitor having the adhesiveness to the cell as a result of the cell adhesion inhibiting material being decomposed or modified, and the cell having a low adhesiveness to the cell without being decomposed or modified. Since it has an adhesion-inhibiting part, the cell culture patterning substrate is capable of adhering cells with high precision only to the cell adhesion part without using complicated processes or treatment solutions that adversely affect the cells. It can be.

  The present invention also provides a cell culture substrate, wherein cells are adhered on the cell adhesion part of the cell culture patterning substrate.

  According to the present invention, the cell culture patterning substrate is formed with a cell adhesion portion having adhesion to cells and a cell adhesion inhibition portion having low adhesion to cells. In addition, cells can be adhered only on the cell adhesion part, and a cell culture substrate having cells adhered in a highly fine 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 inhibitory material that inhibits cell adhesion and that is decomposed or denatured by the action of a photocatalyst associated with energy irradiation, and at least the cell after energy irradiation. And a cell adhesion material having adhesiveness, and a coating liquid for a patterning substrate.

  According to the present invention, the cell adhesion-inhibiting material is easily decomposed by applying the coating liquid for patterning substrate on, for example, a base material and irradiating energy using a photocatalyst-containing layer containing a photocatalyst. And can have adhesiveness to cells. In addition, since the cell-adhesive material is contained in the energy-irradiated region, the adhesiveness with the cells can be further improved. On the other hand, since the region not irradiated with energy is inhibited from adhering to cells by the cell adhesion inhibiting material, it can be a region having low adhesion to cells. 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.

The present invention also provides a cell adhesion inhibitor comprising a cell adhesion inhibitory material that has a cell adhesion inhibitory property that inhibits adhesion to cells and is decomposed or modified by the action of a photocatalyst associated with energy irradiation on a substrate. A cell adhesion inhibition layer forming step of forming a layer;
After arranging the cell adhesion inhibition layer, the photocatalyst containing layer containing the photocatalyst and the photocatalyst containing layer side substrate having the base so that the cell adhesion inhibition layer and the photocatalyst containing layer face each other, from a predetermined direction An energy irradiation step of forming a pattern including a cell adhesion portion in which the cell adhesion inhibition material contained in the cell adhesion inhibition layer is decomposed or denatured and a cell adhesion inhibition portion other than the cell adhesion portion, which is irradiated with energy; A method for producing a patterning substrate for cell culture, comprising:

  According to the present invention, since the cell adhesion-inhibiting material is contained in the cell adhesion-inhibiting layer formed in the cell adhesion-inhibiting layer forming step, in the energy irradiation step, a pattern is formed using the photocatalyst-containing layer side substrate. The cell adhesion inhibiting material is decomposed or denatured by irradiating energy in the form of a cell adhesion part having adhesiveness to cells, and the cell adhesion inhibiting part having low adhesion to cells not irradiated with energy. It can be an easily formed patterning substrate for cell culture. 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 adhesion to cells and a cell adhesion inhibition portion having low adhesion to cells are formed. It is possible to easily produce a cell culture substrate that is adhered only on the cell adhesion part with high definition.

  According to the present invention, by performing energy irradiation in a pattern using a photocatalyst-containing layer containing a photocatalyst, a region having adhesion to cells and a region having low adhesion to cells are easily formed. It is possible to obtain a patterning substrate that can be used.

  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 inhibitory property that inhibits adhesion to cells and is decomposed or denatured by the action of a photocatalyst associated with energy irradiation, and at least energy irradiation. After that, it contains cells and a cell adhesion material having adhesiveness.

Since the patterning substrate coating liquid of the present invention contains the cell adhesion inhibitory material having the cell adhesion inhibitory property, a layer obtained by applying the patterning substrate coating liquid on a base material or the like is used. The adhesiveness with the cells can be low. Further, by irradiating this layer with energy using a photocatalyst-containing layer containing a photocatalyst or the like, the cell adhesion-inhibiting material is decomposed or denatured so that adhesion to cells is not inhibited. Furthermore, since the cell adhesion material is contained, the adhesion to cells can be improved. Therefore, it is possible to easily form a region having good adhesion to cells and a region having low adhesion to cells without using a complicated process or a treatment solution that adversely affects the cells. .
Hereinafter, each material used for the coating liquid for patterning substrates as described above will be described.

1. Cell Adhesion Inhibiting Material First, 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 in the coating solution for a patterning substrate of the present invention has a cell adhesion-inhibiting property that inhibits adhesion to cells and is decomposed or modified by the action of a photocatalyst associated with energy irradiation. If there are, the kind etc. are not specifically limited.

  Here, having the cell adhesion inhibitory property means having a property of inhibiting the cell from adhering to the cell adhesion-inhibiting material. If the adhesion with the cell differs depending on the cell type, It has the property of inhibiting adhesion to cells.

  The cell adhesion-inhibiting material used in the present invention has such cell adhesion-inhibiting properties, and is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation, and has no cell-adhesion inhibiting properties, Those having good adhesive properties are used.

  As the cell adhesion inhibiting material, for example, a material having high hydration ability can be used. When a material with high hydration ability is used, a hydrated layer in which water molecules gather around the cell adhesion inhibiting material is formed. Usually, such a substance with high hydration ability has higher adhesion with water molecules than adhesion with cells, so that the cells cannot adhere to the material with high hydration ability. This results in low adhesion. Here, the hydration ability means the property of hydrating with water molecules, and the high hydration ability means that it easily hydrates with water molecules.

  Examples of the material having a high hydration ability and used as a cell adhesion inhibiting material include polyethylene glycol, zwitterionic materials having a betaine structure, and phospholipid-containing materials. When such a material is used as the cell adhesion-inhibiting material and irradiated with energy, the cell adhesion-inhibiting material is decomposed or altered by the action of a photocatalyst, and the hydration layer on the surface is separated. The cell adhesion inhibitory property can be eliminated.

  A surfactant having a water-repellent or oil-repellent organic substituent that can be decomposed by the action of a photocatalyst can also be used. Examples of such surfactants include hydrocarbons such as NIKKOL BL, BC, BO, and BB series manufactured by Nikko Chemicals Co., Ltd., ZONYL FSN, FSO manufactured by DuPont, and Surflon S- manufactured by Asahi Glass Co., Ltd. 141, 145, Mega Japan F-141, 144, manufactured by Dainippon Ink & Chemicals, Inc., Neogents, F-200, F251, Daikin Industries, Ltd. Unidyne DS-401, 402, 3M, Inc. ) Fluoro- or silicone-based nonionic surfactants such as Fluorad FC-170 and 176 manufactured by the company, and cationic surfactants, anionic surfactants and amphoteric surfactants can also be used. .

  When such a material is used as a cell adhesion inhibiting material, the cell adhesion inhibiting material is unevenly distributed on the surface when a layer is formed by applying the patterning substrate coating solution. As a result, the water repellency and oil repellency of the surface of the cell adhesion inhibiting layer can be increased, the interaction with the cells can be reduced, and the adhesion with the cells can be decreased. When this layer is irradiated with energy, it can be easily decomposed by the action of the photocatalyst to expose the photocatalyst and not have the cell adhesion inhibitory property.

  Here, in the present invention, as the cell adhesion inhibiting material, it is particularly preferable to use a material that has good adhesion to cells by the action of a photocatalyst accompanying energy irradiation. As such cell adhesion inhibiting material, it has oil repellency and water repellency in particular, and due to the action of the photocatalyst accompanying energy irradiation, the surface becomes appropriate hydrophilicity and lipophilicity, so that the adhesion to cells is good. It is preferable that

  When the material having the water repellency or oil repellency is used as the cell adhesion inhibiting material, for example, hydrophobic interaction between the cell and the cell adhesion inhibiting material due to the water repellency or oil repellency of the cell adhesion inhibiting material. Because the interaction with the cells can be made small, the adhesion with the cells can be made low, and the interaction with the cells can be made large by making the surface moderately hydrophilic and lipophilic. is there.

  As such a material having water repellency or oil repellency, for example, it has a high binding energy such that the skeleton is not decomposed by the action of the photocatalyst, and the water repellency or oil repellency is decomposed by the action of the photocatalyst. Examples include those having an organic substituent.

  Examples of those having a high binding energy such that the skeleton is not decomposed by the action of the photocatalyst and having water-repellent or oil-repellent organic substituents that are decomposed by the action of the photocatalyst include (1) Sol gel Examples include organopolysiloxanes that exhibit high strength by hydrolysis and polycondensation of chloro or alkoxysilane by reaction or the like, and (2) organopolysiloxanes crosslinked with reactive silicones.

In the case of (1) above, the general formula:
Y _n SiX _(4-n)
(Where Y is an alkyl group, fluoroalkyl group, vinyl group, amino group, phenyl group or epoxy group, or an organic group containing these, X is an alkoxyl group, acetyl group or halogen. It is an integer up 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.

In addition, a polysiloxane containing a fluoroalkyl group can be particularly preferably used as the organic group. Specifically, one or more hydrolyzed condensates or cohydrolytic condensates of the following fluoroalkylsilanes can be used. In general, those known as fluorine-based silane coupling agents can be used.
_{CF 3 (CF 2) 3 CH} 2 CH 2 Si (OCH 3) 3;
_{CF 3 (CF 2) 5 CH} 2 CH 2 Si (OCH 3) 3;
_{CF 3 (CF 2) 7 CH} 2 CH 2 Si (OCH 3) 3;
_{CF 3 (CF 2) 9 CH} 2 CH 2 Si (OCH 3) 3;
(CF ₃ ) ₂ CF (CF ₂ ) ₄ CH ₂ CH ₂ Si (OCH ₃ ) ₃ ;
_{(CF 3) 2 CF (CF} 2) 6 CH 2 CH 2 Si (OCH 3) 3;
_{(CF 3) 2 CF (CF} 2) 8 CH 2 CH 2 Si (OCH 3) 3;
_{CF 3 (C 6 H 4)} C 2 H 4 Si (OCH 3) 3;
_{CF 3 (CF 2) 3 (} C 6 H 4) C 2 H 4 Si (OCH 3) 3;
_{CF 3 (CF 2) 5 (} C 6 H 4) C 2 H 4 Si (OCH 3) 3;
_{CF 3 (CF 2) 7 (} C 6 H 4) C 2 H 4 Si (OCH 3) 3;
_{CF 3 (CF 2) 3 CH} 2 CH 2 SiCH 3 (OCH 3) 2;
_{CF 3 (CF 2) 5 CH} 2 CH 2 SiCH 3 (OCH 3) 2;
_{CF 3 (CF 2) 7 CH} 2 CH 2 SiCH 3 (OCH 3) 2;
_{CF 3 (CF 2) 9 CH} 2 CH 2 SiCH 3 (OCH 3) 2;
_{(CF 3) 2 CF (CF} 2) 4 CH 2 CH 2 SiCH 3 (OCH 3) 2;
_{(CF 3) 2 CF (CF} 2) 6 CH 2 CH 2 Si CH 3 (OCH 3) 2;
_{(CF 3) 2 CF (CF} 2) 8 CH 2 CH 2 Si CH 3 (OCH 3) 2;
_{CF 3 (C 6 H 4)} C 2 H 4 SiCH 3 (OCH 3) 2;
_{CF 3 (CF 2) 3 (} C 6 H 4) C 2 H 4 SiCH 3 (OCH 3) 2;
_{CF 3 (CF 2) 5 (} C 6 H 4) C 2 H 4 SiCH 3 (OCH 3) 2;
_{CF 3 (CF 2) 7 (} C 6 H 4) C 2 H 4 SiCH 3 (OCH 3) 2;
_{CF 3 (CF 2) 3 CH} 2 CH 2 Si (OCH 2 CH 3) 3;
_{CF 3 (CF 2) 5 CH} 2 CH 2 Si (OCH 2 CH 3) 3;
_{CF 3 (CF 2) 7 CH} 2 CH 2 Si (OCH 2 CH 3) 3;
_{CF 3 (CF 2) 9 CH} 2 CH 2 Si (OCH 2 CH 3) 3;
_{CF 3 (CF 2) 7 SO} 2 N (C 2 H 5) C 2 H 4 CH 2 Si (OCH 3) 3.

  By using a polysiloxane containing a fluoroalkyl group as described above as a cell adhesion inhibiting material, water repellency and oil repellency can be increased, interaction with cells is small, and adhesion to cells is low. Can be low. In addition, when energy is irradiated to the material as described above, fluorine and the like can be easily removed to introduce OH groups and the like on the surface, and the interaction with the cells can be increased. It is possible to improve the adhesiveness.

  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.

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

  By using the reactive silicone as described above, water repellency and oil repellency can be increased, interaction with cells can be reduced, and adhesion with cells can be decreased. In addition, when energy is irradiated to the above materials, substituents can be easily removed and OH groups and the like can be introduced on the surface, and the interaction with the cells can be increased. It is possible to improve the adhesiveness.

  In addition, when organopolysiloxane or reactive silicone as described above is used, it is modified by the action of a photocatalyst accompanying energy irradiation, but the skeleton remains undecomposed, so a coating solution for a substrate for patterning is applied. It also has the advantage that it can also serve as a binder for the formed layer.

  Here, when the material having water repellency or oil repellency is used as a cell adhesion inhibiting material, a material having a contact angle with water of 80 ° or more, particularly within a range of 100 ° to 130 °, is usually selected. It is preferable to use as. This is because the adhesiveness with cells can be reduced. The upper limit of the angle is the upper limit of the contact angle with water of the cell adhesion inhibiting material on a flat substrate, for example, with the water of the cell adhesion inhibiting material on the substrate having irregularities. When the contact angle is measured, the upper limit may be about 160 ° as shown in, for example, Japanese Journal of Applied Physics, Part 2, Volume 32, L614-L615, 1993 Ogawa et al. is there.

  In addition, when energy is applied to the cell adhesion-inhibiting material to have adhesiveness with cells, the contact angle with water is in the range of 10 ° to 40 °, particularly 15 ° to 30 °. Thus, it is preferable that energy is irradiated. This is because the adhesiveness with cells can be increased.

  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.

  Such cell adhesion inhibiting materials are in the range of 0.001% to 60% by weight, particularly 0.01% to 40% by weight, particularly 0.1% to 20% by weight in the coating liquid for patterning substrate. It is preferable to be contained within. This is because the region containing the cell adhesion-inhibiting material can be made a region having low adhesion to cells.

In addition, when the cell adhesion inhibiting material as described above is decomposed or denatured by irradiating energy and used as a material having good adhesion to cells, the adhesion to the target cells is improved. It suffices that the cell adhesion-inhibiting material is decomposed or denatured by being irradiated with energy to the extent, and the cell adhesion-inhibiting material need not be completely decomposed or denatured.
Moreover, it is preferable that the said cell adhesion inhibitory material is what has surface activity. This is because the ratio of uneven distribution on the surface of the coating film increases during the application and drying process of the coating liquid for the patterning substrate, and as a result, good cell adhesion inhibitory properties can be obtained.

2. Cell Adhesive Material Next, 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 present invention is not particularly limited as long as it has adhesiveness to cells after at least energy irradiation, and may be used as a binder. Alternatively, it may be used. Further, for example, it may have good adhesion to cells before being irradiated with energy, or may have good adhesion to cells by the action of a photocatalyst accompanying energy irradiation. . Here, having adhesiveness with the cell means that it adheres well to the cell, and if the adhesiveness with the cell differs depending on the type of cell, it means that it adheres well to the target cell. Say.

  In the present invention, at least after the energy irradiation, if the cell adhesion material has good adhesion to cells, adhesion to the cells is, for example, hydrophobic interaction, electrostatic interaction, hydrogen It may be made good by physical interaction such as bonding, van der Waals force, or may be made good by biological characteristics.

  Specific examples of materials that adhere to cells by the above physical interaction include hydrophilic polymers such as hydrophilic polystyrene, poly (N-isopropylacrylamide), basic polymers such as polylysine, aminopropyltriethoxysilane, Examples include basic compounds such as N- (2-aminoethyl) -3-aminopropyltrimethoxysilane and condensates containing them.

  Specific examples of materials that adhere to cells due to the above biological properties include fibronectin, laminin, tenascin, vitronectin, RGD (arginine-glycine-aspartic acid) sequence-containing peptide, YIGSR (tyrosine-isoleucine-glycine-serine- Arginine) sequence-containing peptides, collagen, atelocollagen, gelatin and the like.

  In the present invention, such a cell adhesion material is appropriately optimized depending on the cell adhesion ability of the material, but is usually 0.0001% to 30% by weight, especially 0% in the coating liquid for patterning substrate. It is preferably contained within a range of 0.001% by weight to 10% by weight. This is because, when the patterning substrate coating solution is applied to form a layer, the adhesiveness with the cells in the region irradiated with energy can be improved. In addition, in the case where a material having good adhesiveness with cells is used as a cell adhesion material before energy irradiation, in a region where energy is not irradiated after the coating liquid for patterning substrate is applied and a layer is formed. In addition, it is preferably contained to the extent that the cell adhesion inhibitory property of the cell adhesion inhibitory material is not inhibited.

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, the cell adhesion-inhibiting material and the cell adhesion material can serve 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 has two embodiments depending on the structure of the cell adhesion inhibiting layer. In any of the embodiments, for example, as shown in FIG. 1, the base material 1 is formed on the base material 1 and has an inhibitory effect on adhesion to cells, and is modified by the action of a photocatalyst accompanying energy irradiation. The cell adhesion inhibiting layer 2 is provided. Hereinafter, each embodiment will be described in detail.

1. First Embodiment First, a first embodiment of the patterning substrate of the present invention will be described. The patterning substrate of this embodiment is a substrate and a cell adhesion that is formed on the substrate and has a cell adhesion inhibitory property that inhibits adhesion to cells and is denatured by the action of a photocatalyst associated with energy irradiation. It has a cell adhesion inhibition layer containing an inhibitory material.

  In this embodiment, since the cell adhesion-inhibiting material is denatured by the action of a photocatalyst accompanying energy irradiation, energy is applied to the cell adhesion-inhibiting layer using a photocatalyst-containing layer that contains a photocatalyst, for example. By irradiation, the cell adhesion inhibiting material is denatured, the cell adhesion inhibiting property is reduced, and a region having adhesiveness with cells can be formed. On the other hand, in a region where energy is not irradiated, the cell adhesion inhibitory property of the cell adhesion inhibitory material can reduce the adhesion to cells. Therefore, according to the present embodiment, for example, by irradiating energy using a photocatalyst-containing layer, the cell adhesion-inhibiting material is denatured and the cell adhesion-inhibiting material does not change, It can be set as the patterning board | substrate which can form easily the area | region with bad adhesiveness with a cell.

Here, in the patterning substrate of this embodiment, for example, as shown in FIG. 2, the light shielding part 3 is formed on the base material 1, and the cell adhesion inhibition layer 2 is formed on the light shielding part 3. It may be a thing. Thereby, after arranging the photocatalyst-containing layer or the like and the cell adhesion-inhibiting layer facing each other and then irradiating energy from the substrate side, the photocatalyst containing layer in the region where the light shielding portion is formed is not excited, It is possible to denature the cell adhesion inhibiting material only in the region where the light shielding portion is not formed. Therefore, there is an advantage that a high-definition cell adhesion portion can be easily formed without requiring alignment of a photomask or the like.
Hereinafter, each configuration of the patterning substrate of this embodiment will be described.

(Cell adhesion inhibition layer)
First, the cell adhesion inhibition layer used in this embodiment will be described. The cell adhesion-inhibiting layer used in the present embodiment is a cell that is formed on a substrate to be described later, has cell adhesion-inhibiting properties that inhibit adhesion to cells, and is denatured by the action of a photocatalyst accompanying energy irradiation. It contains an adhesion inhibiting material.

  The cell adhesion-inhibiting layer in this embodiment is not particularly limited as long as it contains at least the cell adhesion-inhibiting material. For example, when the cell adhesion-inhibiting material also serves as a binder, It does not have to contain a binder or the like. When such a cell adhesion inhibiting layer is irradiated with energy using a photocatalyst-containing layer or the like, the cell adhesion inhibiting material is denatured in the energy-irradiated region, so that the adhesion inhibiting property with cells is reduced. In other words, it has adhesiveness with cells. As such a cell adhesion inhibiting material, among the cell adhesion inhibiting materials described in the section of “A. Patterning Substrate Coating Solution”, for example, organopolysiloxane, reactive silicone, etc., due to the action of a photocatalyst accompanying energy irradiation. What is denatured can be used.

  Here, in this embodiment, it is preferable that a cell adhesion material is contained in the cell adhesion inhibition layer. As a result, when the cell adhesion inhibition layer is irradiated with energy, the adhesiveness with the cells in the region irradiated with energy can be improved, and only the region irradiated with energy can be improved. This is because cells can be adhered in a fine pattern.

  In this embodiment, the cell adhesion-inhibiting layer can be formed by applying a coating solution containing the cell adhesion-inhibiting material, for example. A layer can be formed by using a patterning substrate coating solution containing a cell adhesion material in addition to the cell adhesion inhibiting material as described in the section “A. Patterning substrate coating solution”.

  As a method for forming such a cell adhesion-inhibiting layer, it can be formed by applying the coating liquid for patterning substrate or the like by a general application method, and the specific application method is spin coating. Methods, spray coating methods, dip coating methods, roll coating methods, bead coating methods and the like can be used. In addition, a method of forming an ultrathin film such as an adsorption method or an alternate adsorption method can also be suitably used.

  Further, for example, when a concave portion is formed in the base material described later, a casting method for forming a cell adhesion inhibiting layer by dropping a coating solution for a patterning substrate into the concave portion of the base material and drying it, or the above base An adsorption method or the like in which a patterning substrate coating solution or the like is dropped into the concave portion of the material and washed after a predetermined time can also be used.

  Since the cell adhesion inhibiting material and the cell adhesion material used for the cell adhesion inhibiting layer in the present embodiment are the same as those described in the above-mentioned section “A. Patterning substrate coating solution”, Description is omitted.

  The thickness of the cell adhesion-inhibiting layer is appropriately selected depending on the type of the substrate for patterning and the like, but is usually about 0.001 to 1.0 μm, particularly about 0.01 to 0.3 μm. it can.

  In addition, it is preferable that the photocatalyst is not contained in the cell adhesion inhibition layer in this embodiment. This is because, for example, when cells are adhered on the cell adhesion-inhibiting layer, the photocatalyst can be prevented from affecting the cells over time, and the quality can be improved. In addition, as described above, the method for decomposing or denaturing the cell adhesion-inhibiting material of such a cell adhesion-inhibiting layer is performed by irradiating energy with the photocatalyst-containing layer facing the photocatalyst-containing layer as described above. Can do.

(Base material)
Next, the base material used in this embodiment will be described. The base material used in the present embodiment is not particularly limited as long as it is a layer capable of forming the cell adhesion inhibition layer. For example, inorganic materials such as metal, glass, silicon, and organic materials represented by plastics are used. Materials and the like 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 energy applied to decompose or denature the cell adhesion-inhibiting material, and the base material is, for example, a light-shielding portion. In the case where the energy irradiation is performed from the base material side, the base material has transparency.

  In this embodiment, the substrate may be flat, but a recess may be formed in the substrate. As such a base material, for example, as shown in FIG. 10 (a), one recess may be formed, and for example, as shown in FIG. It may be formed.

  Further, at this time, a treatment may be performed so that the cell adhesion-inhibiting layer is not formed on the side wall of the substrate having the recess. 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 embodiment, the substrate may be one that has been 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 inhibiting layer is improved.

  Here, as described above, the light shielding portion may be formed on the base material used in this embodiment. The light shielding portion that can be used in this embodiment is not particularly limited as long as it can block the energy applied to the patterning substrate. For example, a sputtering method, a vacuum evaporation method, or the like. May be formed by forming a metal thin film of chromium or the like 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.
In addition, the said light-shielding part may be formed in the surface by which the said cell adhesion inhibition layer of a base material is formed, and may be formed in the surface on the opposite side.

(Pattern for patterning)
Next, the patterning substrate of this embodiment will be described. The patterning substrate according to this embodiment is not particularly limited as long as the cell adhesion inhibition layer is formed on the above-described base material. For example, the other substrate may be laminated as necessary. It may be.

2. Second Embodiment Next, a second embodiment of the patterning substrate of the present invention will be described. The patterning substrate in this embodiment is formed on the base material and has the cell adhesion inhibitory property that inhibits adhesion to cells, and is decomposed or modified by the action of a photocatalyst accompanying energy irradiation. It has a cell adhesion inhibiting material and a cell adhesion inhibiting layer containing a binder.

  According to this embodiment, since the cell adhesion-inhibiting material is decomposed or modified by the action of a photocatalyst accompanying energy irradiation, for example, a photocatalyst-containing layer containing a photocatalyst is used as the cell adhesion-inhibiting layer. By irradiating with energy, the cell adhesion-inhibiting material is decomposed or denatured, and the cell adhesion inhibitory ability is lowered, whereby a region having adhesiveness with cells can be formed. On the other hand, in a region where energy is not irradiated, the cell adhesion inhibitory property of the cell adhesion inhibitory material can reduce the adhesion to cells. Therefore, according to the present embodiment, for example, by irradiating energy using a photocatalyst-containing layer, the cell adhesion-inhibiting material is denatured and the cell adhesion-inhibiting material does not change, It can be set as the patterning board | substrate which can form easily the area | region with bad adhesiveness with a cell.

  Here, also in the patterning substrate of this embodiment, as in the first embodiment described above, for example, a light shielding portion is formed on a base material, and a cell adhesion inhibition layer is formed on the light shielding portion. It may be a thing. In addition, since the base material, the light-shielding portion, and the like used in this embodiment are the same as those used in the first embodiment, description thereof is omitted, and the following description is used for the patterning substrate of this embodiment. The cell adhesion inhibiting layer to be described will be described.

(Cell adhesion inhibition layer)
The cell adhesion inhibiting layer in the present embodiment is not particularly limited as long as it contains at least the cell adhesion inhibiting material and the binder. Since the cell adhesion inhibiting material used in this embodiment is decomposed or modified by the action of a photocatalyst accompanying energy irradiation, energy irradiation is performed using a photocatalyst-containing layer or the like as the cell adhesion inhibiting layer. In this case, in the region irradiated with energy, the cell adhesion-inhibiting material is denatured or decomposed, so that the cell adhesion inhibiting property is lowered and the cell has adhesiveness. Specifically, as the cell adhesion inhibiting material that is decomposed or modified by the action of the photocatalyst accompanying the irradiation of energy, those described in the section of the cell adhesion inhibiting material in “A. Patterning substrate coating solution” are used. be able to.

  In the present embodiment, since the binder is contained in the cell adhesion inhibiting layer, for example, the binder remains even when the cell adhesion inhibiting material is completely decomposed by the action of the photocatalyst accompanying the energy irradiation. As a result, the cell adhesion inhibition layer remains. Such a binder is not particularly limited as long as it does not have cell adhesion inhibitory properties, and those usually used for a cell culture layer of a cell culture substrate can be used.

  Here, also in this embodiment, it is preferable that the cell adhesion-inhibiting layer contains a cell adhesion material. As a result, when the cell adhesion inhibition layer is irradiated with energy, the adhesiveness with the cells in the region irradiated with energy can be improved, and only the region irradiated with energy can be improved. This is because cells can be adhered in a fine pattern.

  In this embodiment, the cell adhesion-inhibiting layer can be formed by applying a coating solution containing the cell adhesion-inhibiting material and a binder or the like. When the cell adhesion material is contained as described above, A layer can be formed by using a coating liquid for a patterning substrate containing a cell adhesion material in addition to a cell adhesion inhibiting material as described in the section of “A. Patterning substrate coating liquid”.

  Since the method for forming such a cell adhesion inhibition layer, the thickness of the cell adhesion inhibition layer, and the like are the same as those in the first embodiment described above, detailed description thereof is omitted here. Further, since the cell adhesion material, binder, cell adhesion inhibition material and the like used for the cell adhesion inhibition layer are the same as those described in the above-mentioned section “A. Coating liquid for patterning substrate”, here. Description of is omitted.

  In addition, it is preferable that the photocatalyst is not contained in the cell adhesion inhibiting layer in this embodiment. This is because, for example, when cells are adhered on the cell adhesion-inhibiting layer, the photocatalyst can be prevented from affecting the cells over time, and the quality can be improved.

(Pattern for patterning)
Next, the patterning substrate of this embodiment will be described. Also in the patterning substrate of this embodiment, there is no particular limitation as long as the cell adhesion inhibition layer as described above is formed on the base material. These layers may be laminated.

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-inhibiting layer of the patterning substrate described above is formed in a cell adhesion part in which the cell adhesion-inhibiting material is decomposed or denatured in a pattern and in a region other than the cell adhesion part. It has a certain cell adhesion inhibition part.

  For example, as shown in FIG. 3, the cell culture substrate of the present invention has a substrate 1 and a cell adhesion inhibition layer 2 formed on the substrate 1, and the cell adhesion inhibition layer 2 is a cell. The cell adhesion inhibiting material contained in the adhesion inhibiting layer 2 is decomposed or denatured, and has a cell adhesion part 4 having adhesion with cells, and is an area other than the cell adhesion part 4 and has cell adhesion inhibition properties with cells. And a cell adhesion inhibiting part 2 ′ having

  Since the cell culture patterning substrate of the present invention has the cell adhesion part and the cell adhesion inhibition part, cells can be easily adhered only on the cell adhesion part, for example, on the entire surface of the cell adhesion inhibition layer. Even when the cells are applied, the cells can be adhered with high definition only on the cell adhesion portion.

  Here, in the present invention, for example, as shown in FIG. 4, the light shielding portion 3 may be formed on the base material 1 in the same pattern as the cell adhesion inhibiting portion 2 ′ of the cell adhesion inhibiting 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 opposed to the cell adhesion inhibiting layer. By disposing and irradiating energy from the substrate side, the cell adhesion inhibiting material can be decomposed or denatured only on the region where the light shielding portion is not formed, and the cell adhesion portion is easily formed. Because it can be done.

  Here, the cell adhesion inhibiting part is a region containing a cell adhesion inhibiting material that has a cell adhesion inhibiting property that inhibits at least adhesion to cells and is denatured or decomposed and removed by the action of a photocatalyst associated with energy irradiation. This is a region having low adhesion to the target cells.

  On the other hand, the cell adhesion part is an area where the cell adhesion-inhibiting material is decomposed or denatured and has adhesion to cells. Here, the cell adhesion-inhibiting material is decomposed or denatured as compared with the amount of the cell adhesion-inhibiting material that does not contain the cell adhesion-inhibiting material or is contained in the cell adhesion-inhibiting part, It means that a small amount of cell adhesion inhibiting material is contained. For example, when the cell adhesion inhibiting material is decomposed by the action of a photocatalyst accompanying energy irradiation, the cell adhesion inhibiting material does not contain the cell adhesion inhibiting material, or a degradation product of the cell adhesion inhibiting material Etc. will be contained. Here, when the cell adhesion-inhibiting material is to be decomposed, as described above, a binder or the like is usually contained in the cell adhesion-inhibiting layer, and the binder is exposed, etc. It is preferable that the cell adhesion portion has adhesiveness. On the other hand, when the cell adhesion-inhibiting material is denatured by the action of a photocatalyst accompanying energy irradiation, the denatured product or the like is contained in the cell adhesion portion. In this case, the modified substance of the cell adhesion inhibiting material can have adhesiveness with cells, and the cell adhesion inhibiting layer contains a binder other than the cell adhesion inhibiting material. Or any of the cases where a binder etc. are not contained may be sufficient.

  In any case, in the present invention, it is particularly preferable that a cell adhesion material having good adhesion to cells as described in the above section “A. Patterning substrate coating solution” is contained. . This is because the adhesion of the cell adhesion part to the cells can be made better.

  Here, the base material and the cell adhesion-inhibiting layer used in the cell culture patterning substrate of the present invention are the same as those described in the above-mentioned “B. Patterning substrate”, and thus description thereof is omitted here. Hereinafter, a method for forming a cell adhesion inhibiting 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 inhibition layer 2 containing at least a cell adhesion inhibiting material 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 inhibiting layer 2 are prepared. They are arranged facing each other (FIG. 5B). Subsequently, energy 6 is irradiated onto the pattern forming the cell adhesion portion using, for example, a photomask 5 (FIG. 5B). Thereby, the cell adhesion inhibiting material contained in the cell adhesion inhibiting layer 2 is decomposed or denatured by the action of the photocatalyst contained in the photocatalyst containing layer 12, and the cell adhesion part 4 having adhesiveness to cells is obtained. Since no energy is irradiated and the cell adhesion-inhibiting material is not decomposed, a cell adhesion-inhibiting portion 2 'having low adhesion to cells is formed (FIG. 5 (c)). The method for forming the cell adhesion-inhibiting 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 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 is configured to decompose or denature the cell adhesion-inhibiting material in the adjacent cell adhesion-inhibiting layer, It may be composed of a photocatalyst and a binder, or may be a film formed of a single photocatalyst. 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 a cell adhesion part, it is not necessary to irradiate the pattern using a photomask or the like, and the entire surface is irradiated with energy. Thus, a pattern in which the cell adhesion-inhibiting material contained in the cell adhesion-inhibiting layer is decomposed or denatured can be formed.
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-inhibiting material is decomposed or denatured only in the part on the cell adhesion-inhibiting layer that actually faces the photocatalyst-containing layer, the direction of energy irradiation depends on the photocatalyst-containing layer, the cell adhesion-inhibiting layer, and the like. As long as energy is irradiated to the portion facing the surface, the irradiation may be performed 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 composed only of a photocatalyst, the efficiency of decomposition or denaturation of the cell adhesion-inhibiting material in the cell adhesion-inhibiting layer is improved, which is advantageous in terms of cost such as shortening of the processing 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 the cell adhesion-inhibiting material in the cell adhesion-inhibiting layer or Since denaturation can be performed uniformly and it consists only of a photocatalyst, it becomes possible to decompose or denature the cell adhesion-inhibiting material more efficiently than when a binder is used.

  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, the photocatalyst-containing layer-side light-shielding portion is disposed in the vicinity of the arrangement portion of the photocatalyst-containing layer and the cell adhesion-inhibiting layer as compared with the case where a photomask is used. Since it is possible to reduce the influence of energy scattering, the energy pattern irradiation can be performed very accurately.
Here, in the present invention, when the photocatalyst containing layer side light shielding part 14 is formed on the photocatalyst containing layer 12 as shown in FIG. 8, the photocatalyst containing layer and the cell adhesion inhibiting layer are placed at predetermined positions. The photocatalyst containing layer side light-shielding part can be 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. It has the advantage that it can be used.

  That is, when the photocatalyst-containing layer and the cell adhesion-inhibiting layer are arranged facing each other with a predetermined gap, the photocatalyst-containing layer side light-shielding portion and the cell adhesion-inhibiting layer are arranged in close contact with each other. Thus, the predetermined gap can be made accurate, and by irradiating energy in this state, the cell adhesion inhibiting layer in the part where the cell adhesion inhibiting layer and the light shielding part are in contact with each other Since the inhibitory material is not decomposed or denatured, the cell adhesion part can be formed with high accuracy.

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 aspect, 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 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.

  The action and function of this primer layer are not necessarily clear, but by forming a primer layer between the photocatalyst containing layer side light-shielding part and the photocatalyst containing layer, the primer layer can be used as a cell adhesion inhibiting material due to the action of the photocatalyst. Impurities from the openings existing between the photocatalyst containing layer side light shielding part and the photocatalyst containing layer side light shielding part, which are factors that inhibit decomposition or modification, in particular, residues generated when patterning the photocatalyst containing layer side light shielding part, or metal It is considered that it exhibits a function of preventing diffusion of impurities such as metal ions. Therefore, by forming the primer layer, the cell adhesion inhibiting material is decomposed or denatured with high sensitivity, and as a result, a cell adhesion 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 part will be described. Energy irradiation can be performed by arranging the cell adhesion inhibition 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 means a state in which the photocatalyst acts substantially on the surface of the cell adhesion inhibiting layer. In addition to a state in which the photocatalyst actually touches, a predetermined interval is set. The photocatalyst-containing layer and the cell adhesion-inhibiting layer are arranged so as to be spaced apart. 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, considering the point that the efficiency of the decomposition or denaturation of the cell adhesion-inhibiting material of the cell adhesion-inhibiting layer is particularly good, the gap is 0.2 μm to It is preferable to be within the range of 10 μm, preferably within the range of 1 μm to 5 μm. Such a gap range is particularly effective for a cell adhesion inhibition 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 inhibition layer having a large area of, for example, 300 mm × 300 mm or more, the fine gap as described above is formed between the photocatalyst-containing layer side substrate and the cell adhesion inhibition layer without contact. Forming in between is extremely difficult. Therefore, when the cell adhesion inhibition 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, there are problems such as a decrease in pattern accuracy such as a blurred pattern and a problem that the sensitivity of the photocatalyst deteriorates and the efficiency of decomposing or denaturing the cell adhesion inhibiting material deteriorates. This is because there is an effect that unevenness does not occur in the decomposition or denaturation of the cell adhesion inhibiting material.

  Thus, when irradiating energy to a cell adhesion inhibition 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 inhibition layer in the energy irradiation device is in the range of 10 μm to 200 μm. Especially, it is preferable to set in the range of 25 micrometers-75 micrometers. By making the set value within such a range, the pattern accuracy is not significantly reduced and the sensitivity of the photocatalyst is not greatly deteriorated, and the photocatalyst-containing layer side substrate and the cell adhesion inhibiting layer are not in contact with each other. Because it becomes possible to do.

  Thus, by disposing the photocatalyst-containing layer and the cell adhesion inhibition 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-inhibiting layer is narrower than the above range, it becomes difficult to desorb the active oxygen species, and as a result, the rate at which the cell adhesion-inhibiting material is decomposed or denatured is decreased. It is not preferable because there is a possibility that the In addition, when arranged at a distance from the above range, the generated active oxygen species are difficult to reach the cell adhesion-inhibiting layer, and in this case, the rate of decomposition or denaturation of the cell adhesion-inhibiting material may be slowed. This is not preferable.

As a method of arranging such a very narrow gap uniformly and arranging the photocatalyst containing layer and the cell adhesion inhibiting layer, for example, a method using a spacer can be mentioned. 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 inhibiting layer at the portion where the spacer contacts. By having the same pattern as that of the cell adhesion inhibiting part, the cell adhesion inhibiting material can be decomposed or denatured only in the part where the spacer is not formed, and the cell adhesion part can be formed with high definition. It is. In addition, by using such a spacer, the active oxygen species generated by the action of the photocatalyst can reach the cell adhesion inhibition layer surface at a high concentration without diffusing, thereby efficiently forming a high-definition cell adhesion part. 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 ray capable of decomposing or modifying the cell adhesion-inhibiting material by the action of a photocatalyst accompanying energy irradiation, However, the present invention is not limited to this 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 inhibiting portion, it can be performed by irradiating the entire surface with energy from the substrate side. Moreover, when it has a light-shielding part on the photocatalyst containing layer in the same pattern as the cell adhesion inhibiting part, it can be performed by irradiating the entire surface with energy 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 inhibition 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 inhibiting material to be decomposed or denatured by the action of the photocatalyst.
In this case, it is preferable in that the sensitivity can be increased by irradiating the photocatalyst-containing layer with energy while heating, and the cell adhesion-inhibiting 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.

(Pattern for cell culture)
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 inhibition layer having the cell adhesion part and the cell adhesion inhibition part is formed on the base material. Depending on the case, other layers may be appropriately formed.
Further, in the present invention, after forming the cell adhesion part, a part of the cell culture patterning substrate is cut off, and a part of the cell culture patterning substrate is attached to the bottom of a concave base material, etc. 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, the cells 7 adhere only on the cell adhesion part 4 of the cell adhesion inhibition layer 2, and the cells 7 adhere on the cell adhesion inhibition part 2 ′. It is not.

According to the present invention, on the cell culture patterning substrate, a cell adhesion part having adhesion to cells and a cell adhesion inhibition part having no adhesion to cells are formed. Even when applied to the entire surface of the cell culture patterning substrate, the cells on the cell adhesion-inhibiting part can be easily removed by attaching the cells only to the cell adhesion part. 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.
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 adhesion inhibition property to the cell adhesion inhibiting material differs depending on the cell type, it is necessary to select the cell adhesion inhibiting material used in the cell adhesion inhibiting part and the composition ratio thereof 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 inhibitor that inhibits adhesion to cells on a substrate and is decomposed or denatured by the action of a photocatalyst associated with energy irradiation. A cell adhesion inhibition layer forming step of forming a cell adhesion inhibition layer containing a material;
After arranging the cell adhesion inhibition layer, the photocatalyst containing layer containing the photocatalyst and the photocatalyst containing layer side substrate having the base so that the cell adhesion inhibition layer and the photocatalyst containing layer face each other, from a predetermined direction An energy irradiation step of forming a pattern including a cell adhesion portion in which the cell adhesion inhibition material contained in the cell adhesion inhibition layer is decomposed or denatured and a cell adhesion inhibition portion other than the cell adhesion portion, which is irradiated with energy; It is what has.

  In the method for producing a patterning substrate for cell culture according to the present invention, for example, as shown in FIG. 5, cells that form a cell adhesion inhibiting layer 2 that forms a cell adhesion inhibiting layer containing at least a cell adhesion inhibiting material on a substrate 1 are formed. A photocatalyst containing layer side substrate 13 having an adhesion inhibiting layer forming step (FIG. 5 (a)) and a photocatalyst containing layer 12 containing a substrate 11 and a photocatalyst is prepared, and the cell adhesion inhibiting layer 2 and the photocatalyst containing layer 12 are prepared. For example, a photomask 5 or the like is used to irradiate energy 6 (FIG. 5B), and the cell adhesion inhibiting material contained in the cell adhesion inhibiting layer 2 in the region irradiated with energy is decomposed. Or it has the energy irradiation process (FIG.5 (c)) which forms the cell adhesion part 4 which modified | denatured, and cell adhesion inhibition part 2 'which is not irradiated with energy and has low adhesiveness with a cell. .

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

1. Cell Adhesion Inhibition Layer Formation Step First, the cell adhesion inhibition layer formation step of the present invention will be described. The cell adhesion-inhibiting layer forming step of the present invention comprises a cell adhesion-inhibiting material having a cell adhesion-inhibiting property that inhibits adhesion to cells and decomposed or modified by the action of a photocatalyst accompanying energy irradiation on a substrate. It is a process of forming the cell adhesion inhibition layer to contain. In the present invention, it can be carried out by coating on a substrate using at least a coating solution containing the cell adhesion-inhibiting material, and the coating method and the material thereof are particularly limited. However, it is preferable that a cell adhesion material having cell adhesion with good adhesion to cells is contained at least after energy irradiation. Thereby, in the energy irradiation process to be described later, cells that can adhere to the cells of the cell adhesion part, which is the region irradiated with energy, can adhere to the cells in a high-definition pattern. It is because it can be set as the patterning board | substrate for culture | cultivation.
Note that the cell adhesion inhibiting material, cell adhesion material, and base material used in this step, and the method for forming the cell adhesion inhibiting layer are the same as those described in the above section “B. Patterning substrate”. Therefore, 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-inhibiting 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-inhibiting layer and the photocatalyst-containing layer face each other. The cell adhesion inhibiting part in which the cell adhesion inhibiting material contained in the cell adhesion inhibiting layer is decomposed or denatured, and cell adhesion parts other than the cell adhesion inhibiting part, Forming a pattern comprising:

In this step, the cell adhesion inhibition layer formed in the cell adhesion inhibition 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, As long as the cell adhesion material is decomposed or denatured to form a cell adhesion part having adhesion to cells, the method and the like are not particularly limited.
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. The method for producing a cell culture substrate of the present invention comprises a cell adhesion step for adhering cells on the cell adhesion part of the cell culture patterning substrate produced by the method for producing a cell culture patterning substrate.

  According to the present invention, on the cell culture patterning substrate, a cell adhesion portion having adhesion to cells and a cell adhesion inhibition portion having no adhesion to cells are formed on the cell adhesion inhibition layer. Therefore, by applying cells or the like on the cell adhesion-inhibiting layer, a cell culture substrate having cells adhered only on the cell adhesion portion can be easily produced. 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 invention is not particularly limited as long as it is a method capable of adhering cells on the cell adhesion part, and in the above-mentioned section “D. Cell culture substrate”. Since the method described above can be used, detailed 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 inhibition layer)
3 g of isopropyl alcohol, 0.4 g of organosilane TSL8114 (GE Toshiba Silicone), and 0.1 g of fluoroalkylsilane XC95-A9715 (GE Toshiba Silicone) 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. A cell adhesion inhibiting layer made of an organopolysiloxane layer containing a fluoroalkyl group and having a thickness of about 40 nm was formed on the substrate to form 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-inhibiting layer on the patterning substrate face each other, and 6 J / cm ² (measurement wavelength 254 nm) is irradiated with ultraviolet rays through the photomask with a mercury lamp. Thus, a cell culture patterning substrate having a cell adhesive surface in which the unexposed part was patterned to inhibit cell adhesion and the exposed part was patterned to be cell adhesive was obtained.

(Cell adhesion process)
The details of the experimental procedure for culturing cells derived from various tissues are described in, for example, “Tissue Culture Technology 3rd Edition Basics”, 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]
(Formation of cell adhesion inhibition layer)
6 g of isopropyl alcohol, 2 g of toluene, 0.4 g of organosilane TSL8114 (GE Toshiba Silicone) and 0.1 g of poly (2-methoxyethyl) acrylate 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 a fluoroalkyl group having a film thickness of about 60 nm was formed on the substrate to obtain a patterning substrate having a cell adhesion inhibiting layer.

(Patterning of patterning substrate)
The photomask photocatalyst layer is formed, the photocatalyst layer and the cell adhesion layer of the patterned substrate is allowed to stand so as to face, ultraviolet irradiation of 5 J / cm 2 ^(measurement wavelength 254 nm) by a mercury lamp through a photomask Thus, a cell culture patterning substrate having a cell adhesive surface in which the unexposed part was patterned to inhibit cell adhesion and the exposed part was patterned to be cell adhesive was obtained.
(Cell adhesion process)
Experiments were performed in the same procedure as in Example 1, and it was confirmed that the cells were adhered along the cell adhesion part on the cell culture patterning substrate.

[Example 3]
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.

[Example 4]
A cell culture patterning substrate having a cell adhesion inhibition layer was formed in the same manner as in Example 2 using a glass substrate having a thickness of about 0.1 mm, 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 by the same procedure as in Example 3.
(Cell culture)
When cells were cultured in a plastic dish by the same method as in Example 3, it was confirmed that the cells were adhered along the cell adhesion part in the plastic dish.

[Example 5]
(Formation of patterning substrate)
3 g of isopropyl alcohol, 0.2 g of organosilane TSL8114 (GE Toshiba Silicone), and 0.2 g of PEG-silane (Methoxypolyethylene glycol 5,000 trimethylsilyl ether, Fluka) were mixed and heated at 100 ° C. for 20 minutes with stirring. This solution was applied onto a cleaned glass substrate (thickness 0.7 mm) by spin coating, and then the substrate was subjected to heat treatment at 150 ° C. for 10 minutes. This obtained the patterning board | substrate with which the cell adhesion inhibition layer which consists of an organopolysiloxane layer containing polyethyleneglycol with a film thickness of 160 nm was formed on the board | substrate.
(Patterning of patterning substrate and cell adhesion)
In the same manner as in Example 1, the patterning substrate was patterned to form a cell culture patterning substrate. Thereafter, cells were adhered onto the cell culture patterning substrate in the same manner as in Example 1. As a result, it was confirmed that cells were adhered along the cell adhesion part on the cell culture patterning substrate in this example. confirmed.

[Example 6]
A cell culture patterning substrate having a cell adhesion-inhibiting layer was formed in the same manner as in Example 5 using a glass substrate having a thickness of about 0.1 mm, and this 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 by the same procedure as in Example 3.
(Cell culture)
When cells were cultured in a plastic dish by the same method as in Example 3, it was confirmed that the cells were adhered along the cell adhesion part 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 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 inhibition layer 2 '... Cell adhesion inhibition part 3 ... Light-shielding part 4 ... Cell adhesion part 5 ... Photomask 6 ... Energy 11 ... Base | substrate 12 ... Photocatalyst containing layer 13 ... Photocatalyst containing layer side board | substrate

Claims (6)

Cell adhesion-inhibiting layer comprising a substrate and a cell adhesion-inhibiting material which is formed on the substrate and has a cell adhesion-inhibiting property which inhibits adhesion to cells and which is denatured by the action of a photocatalyst associated with energy irradiation And
Shielding portion is formed on the substrate,
Patterning characterized in that the cell adhesion-inhibiting layer contains a cell adhesion material having good adhesion to cells before being irradiated with energy and having adhesion to cells even after being irradiated with energy. Substrate. Contains a base material, a cell adhesion inhibitory material that is formed on the base material and has a cell adhesion inhibitory property that inhibits adhesion to cells and that is decomposed or modified by the action of a photocatalyst associated with energy irradiation, and a binder Cell adhesion inhibition layer
The binder has a high binding energy such that the main skeleton is not decomposed by photoexcitation of the photocatalyst,
Shielding portion is formed on the substrate,
Patterning characterized in that the cell adhesion-inhibiting layer contains a cell adhesion material having good adhesion to cells before being irradiated with energy and having adhesion to cells even after being irradiated with energy. Substrate. The cell adhesion inhibition layer of the patterning substrate according to claim 1 or 2 , wherein the cell adhesion inhibition layer is a cell adhesion part in which the cell adhesion inhibition material is decomposed or denatured, and a cell other than the cell adhesion part. A patterning substrate for cell culture, comprising an adhesion inhibiting part. The cell culture substrate according to claim 3 , wherein cells are adhered on the cell adhesion part of the patterning substrate for cell culture. A cell adhesion inhibiting layer comprising a cell adhesion inhibiting layer containing a cell adhesion inhibiting material having a cell adhesion inhibiting property that inhibits adhesion to cells and denatured by the action of a photocatalyst associated with energy irradiation on a substrate. Forming process;
After arranging the cell adhesion inhibition layer, the photocatalyst containing layer containing the photocatalyst, and the photocatalyst containing layer side substrate having the base so that the cell adhesion inhibition layer and the photocatalyst containing layer face each other, from a predetermined direction An energy irradiation step of forming a pattern consisting of a cell adhesion portion in which the cell adhesion inhibition material contained in the cell adhesion inhibition layer is decomposed or denatured and a cell adhesion inhibition portion other than the cell adhesion portion; Have
Shielding portion is formed on the substrate,
A cell characterized in that the cell adhesion-inhibiting layer contains a cell adhesion material that has good adhesiveness with cells before being irradiated with energy and has adhesiveness with cells even after being irradiated with energy. A method for producing a patterning substrate for culture. 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 5. Method.

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