KR100886291B1 - Patterning Substrate for Cell Culture - Google Patents

Abstract

The present invention aims to provide a cell culture patterning substrate that can arrange cells efficiently over a large area on a substrate and form tissues and the like. In order to achieve the above object, the present invention provides a substrate and a cell culture region formed on the substrate, the cell culture region containing a cell adhesion layer having adhesion to the cells, wherein the cell culture region is the cell The cell adhesion part is formed in a pattern form with the cell adhesion part formed, and has a cell adhesion aid that inhibits adhesion with the cells, wherein the cell adhesion aid is adjacent to the cell adhesion aid when the cell is attached to the cell adhesion part. It provides a patterning substrate for cell culture, characterized in that the cells on the cell adhesion portion is formed to be able to bind to each other on the cell adhesion assistant.

Substrate, cell culture patterning substrate, cell culture region, cell adhesion layer, cell adhesion part, cell adhesion aid

Description

Patterning Substrate for Cell Culture

The present invention relates to a cell culture patterning substrate used for culturing cells such as blood vessels, for example.

Currently, cell cultures of various animals and plants are carried out, and new cell culture methods have been developed. Cell culture technology is used for the purpose of elucidating the biochemical phenomena and properties of cells and producing useful substances. In addition, attempts have been made to investigate 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 attaching to something and cannot survive long-term in a floating state outside of the body. The culture | cultivation of the cell which has such adhesive dependence requires the carrier for cell adhesion, and the plastic culture dish which apply | coated uniformly cell adhesive protein, such as collagen and fibronectin, is used generally. These cell adhesive proteins are known to act on cultured cells to facilitate cell adhesion or to affect the shape of the cells.

On the other hand, a technique for adhering and arranging cultured cells to a minute portion on a substrate has been reported. With this technique, cultured cells can be applied to artificial organs, biosensors, bioreactors and the like. As a method for arranging cultured cells, cells are cultured on this surface using a substrate on which a surface that is easily adhered to the cells is patterned, and the cells are arranged by adhering the cells only to a surface that has been processed to adhere to the cells. The method is taken.

For example, Patent Document 1 applies a charge retention medium in which a static charge pattern is formed for cell culture, for the purpose of proliferating neurons in a circuit. In addition, Patent Document 2 attempts to arrange cultured cells on a surface on which a cell non-adhesive or cell adhesive photosensitive hydrophilic polymer is patterned by photolithography.

In addition, Patent Document 3 discloses a cell culture substrate on which a substance such as collagen, which affects the adhesion rate and shape of cells, is patterned, and a method of producing the substrate by a photolithography method. By culturing the 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, in the cell culture method, when the cell culture pattern has a large area, the cells can be regularly arranged at the end of the cell culture pattern, but the arrangement of the cells becomes poor at the center of the cell culture pattern, or the cells There is a problem such as a case that does not adhere. In addition, a general cell is a cell that is adhered to a cell adhesion part having adhesiveness to the cell when the cell culture is performed in the cell culture pattern or the like so that individual cells change in form to form a tissue and form such a tissue. Non-Patent Document 1 and the like show that a boundary region with an inhibitory cell adhesion inhibiting portion stimulates a cell, thereby causing a change in the morphology of the cell and gradually propagating the central portion of the cell culture pattern. However, when the area of the cell culture pattern is widened, it is difficult for the morphology of the cell to be transmitted to the center part, so that the morphology of the cell in the center part is less likely to occur and the tissue is not formed in the center part. Moreover, when seeding a cell and adhering it to a board | substrate in this way, there also existed a problem that adhesion of a cell took time.

Patent Document 1: Japanese Patent Laid-Open No. 2-245181

Patent Document 2: Japanese Patent Laid-Open No. 3-7576

Patent Document 3: Japanese Patent Laid-Open No. 5-176753

Non-Patent Document 1: [Spargo et al., Proceedings of the National Academy of Sciences of the United States of America (1994) p. 11070 and below]

Problems to be Solved by the Invention

Therefore, it is desired to provide a cell culture patterning substrate capable of efficiently arranging cells in a large area on a substrate and forming tissues and the like.

Means for solving the problem

The present invention is a cell culture patterning substrate having a substrate and a cell culture region formed on the substrate, the cell culture region containing a cell adhesion layer having adhesion to the cells, wherein the cell culture region is the cell adhesion layer. The formed cell adhesion part and a cell adhesion aiding part which is formed in a pattern shape and inhibits adhesion with the cells, wherein the cell adhesion aiding part is adjacent to the cell adhesion aiding part when attaching the cell to the cell adhesion part. It provides a patterning substrate for cell culture, characterized in that the cells on the cell adhesion portion is formed to be able to bind to each other on the cell adhesion assistant.

According to the present invention, since the cell adhesion region is formed in the cell culture region, when cells adhere to the cell adhesion portion, the cells can be activated, and the cells can be cultured in a short time efficiently. In addition, since the cells are cultured for each of the regions sandwiched between the cell adhesion assistants, the number of cells stimulated in the boundary region can be increased compared with the case where the cells are cultured in the entire cell culture region without the cell adhesion assistants. Can be. As a result, the arrangement of the cells can be improved, and the morphology of the cells can also be uniformly performed. In addition, in the present invention, when the cell adhesion assistant is attached to the cell adhesion layer, the cell adhesion assistant is formed so as not to inhibit the binding of the cells attached to the adjacent cell adhesion units. The cells can be bonded to each other, and the resulting tissue can be made large.

In the present invention, the cell culture assistant may be formed linearly in the cell culture region. In this case, the design at the time of forming the said cell culture area | region becomes easy, and it has the advantage that it is easy to arrange | position a cell regularly in the case of attaching a cell.

In addition, in the present invention, the boundary between the cell adhesion assistant and the cell adhesion portion may be formed in a pattern having irregularities. As described above, when the cells are attached in accordance with the pattern having the unevenness, the stimuli that the cells take up in the boundary region increase, so that the cells can be arranged more aligned. In addition, the adhesion to the cell adhesion portion of the cells can be activated, and the cell culture patterning substrate can be efficiently adhered to the substrate on the substrate in a short time.

In addition, the present invention is a cell culture patterning substrate having a substrate and a cell culture region formed on the substrate, the cell culture region containing a cell adhesion layer having adhesion to the cells, the cell adhesion layer is Provided is a patterning substrate for cell culture, which is formed in a pattern form having irregularities.

According to the present invention, since the end portion of the cell adhesion layer is formed in a pattern shape having irregularities, when the cells are adhered on the cell adhesion layer, the stimulation of the cells is increased in the boundary region, and the end of the cell adhesion layer is increased. Thus, cells can be arranged to be more aligned. In addition, since the adhesion to the cell adhesion part of the cells can be activated, the cell culture patterning substrate can be efficiently adhered to the substrate on the substrate in a short time.

In the above invention, it is preferable that the distance from the concave end of the concave-convex portion to the convex end is a size in which cells align linearly when attaching the cells on the cell adhesion layer. This is because with this size of irregularities, cells can be well aligned.

In the said invention, it is preferable that the average of distance from the recessed part edge part of the said uneven | corrugated part to a convex part edge part exists in the range of 0.5 micrometer-30 micrometers. This is because by making the size of the unevenness in this range, it is possible to align the cells well and to activate the cells.

Effect of the Invention

According to the present invention, it is possible to provide a cell culture patterning substrate capable of activating the cells when attaching the cells on the cell adhesion part and culturing the cells in a large area efficiently in a short time. Further, at this time, the arrangement of the cells can be improved, and the change in the morphology of the cells can also be achieved.

BRIEF DESCRIPTION OF THE DRAWINGS It is a schematic sectional drawing which shows an example of the patterning substrate for cell culture of this invention.

2 is a schematic cross-sectional view showing another example of the patterning substrate for cell culture of the present invention.

3 is a schematic cross-sectional view showing another example of the patterning substrate for cell culture of the present invention.

4 is a schematic cross-sectional view showing another example of the patterning substrate for cell culture of the present invention.

5 is a schematic cross-sectional view showing another example of the patterning substrate for cell culture of the present invention.

Fig. 6 is a process chart showing an example of a method for forming a cell adhesion auxiliary part in a cell culture patterning substrate of the present invention.

Fig. 7 is a schematic cross-sectional view showing an example of the photocatalyst-containing layer side substrate used in the present invention.

8 is a schematic cross-sectional view showing an example of the photocatalyst-containing layer side substrate used in the present invention.

9 is a schematic cross-sectional view showing an example of the photocatalyst-containing layer side substrate used in the present invention.

10 is a flowchart showing another example of the method for forming a cell culture auxiliary part in the cell culture patterning substrate of the present invention.

FIG. 11 is a flowchart showing an example of a method for forming a cell adhesion layer in a cell culture patterning substrate of the present invention. FIG.

12 is a schematic cross-sectional view showing another example of the patterning substrate for cell culture of the present invention.

<Description of the code>

1 ... mention

2 cell culture area

3 ... cell adhesion

4 cell culture assistant

5 Photo masks

6 ... Energy

The present invention relates to a cell culture patterning substrate used for culturing cells, and the cell culture patterning substrate of the present invention has two embodiments. Hereinafter, each embodiment is demonstrated.

A. First Embodiment

First, the first embodiment of the patterning substrate for cell culture of the present invention will be described. A first embodiment of a cell culture patterning substrate of the present invention is a cell culture patterning pattern having a substrate and a cell culture region formed on the substrate, wherein the cell culture region contains a cell adhesion layer having adhesion to the cells. Substrate,

The cell culture region has a cell adhesion part in which the cell adhesion layer is formed, and a cell adhesion assistant part formed in a pattern shape and inhibits adhesion to the cell, and wherein the cell adhesion assistant part attaches the cell to the cell adhesion part. Cells on two of the cell adhesion portions adjacent to the adhesion aid are formed to bind to each other on the cell adhesion aid.

The patterning substrate for cell culture of this embodiment has the base material 1 and the cell culture area | region 2 formed on this base material 1, for example, as shown in FIG. Has a cell adhesion part 3 which has adhesion with the cell in which the cell adhesion layer was formed, and the cell adhesion | attachment assistance part 4 which inhibits adhesion | attachment with a cell.

Here, generally, when cells are cultured by attaching cells to a cell culture region to form a tissue, the cells are gradually arranged from outside to inside of the cell culture region. In addition, for the formation of tissues, individual cells need to be changed in shape and arranged, and the shape change of these cells is also gradually performed from the end of the cell culture area to the center.

Therefore, when culturing cells using a general cell culture patterning substrate, when the cell culture area for culturing the cells is large, the arrangement of the cells in the central portion is poor, or when tissues are not formed, There is a case where the cells do not adhere to the center of the culture region. Moreover, there also existed a problem that the shape change of the cell in the center part was bad, or the target tissue was not formed.

On the other hand, according to the present invention, a cell adhesion assistant is formed in the cell culture region, and the cells are cultured in the cell adhesion unit 3 sandwiched between the cell culture assistants 4, for example, as shown in FIG. In other words, the cell arrangement and the cell morphology can be changed at the boundary between the cell adhesion assistant 4 and the cell adhesion unit 3, and the cell culture is compared with the case where the cell adhesion assistant 4 is not formed. A boundary region can also be provided inside the region 2. Therefore, the cells adhered to the cell culture region 2 can be stimulated at the boundary between the cell adhesion portion 3 existing inside the cell culture region 2 and the cell adhesion assistance portion 4. As a result, the arrangement and shape change of the cells can be improved in the entire region of the cell culture region 2.

Further, in the present embodiment, the cell adhesion assistant is formed so that cells attached on two adjacent cell adhesion layers can bind to each other on the cell adhesion assistant, for example, as shown in FIG. The cell adhesion aiding portion 4 is formed so that the cells attached to the cell adhesion portion 3 of the region and the cell adhered to the cell adhesion portion 3 of the region b can bind on the cell adhesion assistance portion 4. As a result, the cells can be cultured in the same area as in the case of being cultured in the entire surface of the cell culture region 2. This means that the cells can interact with each other, even in areas that inhibit adhesion to the cells, and in areas where the cells are present on both sides and the cells are close enough to affect each other. Because there is.

In addition, when there are defects or the like in the cell adhesion layer, it is known that the cells are activated and the cells easily adhere to the regions. In this embodiment, since the cell adhesion aid formed in the cell culture region exerts the same effect as this defect and the cells are activated, the cells can be efficiently adhered to the substrate in a short time.

Hereinafter, each structure of the cell culture patterning board | substrate of this embodiment is demonstrated.

1. Cell Culture Area

First, the cell culture region in the cell culture patterning substrate of the present embodiment will be described. The cell culture region in the present embodiment is a region formed for culturing a cell, and includes a cell adhesion portion in which a cell adhesion layer having adhesion with the cell is formed, and a cell adhesion assistant portion formed in a pattern shape and inhibiting adhesion to the cells. It has an area.

In this embodiment, the cell culture region may be formed in a part of the substrate 1, for example, as shown in FIG. 1, or the entire surface of the substrate may be a cell culture region. Here, for example, as shown in FIG. 1, when the cell culture region 2 is formed in a part of the substrate 1, a region other than the cell culture region on the substrate 1 inhibits adhesion to the cells. It becomes an uncultured area. In addition, in this embodiment, the number of cell culture regions formed on one substrate is not limited to one, for example, as shown in FIG. 2, the cell culture regions 2 are formed on the substrate 1. A plurality may be formed. Also in this case, the area | region other than each cell culture area | region on the base material 1 becomes the said cell non-cultivation area | region.

In addition, although the size and type of tissues of interest are also varied, the size of one cell culture region is usually in the range of 0.05 mm ² to 8000 mm ² , particularly 0.1 mm ² to 10 mm ² .

Here, in the cell culture region as described above, the cell adhesion assistant is formed in a pattern form in the cell adhesion portion. In the present embodiment, the cell adhesion assistant is formed so that cells attached to two cell adhesion units adjacent to the cell adhesion assistant can bind to each other on the cell adhesion assistant, and the cells attached on the cell adhesion layer are regularly It will not specifically limit, if it arrange | positions and is formed so that the change of the shape of a cell may arise uniformly. For example, as shown in FIG. 1, the cell adhesion assistant 4 may be linearly formed in the cell culture region 2, or, for example, as shown in FIG. 3, the cell may be formed in the cell culture region 2. The adhesion assistant 4 may be formed at random.

Although the width | variety of the said cell culture support part changes also with the kind, size, etc. of the cell to culture, Usually, the inside of the range of 0.5 micrometer-10 micrometers, especially 1 micrometer-5 micrometers is preferable. When the width is wider than the above range, cells adhering on two cell adhesion parts adjacent to the cell adhesion aid become difficult to interact with each other on the cell adhesion aid, and when the width is narrower than the range, the patterning technique described below It is difficult to obtain a pattern of such a size precisely, and it is difficult for the cell adhesion aid to affect the arrangement and shape change as described above.

In this case, the width of the cell adhesion portion sandwiched between the cell adhesion assistants (for example, the distance indicated by x in FIG. 1), or the width of the cell adhesion portion sandwiched between the cell adhesion assistant and the cell non-culture region (eg, For example, the distance indicated by y in FIG. 1 is appropriately selected depending on the size and type of cells to be cultured, the type of tissue to be used, etc., but is usually within the range of 1 μm to 200 μm, particularly 40 μm to 80 μm. Do. Thereby, the cells adhering to the cell adhesion part can be arranged regularly, and the shape change can be satisfactorily formed to form the tissue.

Especially in this embodiment, it is preferable that the cell adhesion | attachment auxiliary part is formed linearly. This is because the design at the time of forming the cell culture region can be facilitated, and the arrangement of the cells to be cultured can be improved. Linear means that the cell adhesion assistant is formed in a straight line. For example, as shown in FIG. 1, not only the cell adhesion assistant 4 is continuously formed, but also the cell adhesion assistant is broken, for example. It also includes the case where it is formed. In addition, in this embodiment, not only the case where the cell adhesion aid is linearly formed in one direction, but also when the cell adhesion aid 4 is linearly formed in a plurality of directions as shown in FIG. 4, for example. Include.

In addition, in the present embodiment, the boundary between the cell adhesion portion and the cell adhesion assistance portion may be formed in a pattern having irregularities. By arranging cells according to the pattern having such irregularities, the cells can be arranged more regularly. In this case, the attached cells are more activated, and there is an advantage that the cells can be cultured efficiently. Here, the pattern image having irregularities is not particularly limited as long as it is a pattern in which cells can be arranged regularly. For example, as shown in FIG. 5, the boundary between the cell adhesion part 3 and the cell culture auxiliary part 4 is shown. May have right-angled irregularities, and may also have irregularities such as wavy shapes. For example, even when the cell adhesion assistant is formed in a broken line shape or when the cell adhesion assistant is formed in a random pattern, the boundary between the cell adhesion assistant and the cell adhesion part is a pattern having irregularities. Can be formed. This is because the same effect can be obtained only in this case.

Here, the distance from the concave end of the concave-convex to the convex end is preferably the size of linear alignment of the cells when attaching the cells on the cell adhesion layer. Specifically, the size is appropriately selected depending on the shape of the cultured cell, etc., but in general, the average distance from the concave end to the convex end of the unevenness is preferably in the range of 0.5 µm to 30 µm, particularly 1 µm to 5 µm. . This is because when the cells are cultured, the cells can be cultured in the desired shape to form tissues without running out of cells at the end of the cell culture region. Here, the measurement of the average of the distance from the concave end to the convex end of the pattern having the unevenness measures the distance from the lowest end to the highest end of each unevenness within the range of 200 μm between the cell adhesion part and the cell adhesion auxiliary part, The average is calculated.

Hereinafter, the cell adhesion part and the cell adhesion assistance part which comprise such a cell culture area are demonstrated, respectively.

(Cell adhesion part)

First, the cell adhesion part used in this embodiment is demonstrated. The cell adhesion part in this embodiment is an area | region in which the cell adhesion layer which has adhesiveness with a cell was formed on the base material in the cell culture area | region. The cell adhesive layer is not particularly limited as long as it has adhesiveness with cells, and a layer having adhesiveness with cells used in a general cell culture patterning substrate can be used. In this embodiment, a cell adhesion part can be formed by forming such a cell adhesion layer in a pattern form, for example, a cell adhesion part is formed by apply | coating the coating liquid for cell adhesion layer formation containing the material which has adhesiveness with a cell in a pattern form. Can be formed. In addition, the coating solution for forming the cell adhesion layer can be formed on the entire surface of the cell culture region to form a cell adhesion part by a photolithography method or the like.

In addition, in this embodiment, the said cell adhesion layer is a layer containing the cell adhesion material which has adhesiveness with a cell, and decompose | disassembles or denatures by the action of a photocatalyst according to energy irradiation, and irradiates this cell adhesion layer with energy The cell adhesion may be formed by patterning. In this case, for example, by forming a cell adhesion layer on the entire surface of the cell culture region, and then irradiating energy in a pattern to form a cell adhesion assistant, the cell adhesion material can be decomposed or denatured by the action of a photocatalyst, A cell adhesion aid that inhibits adhesion with cells and a cell adhesion portion having adhesion to cells can be formed. In addition, the cell adhesion layer containing such a cell adhesion material, the formation method of the cell adhesion | attachment auxiliary part at that time, etc. are demonstrated in detail below.

In addition, the cell adhesion layer used in the present embodiment has a cell adhesion inhibitory layer containing a cell adhesion inhibitory material which has adhesion inhibition with the cells and which is degraded by the action of a photocatalyst according to energy irradiation, on the entire surface of the cell culture region. By applying and then irradiating energy to a region other than the cell adhesion aid, the cell adhesion inhibiting material can be formed to be decomposed or denatured to have adhesion with the cells. In this case, since it is an area | region which inhibits adhesion | attachment with a cell other than the area | region which became the cell adhesion part by energy irradiation, it can use as a cell adhesion | attachment auxiliary part. The cell adhesion inhibitory layer containing such a cell adhesion inhibitory material, the formation method of the cell adhesion layer at that time, etc. are also demonstrated in detail below.

(Cell adhesion assistant)

Below, the cell adhesion aid of the cell culture region in the present embodiment will be described. The cell adhesion assistant in the present embodiment is formed in a pattern in the cell culture region and inhibits adhesion with the cells, thereby adhering the cells on the cell adhesion assistant to the two cell adhesion assistants adjacent to the cell adhesion assistant. The cells are not particularly limited as long as they are formed so as to be able to bind to each other on the cell culture assistant.

The cell adhesion aid in the present embodiment may be, for example, a region to which a substrate to be described later is exposed, or the like, and a cell adhesion inhibiting layer or the like that inhibits adhesion with cells generally used may be formed. As a method of forming the cell adhesion inhibiting layer, a general printing method, a photolithography method, or a patterning method using the action of a photocatalyst by energy irradiation may be mentioned. Since the patterning method using the action of the photocatalyst according to the energy irradiation is described in combination with the description item of the cell adhesion layer using the cell adhesion inhibitory layer having the cell adhesion inhibitory material described later, the description thereof is omitted.

In addition, as described above, when the cell adhesion layer is a layer containing a cell adhesion material decomposed or denatured by the action of a photocatalyst in response to energy irradiation, the cell adhesion assistant is a region in which the decomposed products or denatured substances of the cell adhesion material remain. Or the like. In this case, the method of forming the cell adhesion assistant is described in combination with the description item of the cell adhesion layer containing the cell adhesion material decomposed by the action of the photocatalyst due to energy irradiation.

2. Description

Below, the description used for this embodiment is demonstrated. The substrate used in the present embodiment is not particularly limited as long as the cell culture region can be formed. For example, inorganic materials such as metal, glass, silicon, organic materials such as plastics, and the like can be used. In addition, flexibility, transparency, etc. of a base material are suitably selected by the kind, application, etc. of the patterning substrate for cell culture.

Here, in this embodiment, since the area | region other than the cell culture area | region on a base material becomes a cell non-cultivation area | region which does not culture a cell, it is preferable to inhibit adhesion | attachment with a cell, For example, except for the said cell culture area | region In the cell non-culture region, a layer or the like may be formed that inhibits adhesion with the cells.

3. Patterning Substrate for Cell Culture

Below, the patterning substrate for cell culture of this embodiment is demonstrated. The cell culture patterning substrate of the present embodiment is not particularly limited as long as the cell culture region is formed on the substrate described above, and a member such as a light shielding portion may be formed, if necessary.

4. Other

As described above, the cell adhesion layer used in the cell culture region of the cell culture patterning substrate of the present embodiment may (1) contain a cell adhesion material decomposed or denatured by the action of a photocatalyst according to energy irradiation. (2) forming a cell adhesion inhibitory layer containing a cell adhesion inhibitory material which inhibits adhesion to cells and which is decomposed or denatured by the action of a photocatalyst according to energy irradiation, and then energy irradiation; And by degrading or denaturing the cell adhesion inhibiting material.

Hereinafter, each will be described separately.

I. For (1)

First, the case where the cell adhesive layer contains a cell adhesive material decomposed or denatured by the action of a photocatalyst according to energy irradiation will be described. The following three embodiments are mentioned as a cell adhesion layer containing such a cell adhesion material.

In a first embodiment, the cell adhesion layer is a photocatalyst-containing cell adhesion layer containing a photocatalyst and a cell adhesion material, and when the photocatalyst-containing cell adhesion layer is irradiated with energy, the action of the photocatalyst contained in the photocatalyst-containing cell adhesion layer itself causes This is the case when the adhesive material is decomposed or modified.

The second embodiment is a photocatalyst in which a cell adhesive layer containing at least a cell adhesive material is formed on a photocatalytic treatment layer containing at least a photocatalyst and the cell adhesive material in the cell adhesive layer is adjacent when energy is irradiated to the cell adhesive layer. It is a case where it decomposes or denatures by the action of the photocatalyst in a process layer.

In the third embodiment, a cell adhesive material is formed by forming a cell adhesive layer containing at least a cell adhesive material on a substrate and irradiating energy by facing a cell adhesive layer with a photocatalyst-containing layer or the like containing at least a photocatalyst at the time of energy irradiation. This is the case where is decomposed or modified by the action of the photocatalyst in the opposing photocatalyst-containing layer. Hereinafter, each embodiment is demonstrated.

(1) First Embodiment

First, the cell adhesive layer is a photocatalyst-containing cell adhesive layer containing a photocatalyst and a cell adhesive material, and when energy is irradiated to the photocatalyst-containing cell adhesive layer, the cell adhesive material is decomposed by the action of the photocatalyst contained in the photocatalyst-containing cell adhesive layer itself. Or the case of denaturation is demonstrated.

According to the present embodiment, since the photocatalyst-containing cell adhesive layer contains the photocatalyst and the cell adhesive material, by irradiating energy to the photocatalyst-containing cell adhesive layer, the cell adhesive material can be decomposed or denatured by the action of the photocatalyst, resulting in energy The irradiated area can be a cell adhesion aid where the cells do not adhere. Moreover, since the cell adhesion material remains in the area | region to which energy is not irradiated, it can be set as the cell adhesion part with favorable adhesiveness with a cell. Therefore, by irradiating energy in a pattern form without the need for a special apparatus or complicated process, the cell adhesion assistant can easily be formed in the cell adhesion part that inhibits adhesion with the cells.

Such a photocatalyst-containing cell adhesive layer can be formed by applying a cell adhesive material decomposed or modified by the action of the photocatalyst in response to energy irradiation, and a coating solution for forming a photocatalyst-containing cell adhesive layer containing the photocatalyst. The coating liquid for forming the photocatalyst-containing cell adhesive layer can be applied using a general coating method. For example, a spin coating method, a spray coating method, a dip coating method, a roll coating method, a bead coating method, or the like can be used.

At this time, the film thickness of the photocatalyst-containing cell adhesive layer is appropriately selected depending on the type of cell culture patterning substrate or the like, but may be usually about 0.01 μm to 1.0 μm, particularly about 0.1 μm to 0.3 μm.

Hereinafter, the cell adhesion material contained in the photocatalyst containing cell adhesion layer used for this embodiment, and a photocatalyst are demonstrated, and the formation method of a cell adhesion | attachment auxiliary part is demonstrated.

a. Cell adhesion material

First, the cell adhesion material contained in the photocatalyst-containing cell adhesion layer of the present embodiment will be described. The cell adhesion material contained in the photocatalyst-containing cell adhesion layer of the present embodiment is not particularly limited as long as it is adhesive to the cells and decomposed or denatured by the action of the photocatalyst upon energy irradiation. Here, it means that the cell adheres well to the cell having adhesiveness, and when the cell has different types of adhesion, the cell adheres well to the target cell.

The cell adhesion material used in the present embodiment has adhesiveness with such cells, and is degraded or denatured by the action of a photocatalyst according to energy irradiation, thereby losing adhesion to the cells or inhibiting adhesion with the cells. It is used for changing the cell adhesion inhibitory effect.

Here, there are two kinds of materials having adhesion to the cells, a material having adhesion with the cells by physicochemical properties, and a material having adhesion with the cells by biochemical properties.

Examples of physicochemical factors that determine the adhesion of cells with the cells by their physical and chemical properties include surface free energy and electrostatic interaction. For example, in the case where the adhesion to cells is determined by the surface free energy of the material, if the material has surface free energy within a predetermined range, the adhesion between the cell and the material is good, but if it is below the range, the cells and material Adhesion with is reduced. Such changes in cell adhesion due to surface free energy are described, for example, in CMC Publishing Co., Ltd. "Biomaterial no Saisentan", the lower part of Yoshito (Response) p.109] are known. As a material which has adhesiveness with a cell by such a factor, hydrophilized polystyrene, poly (N-isopropyl acrylamide), etc. are mentioned, for example. In the case of using such a material, the surface free energy changes due to the action of a photocatalyst according to energy irradiation, for example, by the substitution or decomposition of a functional group on the surface of the material, and thus does not have adhesion to cells. Or inhibit cell adhesion.

In addition, when the adhesion between the cell and the material is determined by electrostatic interaction or the like, the adhesion with the cell is determined by, for example, the amount of positive charge of the material. Examples of materials having adhesion to cells by electrostatic interactions include basic polymers such as polylysine, aminopropyltriethoxysilane, and N- (2-aminoethyl) -3-aminopropyltrimethoxysilane. Basic compounds, such as these, and condensate containing these, etc. are mentioned. In the case of using such a material, the material is decomposed or denatured by the action of a photocatalyst according to energy irradiation, for example, to change the amount of positive charge present on the surface, so that the cell does not have adhesion to the cell, or It may have adhesion inhibition.

In addition, materials having adhesion to cells due to biological properties include those having good adhesion with specific cells, or those having good adhesion with many cells, and specifically, fibronectin, laminin, tenacin, and vitro. Nectin, RGD (arginine-glycine-aspartic acid) sequence-containing peptide, YIGSR (tyrosine-isoleucine-glycine-serine-arginine) sequence-containing peptide, collagen, atelocollagen, gelatin and the like. When such a material is used, it does not have adhesiveness to cells or inhibits cell adhesion by the action of a photocatalyst according to energy irradiation, for example, by destroying part of the structure of the material, or breaking the main chain. May have a last name

Although such a cell adhesion material varies depending on the kind of the material and the like, it is preferable that the cell adhesion layer is usually contained in an amount of 0.01% by weight to 95% by weight, in particular 1% by weight to 10% by weight. It is because the area | region containing a cell adhesive material can be made into the area | region with favorable adhesiveness with a cell by this.

b. Photocatalyst

Below, the photocatalyst contained in the photocatalyst containing cell adhesion layer of this embodiment is demonstrated.

The photocatalyst used in the present embodiment is not particularly limited as long as it can decompose or denature the cell adhesive material described above by the action of the photocatalyst according to energy irradiation.

Here, the mechanism of action of the photocatalyst represented by titanium oxide, which will be described later, is not necessarily clear, but the organic substance is formed by the active oxygen species generated in the presence of oxygen or water in the direct reaction of a carrier produced by light irradiation with a compound nearby. It is believed to change the chemical structure of the compound. In this embodiment, it is believed that this carrier affects the action of the cell adhesion material described above.

As the photocatalyst used in the present embodiment, specifically known as an optical semiconductor, for example, titanium dioxide (TiO ₂ ), zinc oxide (ZnO), tin oxide (SnO ₂ ), strontium titanate (SrTiO ₃ ), tungsten oxide (WO ₃ ), bismuth oxide (Bi ₂ O ₃ ), and iron oxide (Fe ₂ O ₃ ) may be mentioned, and one or two or more thereof may be selected and used.

In the present embodiment, titanium dioxide is particularly preferably used because of its high band gap energy, chemical stability, no toxicity, and easy availability. There are anatase type and rutile type in titanium dioxide, and both can be used in this embodiment, but anatase type titanium dioxide is preferable. The anatase titanium dioxide has an excitation wavelength of 380 nm or less.

As such anatase titanium dioxide, for example, anatase-type titania sol of hydrochloric acid bridge type (STS-02 by Ishihara Industries, Ltd. (average particle size 7 nm), ST-K01 by Ishihara Industries, Ltd.), nitric acid, for example. The anatase type titania sol (the TA-15 (average particle diameter 12nm) made from Ilsan Chemical Co., Ltd.) of a bridge | bridging type | mold is mentioned.

The smaller the particle diameter of the photocatalyst, the more effectively the photocatalytic reaction occurs. Preferably, the average particle diameter is preferably 50 nm or less, particularly preferably 20 nm or less.

Content of the photocatalyst in the photocatalyst containing cell adhesion layer of this embodiment can be set to 5-95 weight%, Preferably it is 10-60 weight%, More preferably, it is 20-40 weight%.

This is because the cell adhesive material in the energy irradiated region of the photocatalyst-containing cell adhesive layer can be decomposed or denatured.

Here, it is preferable that the photocatalyst used for this embodiment has low hydrophilicity, for example by having high hydrophilicity. It is because the area | region which the cell adhesion material mentioned above decomposes and the photocatalyst exposed by this can be used as a area | region with low adhesiveness with a cell.

c. Etc

In this embodiment, not only the said cell adhesive material and a photocatalyst but a binder etc. which improve strength, tolerance, etc. as needed may be contained in a photocatalyst containing cell adhesion layer. In this embodiment, especially as a binder, it is preferable to use the material which has the cell adhesion inhibitory property which inhibits adhesion with a cell at least after energy irradiation. This is because the adhesiveness with the cells of the cell adhesion assistant that is the energy irradiated region can be lowered. As such a material, it may have the said cell adhesion inhibitory property before energy irradiation, for example, and may have cell adhesion inhibition by the action of the photocatalyst by energy irradiation.

In this embodiment, it is preferable to use the material which has cell adhesion inhibition as a binder by the action of the photocatalyst by energy irradiation especially. This is because in the region before the energy irradiation, only the region irradiated with the energy can lower the adhesion with the cells without inhibiting the adhesion of the cell adhesion material to the cells.

As a material used as such a binder, for example, the main skeleton has a high binding energy that is not decomposed by photoexcitation of the photocatalyst, and it is preferable to have an organic substituent decomposed by the action of the photocatalyst, for example, (1) organopolysiloxanes which hydrolyze and polycondense chloro or alkoxysilanes and the like by sol-gel reactions to exhibit great strength, and (2) organopolysiloxanes crosslinked with reactive silicones excellent in water and oil repellency.

In the case of said (1), the organopolysiloxane which is 1 type, or 2 or more types of hydrolysis-condensation products of a silicon compound represented by a following formula, or all hydrolysis-condensation products is preferable.

Y _n SiX _(4-n)

In the formula, Y is an alkyl group, a fluoroalkyl group, a vinyl group, an amino group, a phenyl group or an epoxy group, or an organic group containing them, and X represents an alkoxyl group, an acetyl group or a halogen. n is an integer of 0-3. Moreover, it is preferable that carbon number of the organic group represented by Y is here in the range of 1-20, and it is preferable that the alkoxy group represented by X is a methoxy group, an ethoxy group, a propoxy group, butoxy group.

Moreover, the compound which has a skeleton represented by following General formula (1) is mentioned as reactive silicone of said (2).

n is an integer of 2 or more, R ¹ and R ² each represent a substituted or unsubstituted alkyl, alkenyl, aryl, or cyanoalkyl group having 1 to 20 carbon atoms, and 40% or less of the total in molar ratio is vinyl, phenyl or halogenated phenyl. . Moreover, it is preferable that R <^1> , R <^2> is a methyl group because surface energy becomes smallest, and it is preferable that a methyl group is 60% or more by molar ratio. In addition, the chain terminal or side chain has a reactive group such as at least one or more hydroxyl groups in the molecular chain. By using the material, the surface of the energy irradiated region can have high hydrophilicity by the action of the photocatalyst according to the energy irradiation. This is because the adhesion with the cells is inhibited and the cells can be prevented from adhering to the energy irradiated region.

When the material is used as a material having a cell adhesion inhibitory property, the contact angle with water before energy is irradiated is preferably in the range of 15 ° to 120 °, particularly 20 ° to 100 °. This is because the adhesion with the cells can be improved.

Moreover, when energy is irradiated to the material which has the cell adhesion inhibitoryness, it is preferable that the contact angle with water is 10 degrees or less. It is because it can have high hydrophilicity and can lower adhesiveness with a cell by setting it as the said range.

In addition, the contact angle with water mentioned here measures the contact angle with water or the liquid which has an equivalent contact angle, using a contact angle measuring device (type CA-Z by Kyowa Interface Science, Inc.) (dropping a droplet from a micro syringe). 30 seconds later), and the results are obtained graphically.

In addition, at the same time as the organopolysiloxane, a stable organosilicon compound that does not undergo a crosslinking reaction such as dimethylpolysiloxane can be mixed with a binder.

In addition, in this embodiment, it may contain the degradation substance etc. which reduce adhesiveness with a cell, or assists such a change, for example, by causing the change of the wettability of the area | region to which energy was irradiated.

Examples of such a decomposable substance include surfactants that are decomposed by the action of a photocatalyst due to energy irradiation and are hydrophilic, and the like, resulting in decreased adhesion to cells. Specifically, hydrocarbons such as NIKKOL BL, BC, BO, and BB series manufactured by Nikko Chemicals, ZONYL FSN, FSO, manufactured by DuPont, Saflon S-141, 145, Dainippon Ink Mega Pack F-141, 144, manufactured by Kagaku Kogyo Co., Ltd. Psentant F-200, F-251, manufactured by Neos Co., Ltd. Manufacture DS-401, 402, 3M, manufactured by Daikin Kogyo Co., Ltd. And silicone-based nonionic surfactants such as Floyd FC-170 and 176. Cationic surfactants, anionic surfactants, and amphoteric surfactants can also be used.

In addition to the surfactant, polyvinyl alcohol, unsaturated polyester, acrylic resin, polyethylene, diaryl phthalate, ethylene propylene diene monomer, epoxy resin, phenol resin, polyurethane, melamine resin, polycarbonate, polyvinyl chloride, poly Amide, polyimide, styrenebutadiene rubber, chloroprene rubber, polypropylene, polybutylene, polystyrene, polyvinyl acetate, nylon, polyester, polybutadiene, polybenzimidazole, polyacrylonitrile, epicrolohydrin, poly Oligomers, polymers such as sulfide and polyisoprene, and the like.

In this embodiment, such a binder is preferably included in the range of 5 wt% to 95 wt%, especially 40 wt% to 90 wt%, more particularly 60 wt% to 80 wt%, in the photocatalyst-containing cell adhesion layer.

Moreover, in this embodiment, a light shielding part can be formed on the cell culture area | region of a base material as needed. Accordingly, when the entire surface of the photocatalyst-containing cell adhesive layer is irradiated with energy from the substrate side, the photocatalyst on the region where the light shielding portion is formed is not excited, and the cell adhesive material contained in the cell adhesive layer other than the region where the light shielding portion is formed is decomposed or Can be denatured.

The light shielding portion is not particularly limited as long as it can block energy irradiated at the time of forming the cell adhesion auxiliary portion, and for example, a metal thin film such as chromium having a thickness of about 1000 to 2000 GPa is formed by, for example, sputtering or vacuum deposition. It can be formed by patterning this thin film. As a method of this patterning, normal patterning methods, such as sputter | spatter, can be used.

Moreover, the method of forming in a resin pattern the layer which contained light-shielding particle | grains, such as carbon microparticles | fine-particles, a metal oxide, an inorganic pigment, and an organic pigment, may be sufficient. As a resin binder used, 1 type (s) or 2 or more types of resins, such as a polyimide resin, an acrylic resin, an epoxy resin, polyacrylamide, polyvinyl alcohol, gelatin, casein, and a cellulose, or a photosensitive resin, and O / on it A resin composition of the W emulsion type, for example, an emulsion of reactive silicone can be used. The thickness of such resin light shielding portion can be set within the range of 0.5 to 10 µm. As a method of patterning such a resin light shielding part, the method generally used, such as the photolithography method and the printing method, can be used.

d. How to Form Cell Adhesion Auxiliaries

Below, the formation method of the cell adhesion | attachment auxiliary part in this embodiment is demonstrated. In this embodiment, for example, as shown in Fig. 6, energy 6 is applied to the photocatalyst-containing cell adhesive layer 7 containing the cell adhesive material and the photocatalyst, for example, using a photomask 5 or the like. By irradiating in a pattern form to form a cell adhesion assistant (FIG. 6 (a)), the cell adhesion material 4 is decomposed or denatured in the cell adhesion layer 7 to inhibit the adhesion to the cells. It can be formed (Fig. 6 (b)). At this time, the cell adhesion assistant contains a photocatalyst and a decomposed product or modified product of the cell adhesive material.

Here, energy irradiation (exposure) in this embodiment is a concept including irradiation of any energy ray which can decompose or denature a cell adhesive material by the action of a photocatalyst according to energy irradiation, and is not limited to irradiation of light. .

Usually, the wavelength of light used for such energy irradiation is set in the range of 400 nm or less, Preferably it is the range of 380 nm or less. This is because the preferred photocatalyst used as the photocatalyst as described above is titanium dioxide, and light of the above-described wavelength is preferable as energy for activating the photocatalytic action by the titanium dioxide.

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, a method of drawing irradiation in a pattern form using a laser such as excimer, YAG or the like may be used. As described above, when the substrate has the light shielding portion in the same pattern form as the cell adhesion portion, it can be performed by irradiating the entire surface with energy from the substrate side. In this case, there is no need for a photomask or the like, and there is no need for a process such as positional alignment.

In addition, the irradiation amount of energy in energy irradiation is an irradiation amount required for decomposition | disassembly or denaturation of a cell adhesive material by the action of a photocatalyst.

At this time, by irradiating energy while heating the layer containing a photocatalyst, since a sensitivity can be raised and a cell adhesive material can be decomposed | disassembled or denatured, it is preferable. Specifically, heating in the range of 30 ° C to 80 ° C is preferable.

As for the direction of energy irradiation performed through the photomask in this embodiment, when the above-mentioned base material is transparent, energy irradiation can be performed in either direction of a base material side and the photocatalyst containing cell adhesion layer side. On the other hand, when a base material is opaque, it is necessary to perform energy irradiation from the photocatalyst containing cell adhesion layer side.

(2) Second Embodiment

Below, the cell adhesive layer containing at least the cell adhesive material is formed on the photocatalyst treatment layer containing at least the photocatalyst, and when energy is irradiated to the cell adhesive layer, the cell adhesive material in the cell adhesive layer is adjacent to the photocatalyst treatment layer. The case where it decomposes or denatures by the action of the photocatalyst in the inside is demonstrated.

In the present embodiment, since the cell adhesion layer is formed on the photocatalytic treatment layer by irradiating energy in a pattern to form a cell adhesion assistant, the cell adhesion material in the cell adhesion layer is decomposed by the action of the photocatalyst in the adjacent photocatalytic treatment layer or It can denature | denaturate and reduce adhesiveness with the cell of the area | region, and can use it as a cell adhesion | attachment assistance part. In this case, for example, when the cell adhesive material is decomposed by the action of the photocatalyst due to energy irradiation, the cell adhesion assistant contains a small amount of the cell adhesive material, or a degradation product of the cell adhesive material, or the cell. The adhesive layer is completely decomposed and removed to expose the photocatalyst treatment layer. In addition, when the cell adhesive material is denatured by the action of the photocatalyst due to energy irradiation, the modified cell or the like is contained in the cell adhesion assistant.

Hereinafter, the cell adhesion layer and photocatalyst treatment layer used for this embodiment are demonstrated. In addition, since the formation method of the cell adhesion | attachment auxiliary part in this embodiment is the same as that of 1st Embodiment mentioned above, description here is abbreviate | omitted.

a. Cell adhesion layer

First, the cell adhesion layer used for this embodiment is demonstrated. The cell adhesion layer used in the present embodiment is a layer having a cell adhesion material having at least adhesion with the cells, and in general, a layer used as a layer having adhesion with the cells can be used.

As a specific cell adhesion material, the same thing as the cell adhesion material used for the photocatalyst containing cell adhesion layer demonstrated in 1st Embodiment can be used, A detailed description here is abbreviate | omitted. Moreover, it is preferable that the cell adhesion layer of this embodiment also contains the material which has the cell adhesion inhibitory property demonstrated by the photocatalyst containing cell adhesion layer of 1st Embodiment. This is because the adhesiveness with the cells of the cell adhesion assistant that is the energy irradiated region can be lowered.

In addition, formation of such a cell adhesion layer can be performed by apply | coating the coating liquid for cell adhesion layer formation containing the said cell adhesion material by a general application | coating method, etc., and the formation method of the photocatalyst containing cell adhesion layer of 1st Embodiment. Since it may be the same as, the description thereof is omitted.

In addition, the film thickness of the cell adhesion layer is appropriately selected depending on the type of the cell culture patterning substrate or the like, but may generally be about 0.001 μm to 1.0 μm, particularly about 0.005 μm to 0.3 μm.

b. Photocatalyst treatment layer

Below, the photocatalyst treatment layer used for this embodiment is demonstrated. The photocatalyst treated layer used in the present embodiment is not particularly limited as long as it is a layer containing at least a photocatalyst, and may be a layer composed of only a photocatalyst, or a layer containing other components such as a binder.

The photocatalyst used in the present embodiment may be the same as that used for the photocatalyst-containing cell adhesion layer in the first embodiment, and titanium oxide is particularly preferably used in the present embodiment.

Here, when using the photocatalyst treatment layer which consists only of a photocatalyst, the efficiency regarding disassembly or denaturation of the cell adhesive material in the said cell adhesion layer improves, and it is advantageous in terms of cost, such as shortening of processing time. On the other hand, in the case of using a photocatalyst treatment layer composed of a photocatalyst and a binder, it has the advantage that the formation of the photocatalyst treatment layer is easy.

As a formation method of the photocatalyst treatment layer which consists only of a photocatalyst, the method of using vacuum film forming methods, such as a sputtering method, a CVD method, and a vacuum deposition method, is mentioned, for example. Since the photocatalytic treatment layer is formed by the vacuum film forming method, the photocatalyst treatment layer can be made into a uniform film and contain only the photocatalyst, whereby the cell adhesive material can be uniformly decomposed or denatured, and the photocatalyst is made of only the photocatalyst. In comparison with the case where a binder is used, the cell adhesion material can be decomposed or denatured efficiently.

As another example of the method for forming a photocatalyst treatment layer composed solely of a photocatalyst, for example, when the photocatalyst is titanium dioxide, amorphous titania is formed on a substrate, and then a phase change is made to crystalline titania by firing. have. Examples of the amorphous titania used herein include hydrolysis, dehydration and condensation of inorganic salts of titanium such as titanium tetrachloride and titanium sulfate, tetraethoxy titanium, tetraisopropoxy titanium, tetra-n-propoxy titanium, tetrabutoxy Organic titanium compounds, such as titanium and tetramethoxy titanium, can be obtained by hydrolysis and dehydration condensation in the presence of an acid. Subsequently, it can be modified to anatase titania by firing at 400 ° C to 500 ° C, and to rutile titania by firing at 600 ° C to 700 ° C.

In the case of using a binder, it is preferable that the main skeleton of the binder has a high binding energy that is not decomposed by the photoexcitation of the photocatalyst. For example, as the binder, the organopolysiloxane described in the item of the cell adhesion layer may be used. Can be heard

As described above, in the case of using the organopolysiloxane as a binder, the photocatalyst treatment layer may be formed by dispersing the organopolysiloxane, which is a photocatalyst and a binder, in a solvent simultaneously with other additives to prepare a coating liquid, and applying the coating liquid onto a substrate. Can be. As a solvent to be used, alcohol-based organic solvents, such as ethanol and isopropanol, are preferable. Coating can be performed by well-known coating methods, such as spin coating, spray coating, dip coating, roll coating, and bead coating. When it contains the ultraviolet curable component as a binder, a photocatalyst treatment layer can be formed by irradiating an ultraviolet-ray and hardening process.

It is also possible to use amorphous silica precursors as binders. This amorphous silica precursor is represented by the chemical formula SiX ₄ , and X is preferably 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.

Specifically, tetraethoxysilane, tetraisopropoxysilane, tetra-n-propoxysilane, tetrabutoxysilane, tetramethoxysilane, etc. are mentioned. In this case, the precursor of amorphous silica and the particles of the photocatalyst are uniformly dispersed in a non-aqueous solvent, hydrolyzed by moisture in the air on a transparent substrate, and silanol is formed, followed by dehydration condensation polymerization at room temperature. A treatment layer can be formed. The dehydration polycondensation of silanol can be performed at 100 ° C. or higher to increase the degree of polymerization of silanol and to improve the strength of the membrane surface. In addition, these binders can be used individually or in mixture of 2 or more types.

Content of the photocatalyst in a photocatalyst treatment layer can be set to 5-60 weight%, Preferably it is 20-40 weight%. In addition, the thickness of the photocatalyst treatment layer is preferably in the range of 0.05 to 10 μm.

In addition, the photocatalyst treatment layer may contain, in addition to the photocatalyst and the binder, a surfactant used for the cell adhesion layer described above.

Here, in this embodiment, the surface of the photocatalyst treatment layer is preferably low in adhesion to cells, for example, in that the surface is hydrophilic. This is because when the cell adhesion layer is decomposed and the photocatalytic treatment layer is exposed, the region can be a region with low adhesion to the cells.

In addition, in the present embodiment, as described above, a light shielding portion may be formed on the photocatalyst treatment layer. Accordingly, when energy is irradiated on the entire surface of the cell adhesive layer, the photocatalyst on the region where the light shielding portion is formed is not excited, and the cell adhesive material contained in the cell adhesive layer other than the region where the light shielding portion is formed can be decomposed or denatured. to be. In addition, in this case, since the photocatalyst of the area | region in which the light shielding part is formed is not excited, it has the advantage that the direction to which energy is irradiated is not specifically limited.

Since the same thing as what was demonstrated in 1st Embodiment can be used as such a light shielding part, detailed description here is abbreviate | omitted.

(3) Third Embodiment

Below, a cell adhesive layer containing at least a cell adhesive material is formed on a substrate, and when the energy is irradiated, the cell adhesive material is opposed by opposing a photocatalyst-containing layer containing at least a photocatalyst with the cell adhesive layer and irradiating energy. The case where it decomposes or denatures by the action of the photocatalyst in a photocatalyst containing layer is demonstrated.

In this embodiment, the cell adhesion material in the cell adhesion layer is decomposed or decomposed by the action of the photocatalyst in the photocatalyst-containing layer by irradiating energy in a pattern to form the cell adhesion layer and the photocatalyst-containing layer so as to face the cell adhesion layer. It can be denatured to form cell adhesion aids.

Hereinafter, the method of forming a cell adhesion | attachment auxiliary part using the photocatalyst containing layer side board | substrate used for this embodiment and this photocatalyst containing layer side board | substrate is demonstrated. In addition, since the cell adhesion layer used for this embodiment is the same as that of the cell adhesion layer used by 2nd Embodiment mentioned above, description here is abbreviate | omitted.

a. Photocatalyst-containing layer side substrate

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

(i) Photocatalyst-containing layer

First, the photocatalyst-containing layer used for the photocatalyst-containing layer side substrate will be described. The photocatalyst-containing layer used in the present embodiment is not particularly limited as long as the photocatalyst in the photocatalyst-containing layer decomposes or denatures the cell adhesive material in the adjacent cell adhesion layer, and may be made of a photocatalyst and a binder, or may be formed only with a photocatalyst. have. In addition, the properties of the surface may be particularly lyophilic or repellent to liquid.

The photocatalyst-containing layer used in this embodiment may be formed on the entire surface on the substrate, but for example, as shown in FIG. 7, the photocatalyst-containing layer 12 may be formed on the substrate 11 on the pattern. .

Thus, by forming the photocatalyst-containing layer in a pattern form, it is not necessary to perform a pattern irradiation using a photomask or the like when irradiating energy to form a cell adhesion assistant, and irradiates the whole surface to thereby form cell adhesion contained in the cell adhesion layer. The material may form a degraded or denatured cell culture aid.

Although the patterning method of this photocatalyst containing layer is not specifically limited, For example, it can carry out by the photolithographic method etc ..

Further, since only the portion on the cell adhesion layer that actually faces the photocatalyst-containing layer decomposes or denatures the cell adhesive material, the direction of energy irradiation can be irradiated in any direction if energy is irradiated on the portion that faces the photocatalyst-containing layer and the cell adhesion layer. In addition, the energy irradiated thereon also has the advantage that it is not particularly limited to parallel ones such as parallel light.

Here, about the photocatalyst-containing layer used by this embodiment, since the same thing as the photocatalyst treatment layer demonstrated in 2nd Embodiment mentioned above can be used, the detailed description here is abbreviate | omitted.

(ii) gas

Below, the base used for a photocatalyst containing layer side board | substrate is demonstrated. Usually, the photocatalyst-containing layer-side substrate has at least a substrate and a photocatalyst-containing layer formed on the substrate. At this time, the material constituting the substrate to be used is appropriately selected depending on the direction of irradiation of energy to be described later, or whether the resulting pattern forming body needs transparency.

In addition, the gas used for this embodiment may be flexible, for example, a resin film, etc., may not have flexibility, or may be a glass substrate, for example. This is appropriately selected by the energy irradiation method.

In addition, in order to improve the adhesion between the surface of the substrate and the photocatalyst-containing layer, it is possible to form an anchor layer on the substrate. As such an anchor layer, a silane type, a titanium coupling agent, etc. are mentioned, for example.

(iii) Photocatalyst-containing layer side light shielding portion

As the photocatalyst-containing layer side substrate used in the present embodiment, a photocatalyst-containing layer side light shielding portion formed in a pattern may be used. By using the photocatalyst-containing layer side substrate having the photocatalyst-containing layer side light shielding portion in this manner, it is not necessary to use a photomask or to perform drawing irradiation by laser light in energy irradiation. Therefore, since the alignment between the photocatalyst-containing layer side substrate and the photomask is unnecessary, a simple process can be performed, and an expensive device necessary for drawing irradiation is also unnecessary, which is advantageous in terms of cost.

The photocatalyst-containing layer side substrate having such a photocatalyst-containing layer side light-shielding portion can be made into the following two embodiments by the position at which the photocatalyst-containing layer side light-shielding portion is formed.

One forms a photocatalyst-containing layer side light shielding portion 14 on the substrate 11 and forms a photocatalyst-containing layer 12 on the photocatalyst-containing layer side shielding portions 14, for example, as shown in FIG. It is embodiment made into the photocatalyst containing layer side board | substrate. In another embodiment, as shown in FIG. 9, the photocatalyst-containing layer 12 is formed on the substrate 11, and the photocatalyst-containing layer side light shielding portion 14 is formed thereon to form the photocatalyst-containing layer-side substrate. to be.

In any of the embodiments, the photocatalyst-containing layer side light shielding portion is disposed in the vicinity of an arrangement portion between the photocatalyst-containing layer and the cell adhesion layer, compared with the case of using a photomask, and thus, the influence of energy scattering in the gas or the like. Can be reduced, and the pattern irradiation of energy can be performed very accurately.

Here, in this embodiment, when the photocatalyst-containing layer side light shielding portion 14 is formed on the photocatalyst-containing layer 12 as shown in FIG. 9, when the photocatalyst-containing layer and the cell adhesive layer are disposed at a predetermined position, By matching the film thickness of the photocatalyst-containing layer side light-shielding portion with the width between the two layers, the photocatalyst-containing layer-side light shielding portion can be used as a spacer for keeping the gap constant.

That is, when arranging the photocatalyst-containing layer and the cell adhesive layer in a state where the photocatalyst-containing layer and the cell adhesive layer face each other in a predetermined gap, the predetermined gap can be precisely arranged by arranging the photocatalyst-containing layer side light shielding portion and the cell adhesive layer in close contact. By irradiating the energy with the furnace, the cell adhesion layer of the portion where the cell adhesion layer and the light shielding portion are in contact with each other does not decompose or denature the cell adhesion material, so that the accuracy of the cell adhesion assistant can be improved.

The formation method of such a photocatalyst containing layer side light shielding part is not specifically limited, According to the characteristic of the formation surface of a photocatalyst containing layer side light shielding part, the shielding against energy required, etc., it is suitably selected and used and it demonstrated in 1st Embodiment. Since it can be made the same as the light shielding part provided in the image, detailed description here is abbreviate | omitted.

In addition, although two cases of the photocatalyst-containing layer-side light shielding portion are formed between the substrate and the photocatalyst-containing layer and the surface of the photocatalyst-containing layer, the surface of the side on which the photocatalyst-containing layer is not formed is described above. The embodiment which forms a photocatalyst containing layer side light shielding part in can also be employ | adopted. In this embodiment, for example, a case where the photomask is brought into close contact with the extent to which the surface is detachable and the like can be considered, and when the pattern of the cell adhesion aid is changed to a small lot, it can be preferably used.

(iv) primer layer

Below, the primer layer used for the photocatalyst-containing layer side substrate of this embodiment is demonstrated. In the present embodiment, in the case where the photocatalyst-containing layer side light-shielding portion is formed in a pattern shape on the substrate as described above, and the photocatalyst-containing layer is formed thereon to form a photocatalyst-containing layer side substrate, the photocatalyst-containing layer side light-shielding portion and the photocatalyst-containing layer and You may form a primer layer in between.

Although the action and function of this primer layer are not necessarily clear, by forming a primer layer between the photocatalyst-containing layer side light shielding portion and the photocatalyst-containing layer, the primer layer is a factor that inhibits decomposition or denaturation of cell adhesive material by the action of the photocatalyst. Impurity in the openings between the photocatalyst-containing layer side light-shielding portion and the photocatalyst-containing layer-side light shielding portion, in particular, a function of preventing the diffusion of impurities such as residues, metals and metal ions generated when patterning the photocatalyst-containing layer-side light shielding portion. I think. Therefore, by forming the primer layer, the decomposition or denaturation treatment of the cell adhesive material proceeds with high sensitivity, and as a result, a highly accurate cell culture assistant can be obtained.

In addition, in this embodiment, since the primer layer prevents the impurity which exists in not only the photocatalyst containing layer side light shielding part but also the opening formed between the photocatalyst containing layer side light shielding part, affecting the action | action of a photocatalyst, a primer layer contains an opening part. It is preferable that it is formed over the whole surface of the photocatalyst containing layer side light shielding part.

The primer layer in this embodiment will not be specifically limited if it is a structure in which the primer layer was formed so that it might not contact with the photocatalyst containing layer side light shielding part of a photocatalyst containing layer side substrate, and a photocatalyst containing layer.

Although it does not specifically limit as a material which comprises this primer layer, The inorganic material which is hard to decompose by the action of a photocatalyst is preferable. Specifically, amorphous silica is mentioned. In the case of using such amorphous silica, the precursor of the amorphous silica is represented by the chemical formula SiX ₄ , X is a silicon compound such as halogen, methoxy group, ethoxy group, or acetyl group, silanol which is a hydrolyzate thereof, or an average molecular weight of 3000 or less Polysiloxanes are preferred.

In addition, the film thickness of the primer layer is preferably in the range of 0.001 µm to 1 µm, and particularly preferably in the range of 0.001 µm to 0.1 µm.

b. How to Form Cell Adhesion Auxiliaries

Below, the formation method of the cell adhesion | attachment auxiliary part in this embodiment is demonstrated. In this embodiment, as shown, for example in FIG. 10, the cell adhesion layer 7 and the photocatalyst-containing layer 12 of the photocatalyst-containing layer side board | substrate 13 are arrange | positioned at predetermined gaps, For example, a photomask ( 5) etc., the energy 6 is irradiated in a predetermined direction. As a result, the cell adhesion material in the energy-irradiated region is decomposed or denatured, and a cell adhesion assistant 4 which inhibits adhesion with the cells is formed in the cell adhesion layer 7. At this time, when the cell adhesion material is decomposed by the action of the photocatalyst due to energy irradiation, for example, the cell adhesion aid contains a small amount of the cell adhesion material, or a degradation product of the cell adhesion material. Or the cell adhesion layer is completely decomposed and removed to expose the substrate. In addition, when the cell adhesive material is denatured by the action of the photocatalyst due to energy irradiation, the denatured substance or the like is contained in the cell culture assistant.

The arrangement refers to a state in which the action of the photocatalyst is substantially applied to the surface of the cell adhesion layer, and the photocatalyst-containing layer and the cell adhesion are arranged in physical contact with each other, with a predetermined interval therebetween. . It is preferable that the space | interval is 200 micrometers or less.

In the present embodiment, the gap is particularly preferably within the range of 0.2 μm to 10 μm, considering that the pattern precision is very good, the sensitivity of the photocatalyst is high, and therefore the efficiency of decomposition or denaturation of the cell adhesive material in the cell adhesive layer is good. Preferably within the range of 1 μm to 5 μm. Such a range of gaps is particularly effective for small area cell adhesion layers that can control the gap with high precision.

On the other hand, in the case where the treatment is carried out for a large area cell adhesion layer of 300 mm × 300 mm or more, for example, it is very difficult to form a fine gap without contact between the photocatalyst-containing layer side substrate and the cell adhesion layer. Therefore, when the cell adhesion layer is relatively large, it is preferable that the gap is in the range of 10 to 100 µm, particularly in the range of 50 to 75 µm. By setting the gaps within these ranges, there is no problem of deterioration of pattern accuracy such as blurring of the pattern, deterioration of the efficiency of decomposing or denaturing the cell adhesive material due to deterioration of the sensitivity of the photocatalyst, and decomposing of the cell adhesive material or It has the effect that spotting does not occur in degeneration.

Thus, when energy irradiation of the cell adhesion layer of a comparatively large area, setting of the clearance gap in the positioning device of the photocatalyst-containing layer side substrate and cell adhesion layer in an energy irradiation apparatus is in the range of 10 micrometers-200 micrometers, especially 25 micrometers-75 micrometers It is preferable to set in the micrometer range. This is because by setting the set value in such a range, it is possible to arrange the photocatalyst-containing layer side substrate and the cell adhesion layer without causing a significant reduction in pattern accuracy and a significant deterioration in the sensitivity of the photocatalyst.

By separating and arranging the surface of the photocatalyst-containing layer and the cell adhesion layer at predetermined intervals, active oxygen species generated by oxygen, water and the photocatalytic action are easily desorbed. In other words, when the interval between the photocatalyst-containing layer and the cell adhesion layer is made narrower than the above range, the desorption of the active oxygen species becomes difficult, and as a result, the rate of decomposition or denaturation of the cell adhesion material is likely to be slow. In the case where the intervals are separated from each other in the above range, the generated active oxygen species is less likely to reach the cell adhesion layer, and in this case, the decomposition or denaturation of the cell adhesive material may be slowed, which is not preferable.

As a method of forming such a very narrow gap uniformly and arranging the photocatalyst-containing layer and the cell adhesive 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 at the same time, the portion of the spacer contacting the photocatalyst does not occur on the surface of the cell adhesion layer, and the spacer has the same pattern as the cell adhesion portion described above. The cell adhesion material can be decomposed or denatured only in the portion where the spacer is not formed, and the cell adhesion assistant can be formed with high precision. In addition, by using such a spacer, the active oxygen species produced by the action of the photocatalyst does not diffuse and reaches the surface of the cell adhesion layer at a high concentration, thereby efficiently forming a highly precise cell adhesion assistant.

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

Here, energy irradiation (exposure) as used in this embodiment is a concept including the irradiation of any energy ray which can decompose or denature the cell adhesive material by the action of the photocatalyst according to the energy irradiation, and is not limited to the irradiation of light. Do not.

Here, since the kind etc. of energy irradiated in this embodiment are the same as that of what was demonstrated in 1st embodiment mentioned above, detailed description here is abbreviate | omitted.

In addition, as for the direction of energy irradiation performed through the photomask in this embodiment, when the above-mentioned base material is transparent, energy irradiation can be performed in either direction of a base material side and a photocatalyst containing layer side substrate. On the other hand, when the substrate is opaque, it is necessary to perform energy irradiation on the photocatalyst-containing layer side substrate side.

II. In the case of (2)

In the following, the cell adhesion layer has a cell adhesion inhibitory effect that inhibits adhesion to cells, and after forming a cell adhesion inhibitory layer containing a cell adhesion inhibitory material which is degraded or denatured by the action of a photocatalyst according to energy irradiation, The case of forming by decomposing or denaturing a cell adhesion inhibitory material by energy irradiation is demonstrated. In this case, the following three embodiments are mentioned.

The first embodiment is a pattern in which the cell adhesion inhibitory layer is a photocatalyst-containing cell adhesion inhibitory layer containing a cell adhesion inhibitory material and a photocatalyst that inhibits adhesion to cells, and forms a cell adhesion layer on the photocatalyst-containing cell adhesion inhibition layer. This is a case where the cell adhesion layer is decomposed or denatured by the action of the photocatalyst contained in the photocatalyst-containing cell adhesion inhibitory layer itself by irradiating energy with the type.

In the second embodiment, a cell adhesion inhibiting layer containing at least a cell adhesion inhibiting material is formed on a photocatalytic treatment layer containing at least a photocatalyst, and energy is applied in a pattern to form a cell adhesion layer on the cell adhesion inhibiting layer. This is the case where the cell adhesion inhibitory material is decomposed or denatured by the action of the photocatalyst contained in the photocatalyst treatment layer.

In a third embodiment, a cell adhesion inhibitory layer containing at least a cell adhesion inhibitory material is formed on a substrate, and the cell adhesion inhibitory layer and a photocatalyst-containing layer or the like containing at least a photocatalyst are opposed to the cell adhesion inhibitory layer, It is a case where a cell adhesion inhibitory material turns into a decomposed or denatured cell adhesion layer by irradiating energy in the pattern form which forms a cell adhesion layer.

Hereinafter, each embodiment is divided and demonstrated.

(1) First Embodiment

First, the cell adhesion inhibitory layer is a photocatalyst-containing cell adhesion inhibitory layer containing a cell adhesion inhibitory material and a photocatalyst that inhibits adhesion to cells, and the energy is irradiated in a pattern to form a cell adhesion layer on the photocatalyst-containing cell adhesion layer. The case where the cell adhesion inhibiting material is decomposed or denatured by the action of the photocatalyst contained in the photocatalyst treatment layer to form a cell adhesion layer will be described.

According to the present embodiment, the photocatalyst-containing cell adhesion inhibitory layer contains a photocatalyst and the cell adhesion inhibitory material, and energy is irradiated to the photocatalyst-containing cell adhesion inhibitory layer to decompose or denature the cell adhesion inhibitory material by the action of the photocatalyst. The area to which energy is irradiated can be made into the cell adhesion part which has adhesiveness with a cell, ie, a cell adhesion layer. In addition, the area | region where energy is not irradiated can be used as a cell adhesion | attachment assistance part at this time.

The formation of such a photocatalyst-containing cell adhesion inhibitory layer may be performed by applying a coating solution for forming a cell-catalyst inhibitory layer containing a photocatalyst-containing cell adhesion material and a photocatalyst containing a cell adhesion material and photocatalyst decomposed or modified by the action of a photocatalyst upon energy irradiation, and the like. This can be done by. The coating solution for forming the photocatalyst-containing cell adhesion inhibiting layer may be applied using a general coating method, and for example, spin coating, spray coating, dip coating, roll coating, bead coating, or the like may be used. have.

At this time, the film thickness of the photocatalyst-containing cell adhesion inhibitory layer is appropriately selected depending on the type of cell culture patterning substrate, etc., but may generally be about 0.01 μm to 1.0 μm, particularly about 0.1 μm to 0.3 μm.

Hereinafter, the cell adhesion inhibitory material will be described, and the method for forming the cell adhesion layer will be described. In addition, regarding the photocatalyst used for this embodiment, "I. Since it may be the same as the photocatalyst used by 1st aspect of "the case of (1)", detailed description here is abbreviate | omitted.

a. Cell adhesion inhibitory material

First, the cell adhesion inhibiting material contained in the photocatalyst-containing cell adhesion inhibiting layer used in the present embodiment will be described.

The cell adhesion inhibitory material used in the present embodiment is not particularly limited as long as it has a cell adhesion inhibitory property that inhibits adhesion to cells, and is degraded or denatured by the action of a photocatalyst according to energy irradiation.

Herein, the cell having the cell adhesion inhibitory property refers to a property of inhibiting adhesion of the cell adhesion inhibitory material with the cell adhesion inhibitory material. It has a property to inhibit.

The cell adhesion inhibitory material used in the present embodiment has such cell adhesion inhibitory properties, and is degraded or denatured by the action of a photocatalyst according to energy irradiation, resulting in loss of cell adhesion inhibition or good adhesion to cells. Is used.

As the cell adhesion inhibiting material, for example, a material having high hydration ability can be used. A material having high water hydration is formed with a hydration layer in which moisture is collected. Usually, such a high water hydration material has higher adhesion to water than cells, so that cells can adhere to the high water hydration material. There is no adhesion with cells. Here, the said hydration ability means the property which hydrates with a moisture, and "high hydration ability" means that it is easy to hydrate with a moisture.

Examples of the material used as the cell adhesion inhibitory material having high hydration ability include polyethylene glycol, a zwitterionic material having a betaine structure, and the like, and a phospholipid-containing material. When such a material is used as the cell adhesion inhibiting material, when energy is irradiated in an energy irradiation step to be described later, the cell adhesion inhibiting material is decomposed or altered by a photocatalytic action, and the surface hydration layer is removed to remove the cell adhesion. Inhibitors may be lost.

In the present embodiment, a surfactant having a water-repellent or oil-repellent organic substituent decomposed by the action of a photocatalyst can also be used as the cell adhesion inhibiting material. As such a surfactant, For example, hydrocarbon type, such as each series of Nikko Chemicals, Ltd. NIKKOL BL, BC, BO, BB, ZONYL FSN, FSO by Dupont, Saflon S-made by Asahi Glass Co., Ltd. 141, 145, Megapack F-141, 144 made by Dai Nippon Ink Chemical Industries, Ltd., Psentant F-200, F-251, manufactured by Neos Co., Ltd. 402, 3M Co., Ltd. fluorine-type or silicone type nonionic surfactant, such as Floyd FC-170 and 176, A cationic surfactant, anionic surfactant, and amphoteric surfactant can be used.

When such a material is used as a cell adhesion inhibiting material to form a photocatalyst-containing cell adhesion inhibiting layer, the cell adhesion inhibiting material is ubiquitous on the surface. As a result, the water repellency or oil repellency of the surface can be enhanced, the interaction with the cells is small, and the adhesion with the cells can be lowered. In addition, when energy is irradiated to the layer in the energy irradiation step, the photocatalyst can be easily decomposed by the action of a photocatalyst to expose the photocatalyst, thereby preventing the cell adhesion inhibition.

In this embodiment, it is particularly preferable to use the cell adhesion inhibiting material as the cell adhesion inhibiting material having good adhesion to cells by the action of a photocatalyst according to energy irradiation. For example, as the cell adhesion inhibiting material, oil repellency or water repellency is used. The material which has a is mentioned.

As a cell adhesion inhibiting material, in the case of using the material having water repellency or oil repellency, due to the water repellency or oil repellency of the cell adhesion inhibiting material, the interaction between the cell and the cell adhesion inhibiting material, for example, hydrophobic interaction, is small. The adhesion with the cells can be lowered.

As such a material having water repellency or oil repellency, for example, the skeleton has a high binding energy that is not decomposed by the operation of the photocatalyst, and has a water-repellent or oil repellent organic substituent decomposed by the action of the photocatalyst. Can be mentioned.

As the skeleton having a high binding energy that is not decomposed by the action of the photocatalyst, and having a water-repellent or oil-repellent organic substituent decomposed by the action of the photocatalyst, for example, the above-mentioned "I. In the case of (1), organic polysiloxanes exhibiting high strength by hydrolyzing and polycondensing chloro or alkoxysilanes by (1) sol-gel reaction or the like, and (2) organopolysiloxanes crosslinked with reactive silicones Can be.

Such substances are described in I. In the case of (1), when used as a binder, the side chains of the organopolysiloxane and the like are decomposed or denatured at a high rate by the action of a photocatalyst according to energy irradiation, thereby making them superhydrophilic. It is used as a material which has.

On the other hand, in the present embodiment, when used as a cell adhesion inhibitory material, the side chains of the organopolysiloxane and the like are irradiated with energy to such an extent that they are not completely decomposed or denatured by the action of the photocatalyst due to energy irradiation. The regions can be adhered to the cells.

Here, when the material is used as a cell adhesion inhibiting material, it is usually preferable to use a material having a contact angle with water of 80 ° or more, particularly 100 ° to 130 °, as the cell adhesion inhibiting material. This is because the adhesiveness with the cells can be lowered. In addition, the upper limit of the said angle is an upper limit of the contact angle of the cell adhesion inhibitory material with water on a flat base material, for example, when measuring the contact angle with the water of the said cell adhesion inhibitory material on the base material which has an unevenness | corrugation, For example, as shown in Japanese Journal of Applied Physics, Part 2, Vol. 32, L614-L615, 1993 Ogawa et al., The upper limit is sometimes about 160 °.

In addition, it is preferable to irradiate energy on this cell adhesion inhibitory material and to irradiate energy so that the contact angle with water is in the range of 10 ° to 40 °, particularly 15 ° to 30 °, in the case of having adhesiveness with the cells. . This is because the adhesiveness with the cells can be improved. In addition, the contact angle with the said water can be obtained by the method mentioned above.

At the same time as the organopolysiloxane and the like, a stable organosilicon compound which does not undergo a crosslinking reaction such as dimethylpolysiloxane may be mixed separately.

By using the said reactive silicone, water repellency and oil repellency can be improved, interaction with a cell is small and adhesiveness with a cell can be reduced. In addition, when energy is irradiated to the material, the substituents can be easily removed to introduce OH groups or the like into the surface, and the interaction with the cells can be increased, thereby improving adhesion to the cells. have.

Such cell adhesion inhibitory material is preferably contained in the range of 0.01 wt% to 95 wt%, particularly 1 wt% to 10 wt%, in the photocatalyst-containing cell adhesion inhibitory layer. It is because the area | region containing a cell adhesion inhibitory material can be set as the area | region with low adhesiveness with a cell by this.

Moreover, it is preferable that the said cell adhesion inhibitory material has surfactant. For example, after coating the coating liquid for forming a photocatalyst-containing cell adhesion inhibiting layer containing the cell adhesion inhibitory material or the like, and then drying, the ratio of ubiquitous on the surface of the coating film becomes high, resulting in good cell adhesion inhibition. Because you can get.

b. Etc

In addition, the photocatalyst-containing cell adhesion inhibiting layer of the present embodiment may contain a binder or the like depending on the properties required, such as coating property when forming a layer, strength and resistance when forming a layer, or the like. . In addition, the cell adhesion inhibitory material may function as the binder.

As such a binder, for example, one having a high binding energy in which the main skeleton is not decomposed by the action of the photocatalyst can be used. Specifically, the polysiloxane etc. which do not have an organic substituent or the organic substituent of the grade which does not affect adhesiveness are mentioned, These can be obtained by hydrolyzing and polycondensing tetramethoxysilane, tetraethoxysilane, etc.

In this embodiment, the binder is preferably contained in the photocatalyst-containing cell adhesion inhibiting layer in the range of 5% to 95% by weight, in particular 40% to 90% by weight, more particularly 60% to 80% by weight. This is because the formation of the photocatalyst-containing cell adhesion inhibitory layer can be facilitated, or the strength can be imparted to the photocatalyst-containing cell adhesion inhibitory layer.

Moreover, in this embodiment, it is preferable that the cell adhesion material which has adhesiveness with a cell is contained especially at least after energy irradiation in the said photocatalyst containing cell adhesion inhibitory layer. This is because the photocatalyst-containing cell adhesion inhibiting layer can make the adhesion to the cells of the cell adhesion portion, that is, the cell adhesion layer, which is a region to which energy is irradiated, better. Such cell adhesive material may be used as the binder, or may be used separately from the binder. In addition, for example, it may have good adhesion with the cells before being irradiated with energy, and may have good adhesion with the cells by the action of the photocatalyst according to the energy irradiation. Here, it means that the cell adheres well to the cells having adhesiveness, and in the case where the adhesion with the cells differs depending on the type of the cell, it means that the cells adhere well to the target cell.

In the present embodiment, at least after energy irradiation, if the cell adhesion material has good adhesion with the cells, the adhesion with the cells is, for example, hydrophobic interaction, electrostatic interaction, hydrogen bonding, van der Waals, etc. It may be good by the physical interaction of, or may be good by the biological properties.

In this embodiment, it is preferable that such a cell adhesion material is contained in 0.01 weight%-95 weight%, especially 1 weight%-10 weight% in a photocatalyst containing cell adhesion inhibitory layer. This is because the photocatalyst-containing cell adhesion inhibitory layer can make the adhesion of the cell adhesion layer, which is the energy irradiated region, with the cells more favorable. In addition, in the case of using a material having good adhesion to cells before being irradiated with energy as a cell adhesion material, it does not inhibit the cell adhesion inhibition of the cell adhesion inhibitory material in a region where energy is not irradiated, that is, a region that becomes a cell adhesion assistant. It is preferable to be contained to such an extent.

c. Formation method of cell adhesion layer

Below, the formation method of a cell adhesion layer is demonstrated. In this embodiment, for example, as shown in FIG. 11, the photocatalyst-containing cell adhesion inhibitory layer 8 containing the cell adhesion inhibitory material and the photocatalyst formed on the cell culture region on the substrate 1 is, for example, The energy 6 is irradiated in a pattern form to form a cell adhesion layer (cell adhesion part) using a photomask 5 or the like (Fig. 11 (a)). Accordingly, the energy irradiated region obtains the cell adhesion layer (cell adhesion portion) 7 having the cell adhesion inhibiting material decomposed or denatured and adhered to the cells, while the unirradiated region inhibits adhesion with the cells. The cell adhesion aid 4 can be provided. At this time, the cell adhesion portion contains a photocatalyst and a decomposed product or modified product of a cell adhesion inhibitory material.

Here, the energy irradiation (exposure) mentioned in this embodiment is a concept including irradiation of any energy ray which can decompose or denature cell adhesion inhibitory material by the action of a photocatalyst according to energy irradiation, It is not limited.

In addition, regarding the method of such an energy irradiation, etc. mentioned above "I. Since it is the same as that described in 1st Embodiment of "the case of (1), detailed description here is abbreviate | omitted.

(2) Second Embodiment

Below, the cell adhesion inhibitory layer containing at least the cell adhesion inhibitory material is formed on the photocatalyst treatment layer containing at least a photocatalyst, and forms a cell adhesion layer, The cell adhesion inhibitory material decomposes | disassembles by irradiating energy. Or the case where it is set as the cell adhesion layer denatured is demonstrated.

In the present embodiment, since the cell adhesion inhibitory layer is formed on the photocatalytic treatment layer, by irradiating the cell adhesion inhibition layer with energy, the photocatalyst contained in the photocatalytic treatment layer is excited to inhibit cell adhesion in the cell adhesion inhibition layer. The material can be decomposed or denatured to form a cell adhesion part (cell adhesion layer). In addition, the area | region where an energy is not irradiated and a cell adhesion inhibitory material remain | survive can be used as a cell adhesion | attachment assistance part at this time.

Here, the amount of the cell adhesion inhibitory material is less than that of the cell adhesion inhibitory material that the cell adhesion inhibitory material is decomposed or denatured, or that is less than the amount of cell adhesion inhibitory material contained in the cell adhesion auxiliary layer. It refers to what is contained. For example, when the cell adhesion inhibitory material is decomposed by the action of a photocatalyst by energy irradiation, the cell adhesion part contains a small amount of the cell adhesion inhibitory material, or a degradation product of the cell adhesion inhibitory material, or the like. to be. In addition, when the cell adhesion inhibitory material is modified by the action of a photocatalyst by energy irradiation, the modified cell or the like is contained in the cell adhesion part. In this embodiment, it is preferable that the said cell adhesion part contains the cell adhesion material which has adhesiveness with a cell after at least energy irradiation. This is because the adhesion with the cells of the cell adhesion part can be further enhanced, and the cells can be adhered with high precision only to the cell adhesion part.

Hereinafter, the cell adhesion inhibitory layer used in the present embodiment will be described. In addition, regarding the photocatalyst treatment layer used for this embodiment, "I. Since it can be used similarly to what was described in 2nd Embodiment of "in the case of (1)", and the formation method of a cell adhesion layer can be the same as that of said 1st Embodiment, description here is abbreviate | omitted.

(Cell adhesion inhibitory layer)

The cell adhesion inhibitory layer used in the present embodiment is formed on the photocatalytic treatment layer, has a cell adhesion inhibitory effect that inhibits adhesion with cells, and inhibits cell adhesion that is degraded or denatured by the action of a photocatalyst according to energy irradiation. If it contains a material, it will not specifically limit.

In this embodiment, if such a layer can be formed, the formation method etc. are not specifically limited, For example, cell culturing the coating liquid for cell adhesion inhibitory layer containing the said cell adhesion inhibitory material by the general application | coating method is carried out. It can form by apply | coating to an area | region. In addition, the film thickness of such a cell adhesion inhibiting layer is appropriately selected depending on the type of the cell culture patterning substrate or the like, but may generally be about 0.001 μm to 1.0 μm, particularly about 0.005 μm to 0.3 μm.

Here, as a specific cell adhesion inhibitory material used for the cell adhesion inhibitory layer formed in this embodiment, the same thing as the cell adhesion inhibitory material used for the photocatalyst containing cell adhesion inhibitory layer demonstrated in 1st Embodiment can be used. , The detailed description is omitted here. Moreover, it is preferable that the cell adhesion inhibitory layer of this embodiment also contains the material which has the cell adhesiveness demonstrated by the photocatalyst containing cell adhesion inhibitory layer demonstrated in 1st Embodiment. This is because the adhesiveness with the cells of the cell adhesion part (cell adhesion layer) which is an energy irradiated area can be improved.

(3) Third Embodiment

Below, a cell adhesion inhibiting layer containing at least a cell adhesion inhibiting material is formed on a substrate, and the cell adhesion layer is made by opposing the cell adhesion inhibiting layer, a photocatalyst containing layer containing at least a photocatalyst, and the like to the cell adhesion inhibiting layer. The case where the cell adhesion inhibitory material is decomposed or denatured is described by irradiating energy in a pattern form to be formed.

In the present embodiment, since the cell adhesion inhibitory layer contains a cell adhesion inhibitory material which is decomposed or denatured by the action of the photocatalyst according to energy irradiation, the cell adhesion inhibitory layer and the photocatalyst-containing layer are disposed to face each other. By irradiating energy in a pattern to form the cell adhesion part, the cell adhesion inhibitory material in the cell adhesion inhibitory layer is decomposed or denatured by the action of the photocatalyst in the cell photocatalyst-containing layer to form a cell adhesion layer (cell adhesion part). have. At this time, since the cell adhesion inhibitory material remains in the region to which the energy is not irradiated, adhesion with the cells can be inhibited and used as the cell adhesion assistant.

Wherein the cell adhesion inhibitory material is not contained or that the cell adhesion inhibitory material is decomposed or denatured, or contains less amount of the cell adhesion inhibitory material as compared to the amount of cell adhesion inhibitory material contained in the cell adhesion auxiliary layer. Say what it is. For example, when the cell adhesion inhibiting material is decomposed by the action of a photocatalyst by energy irradiation, a small amount of the cell adhesion inhibiting material is contained in the cell adhesion part (cell adhesion layer), or a degradation product of the cell adhesion inhibiting material is contained. It becomes. In addition, when the cell adhesion inhibitory material is modified by the action of a photocatalyst by energy irradiation, the modified cell or the like is contained in the cell adhesion part. In this embodiment, it is preferable that the said cell adhesion part contains the cell adhesion material which has adhesiveness with a cell after at least energy irradiation. This is because the adhesion with the cells of the cell adhesion part can be further enhanced, and the cells can be adhered with high precision only to the cell adhesion part.

In addition, the cell adhesion inhibitory layer used for this embodiment is the same as the cell adhesion inhibitory layer demonstrated in the said 2nd Embodiment, and the photocatalyst containing layer side board | substrate and its arrangement are "I. Since it is the same as that described in 3rd Embodiment of "the case of (1), detailed description here is abbreviate | omitted. In addition, since it is the same as that of said 1st Embodiment about the energy irradiation method etc., description here is abbreviate | omitted.

B. Second Embodiment

Next, a second embodiment of the patterning substrate for cell culture of the present invention will be described. A second embodiment of the cell culture patterning substrate of the present invention is a cell culture patterning pattern having a substrate and a cell culture region formed on the substrate, wherein the cell culture region contains a cell adhesion layer having adhesion to the cells. Substrate,

The cell adhesive layer is formed in a pattern shape having an uneven end portion.

The patterning substrate for cell culture in this embodiment has the base material 1 and the cell culture area | region 2 formed on this base material 1, for example, as shown in FIG. An end a of the cell adhesion layer 7 formed in 2) is formed in a pattern having irregularities.

As described above, in the case of attaching the cells to the cell culture region, the cells are regularly arranged according to the shape change from the end, so that tissue is formed. Here, when culturing the cells, the cell morphology changes in the case of attaching the cells along a straight line and in the case of attaching the cells along the uneven line when the cells are attached along the uneven line. Can be activated and arranged regularly. According to the present embodiment, since the end of the cell adhesion layer where the cells start to be arranged is formed in a pattern with irregularities, the cells attached to the end can be activated, and the cells can be arranged regularly well. have.

Hereinafter, each structure of the cell culture patterning board | substrate in this embodiment is demonstrated. In addition, since the description used for this embodiment is the same as that used for the 1st embodiment mentioned above, detailed description here is abbreviate | omitted.

1. Cell Culture Area

First, the cell culture region in the cell culture patterning substrate of the present embodiment will be described. The cell culture region in the cell culture patterning substrate of the present embodiment is not particularly limited as long as the cell culture region is a region for culturing cells and has a cell adhesion layer formed in a pattern form having irregularities at its ends.

Here, a cell culture region is formed on the substrate, and a portion other than the cell culture region of the substrate is a cell non-culture region that inhibits adhesion with the cells. In addition, the edge part of the said cell adhesion layer is a boundary between this cell culture area | region and a cell non-cultivation area | region normally (it represents with a in FIG. 12).

In this embodiment, all the ends of the cell adhesion layer formed in this cell culture region may have irregularities, and as shown in FIG. 12, only some of the ends may have irregularities.

As such, the irregularities formed at the end of the cell adhesion layer are preferably those capable of regularly arranging the cells attached to the cell adhesion layer, and in particular, the distance from the concave end to the convex end adheres the cells to the cell adhesion layer. In this case, the cells are preferably formed in a linearly aligned size.

Although the specific size of the unevenness is appropriately selected depending on the shape of the cultured cell or the like, it is usually preferable that the average distance from the concave end to the convex end of the unevenness is in the range of 0.5 µm to 30 µm, particularly 1 µm to 5 µm. . This is because when the cells are cultured, the cells can be cultured in the desired shape to form tissues without running out of cells at the end of the cell culture region. Here, the measurement of the average distance from the concave end to the convex end of the concave and convex measures the distance from the lowest part to the uppermost part of each concave-convex in the range of 200 μm of the boundary between the cell adhesion part and the cell adhesion auxiliary part. The average is calculated.

The cell adhesion layer having such an end is not particularly limited as long as it is a layer having adhesion with the cells. In addition, the method for forming the cell adhesive layer is not particularly limited as long as it is a method capable of forming the end portion. For example, a coating solution for forming a cell adhesive layer containing a material having adhesiveness with a cell or the like is generally printed. The method of apply | coating by the method, the method of apply | coating the said coating liquid for cell adhesion layer formation, and the patterning by the photolithographic method etc. are mentioned. In this embodiment, as described in the first embodiment, a cell adhesion layer containing a cell adhesive material that is decomposed or denatured by the action of a photocatalyst according to energy irradiation is formed, and the cell adhesion layer is formed by energy irradiation. Can be patterned. In addition, as described in the above first embodiment, a cell adhesion inhibitory layer containing a cell adhesion inhibitory material having a cell adhesion inhibitory property is formed, and the cell adhesion inhibitory material is formed by the action of a photocatalyst according to energy irradiation. May be degraded or denatured to form a cell adhesion layer.

Since these methods, materials, etc. are the same as that of the said 1st Embodiment, detailed description here is abbreviate | omitted.

Here, also in this embodiment, it is preferable that the cell adhesion | attachment auxiliary part demonstrated in 1st Embodiment mentioned above is formed in the cell culture area mentioned above. This is because cells can be efficiently cultured to form large-area tissues or the like.

2. Patterning Substrate for Cell Culture

Below, the patterning substrate for cell culture of this embodiment is demonstrated. The cell culture patterning substrate of the present embodiment is not particularly limited as long as the cell adhesion layer having the end portion is formed on the substrate described above, and a member such as a light shielding portion may be formed, if necessary.

In addition, this invention is not limited to the said embodiment. The said embodiment is an illustration, Any thing which has a structure substantially the same as the technical idea described in the claim of this invention, and exhibits the same effect is contained in the technical scope of this invention.

An Example is shown to the following and this invention is concretely demonstrated to it.

Example 1

<Formation of a photomask having a photocatalyst-containing layer>

A photomask having a line and a space of 60 µm / 300 µm having a width of the opening of 60 µm and a width of the shielding portion of 300 µm, and the boundary between the opening and the shielding portion having an unevenness of 1 µm was formed. .

Below, 5 g of trimethoxymethylsilane TSL8114 (GE Toshiba Silicone Co., Ltd.) and 2.5 g of 0.5N hydrochloric acid were mixed, and it stirred for 8 hours. This was diluted 10-fold with isopropyl alcohol to make a composition for primer layers. The primer layer was formed by apply | coating this composition for primer layers on the pattern surface of a photomask by spin coating, and drying the board | substrate at the temperature of 150 degreeC for 10 minutes.

Below, 30 g of isopropyl alcohol, 3 g of trimethoxymethylsilane TSL8114 (GE Toshiba Silicone Co., Ltd.), and 20 g of photocatalyst inorganic coating agent ST-K03 (Ishihara Industries) were mixed, and it stirred at 100 degreeC for 20 minutes. This was diluted three times with isopropyl alcohol to obtain a composition for a photocatalyst-containing layer. This composition for photocatalyst-containing layers was applied to a photomask substrate on which a primer layer was formed by a spin coater, and dried at 150 ° C. for 10 minutes to form a photomask having a transparent photocatalyst-containing layer.

<Method of manufacturing patterning substrate for cell culture>

(Formation of Cell Adhesion Inhibition Layer)

5.0 g of organosilane TSL-8114 (manufactured by GE Toshiba Silicone Co., Ltd.), 1.5 g of fluoroalkylsilane TSL-8233 (manufactured by GE Toshiba Silicone Co., Ltd.), and 2.36 g of 0.005N hydrochloric acid were mixed and stirred for 24 hours. After diluting this solution 100 times with isopropyl alcohol, it was applied to an alkali-treated quartz substrate by spin coating, and the substrate was dried at a temperature of 150 ° C. for 10 minutes to proceed with hydrolysis and polycondensation reaction. And a substrate having a cell adhesion inhibitory layer having a thickness of 0.2 μm.

(Patterning of Patterning Substrate)

The cell adhesion inhibiting layer of the substrate and the photocatalyst-containing layer of the photomask having the photocatalyst-containing layer are opposed to each other, and ultraviolet light is exposed at an energy amount of 6 J / cm ² by a mercury lamp through the photomask, and the unexposed portion inhibits cell adhesion. And a patterned substrate for cell culture having a cell adhesive surface patterned such that the exposed portion had cell adhesiveness.

(Culture of cells)

The cell culture patterning substrate was immersed in DMEM medium to which 10% fetal bovine serum was added, and primary human thoracic vein cells (HUVECs) were seeded. Incubated for 16 hours at 37 ° C. in a 5% carbon dioxide environment, and the cells adhered to the cell adhesion part.

When the cells adhered to the cell culture substrate were observed, it was confirmed that the cells were oriented in the direction along the entire region of the cell culture region to show the extension shape.

In addition, the DMEM medium was exchanged with the addition of bFGF (Sigma) at a concentration of 10 ng / ml, and culture was continued for 24 hours in a 37 ° C, 5% carbon dioxide environment to confirm that the cells continuously formed capillary tissue. .

Comparative Example 1

The substrate was cultured in the same manner as in Example 1 except that the photomask had a line and a space of 60 µm / 300 µm and did not have irregularities at the boundary between the opening and the shielding portion. It was confirmed that the adhesion of cells to was less than in Example 1. When the culture was carried out until after 24 hours, the number of cells adhered to the substrate was increased, but the orientation of the cells and the degree of progression shape were confirmed to be inferior to those in Example 1.

Furthermore, when bFGF was added to DMEM medium in the same manner as in Example 1, the cells were organized, and capillary formation of the cells was performed. However, compared with Example 1, the capillary length was shorter and the tissue formation was incomplete. .

Example 2

A photomask having a line and a space of 190 μm / 500 μm having a width of the annular opening of 190 μm and a light shielding portion of 500 μm, and having a light shielding portion of 5 μm formed in each of the openings having a width of 60 μm. Cells were cultured in the same manner as in Example 1 except that the cells were used.

When the cell morphology was observed 16 hours after the seeding of the cells, the orientation and extension of the cells were observed for all cells in the cell culture region.

Comparative Example 2

Cells were cultured in the same manner as in Example 2 except that a photomask with a line and space of 190 µm / 500 µm was used. In this case, only 24 hours after the seeding of the cells, only the cells near the center of the cell adhesion part were adhered to the substrate, but the orientation and extension were not confirmed.

Example 3

(Formation of Photocatalyst-Containing Cell Adhesion Layer)

3 g of isopropyl alcohol, 0.4 g of organosilane TSL8114 (manufactured by GE Toshiba Silicone Co., Ltd.), 0.04 g of N- (2-aminoethyl) -3-aminopropyltrimethoxysilane (manufactured by Huels America), and photocatalyst inorganic 1.5 g of coating agent ST-K01 (manufactured by Ishihara Industries Co., Ltd.) was mixed and heated at 100 ° C. for 20 minutes while stirring.

The solution is applied to a quartz glass substrate which has been alkali-treated beforehand by spin coating, and the substrate is dried at a temperature of 150 ° C. for 10 minutes to proceed with hydrolysis and polycondensation reaction, whereby the photocatalyst is strongly fixed in the organopolysiloxane. A substrate for patterning was formed having a photocatalyst-containing cell adhesion layer that changed from cell adhesion to cell adhesion inhibition by the action of a photocatalyst according to energy irradiation with a thickness of 0.2 μm.

(Patterning of Patterning Substrate)

The patterning substrate has a 60 µm / 300 µm line and space having a width of an opening of 60 µm and a width of the shielding portion of 300 µm, and a boundary between the opening and the shielding portion of 1 µm angle is provided. Using a photomask formed so as to have a UV exposure for 900 seconds using a mercury lamp (wavelength 365 nm) at a light intensity of 300 mW / cm ² , patterning the unexposed portion to have cell adhesion and the exposed portion to inhibit cell adhesion. A patterning substrate for cell culture having a prepared cell adhesive surface was obtained.

(Culture of cells)

When the cells were cultured in the same manner as in Example 1, and the cell morphology was observed 16 hours after the seeding of the cells, the orientation and extension of the cells were observed for all the cells in the cell culture region.

Example 4

(Formation of photocatalyst-containing cell adhesion inhibitory layer)

3 g of isopropyl alcohol, 0.4 g of organosilane TSL8114 (manufactured by GE Toshiba Silicone Co., Ltd.), 0.04 g of fluoroalkylsilane TSL8233 (manufactured by GE Toshiba Silicon Co., Ltd.), 1.5 g of photocatalyst inorganic coating agent ST-K01 (manufactured by Ishihara Industries Co., Ltd.) are mixed and stirred. Warmed at 100 ° C. for 20 min.

The solution was applied to a quartz glass substrate which had been alkali-treated beforehand by spin coating, and the substrate was dried at a temperature of 150 ° C. for 10 minutes to proceed with hydrolysis and polycondensation reaction, whereby the photocatalyst was strongly fixed in the organopolysiloxane. A substrate for patterning was formed having a photocatalyst-containing cell adhesion inhibitory layer having a thickness of 0.2 μm, which changes from cell adhesion inhibition to cell adhesion by the action of a photocatalyst according to energy irradiation.

(Patterning of Patterning Substrate)

Ultraviolet irradiation was carried out to the said patterning board | substrate similarly to Example 3, and the cell culture patterning board | substrate which has a pattern in which an unexposed part is a cell adhesion inhibition part and an exposure part becomes a cell adhesion part was obtained.

(Culture of cells)

When the cells were cultured in the same manner as in Example 1, and the cell morphology was observed 16 hours after the seeding of the cells, the orientation and extension of the cells were observed for all the cells in the cell culture region.

Example 5

(Formation of Photocatalyst-Containing Layer)

3 g of isopropyl alcohol, 0.4 g of organosilane TSL8114 (manufactured by GE Toshiba Silicone Co., Ltd.), and 1.5 g of the photocatalyst inorganic coating agent ST-K01 (Ishihara Industries) were mixed and heated at 100 ° C for 20 minutes while stirring.

The solution is applied to a quartz glass substrate which has been alkali-treated beforehand by spin coating, and the substrate is dried at a temperature of 150 ° C. for 10 minutes to proceed with hydrolysis and polycondensation reaction, and the photocatalyst is firmly fixed in the organopolysiloxane. A photocatalyst-containing layer having a thickness of 0.2 탆 was formed on the substrate.

(Formation of Cell Adhesion Layer)

0.2 mg of fibronectin F-4759 (Sigma) and 200 ml of pure water were mixed, and this aqueous solution was added dropwise to the photocatalyst-containing layer of the substrate provided with the photocatalyst-containing layer at a rate of 300 µl per 1 cm ^{2 of} substrate area. It left still at 4 degrees C or less for 24 hours. Further, the substrate was washed twice with PBS, exposed to nitrogen gas and dried to obtain a patterning substrate having a photocatalyst-containing layer and a cell adhesive layer on the substrate.

(Patterning of Patterning Substrate)

Ultraviolet irradiation was performed to the said patterning board | substrate similarly to Example 3, and the patterning substrate for cell cultures which has a pattern in which an unexposed part is a cell adhesion part and an exposure part becomes a cell adhesion inhibition part is obtained.

(Culture of cells)

When the cells were cultured in the same manner as in Example 1, and the cell morphology was observed 16 hours after the seeding of the cells, the orientation and extension of the cells were observed for all the cells in the cell culture region.

Example 6

(Formation of Photocatalyst-Containing Layer)

3 g of isopropyl alcohol, 0.4 g of organosilane TSL8114 (manufactured by GE Toshiba Silicone Co., Ltd.), and 1.5 g of the photocatalyst inorganic coating agent ST-K01 (Ishihara Industries) were mixed and heated at 100 ° C for 20 minutes while stirring.

The solution is applied to a quartz glass substrate which has been alkali-treated beforehand by spin coating, and the substrate is dried at a temperature of 150 ° C. for 10 minutes to proceed with hydrolysis and polycondensation reaction, and the photocatalyst is firmly fixed in the organopolysiloxane. A photocatalyst-containing layer having a thickness of 0.2 탆 was formed on the substrate.

(Formation of Cell Adhesion Inhibition Layer)

A solution consisting of 5 g of isopropyl alcohol, 0.4 g of organosilane TSL8114 (manufactured by GE Toshiba Silicon Co., Ltd.), and 0.04 g of fluoroalkylsilane TSL8233 (manufactured by GE Toshiba Silicon Co., Ltd.) was applied by spin coating to form a substrate. The cell adhesion inhibition layer was formed by drying at 150 ° C. for 10 minutes.

(Patterning of Patterning Substrate)

Ultraviolet irradiation was carried out to the said patterning board | substrate similarly to Example 3, and the cell culture patterning board | substrate which has a pattern in which an unexposed part is a cell adhesion inhibition part and an exposure part becomes a cell adhesion part was obtained.

(Culture of cells)

When the cells were cultured in the same manner as in Example 1, and the cell morphology was observed 16 hours after the seeding of the cells, the orientation and extension of the cells were observed for all the cells in the cell culture region.

Example 7

(Formation of Cell Adhesion Layer)

3 g of isopropyl alcohol, 0.4 g of organosilane TSL8114 (manufactured by GE Toshiba Silicon Co., Ltd.), and 0.4 g of aminopropyltriethoxysilane were mixed and heated at 100 ° C for 20 minutes with stirring. The solution was applied to a quartz glass substrate which had been alkali-treated beforehand by spin coating, and the substrate was dried at 150 ° C. for 10 minutes to proceed with hydrolysis and polycondensation reactions, and the amino group having a film thickness of about 80 nm. An organopolysiloxane layer containing was formed on a substrate to obtain a patterning substrate.

(Patterning of Patterning Substrate)

Ultraviolet irradiation was performed to the said patterning board | substrate similarly to Example 1, and the patterning substrate for cell culture which has a pattern in which an unexposed part is a cell adhesion part and an exposure part becomes a cell adhesion inhibition part was obtained.

(Culture of cells)

When the cells were cultured in the same manner as in Example 1, and the cell morphology was observed 16 hours after the seeding of the cells, the orientation and extension of the cells were observed for all the cells in the cell culture region.

Claims (6)

A base material 1, and A cell culture region (2) comprising a cell adhesion portion (3) and a cell adhesion inhibition portion (4) formed on the substrate A patterned substrate for cell culture comprising: The cell adhesion layer 3 is formed with a cell adhesion layer, Patterned substrate for cell culture, characterized in that the cells on the two cell adhesion part (3) adjacent to the cell adhesion inhibition part (4) is formed to be able to bind to each other on the cell adhesion inhibition part (4). The patterned substrate for cell culture according to claim 1, wherein the cell adhesion inhibiting portion (4) is formed in a straight line in plan view in the cell culture region (2). The patterned substrate for cell culture according to claim 1 or 2, wherein the boundary between the cell adhesion inhibiting portion (4) and the cell adhesion portion (3) has an uneven shape. Base material (1), and A cell culture region (2) containing a cell adhesion layer having adhesion to cells formed on the base material (1) A patterned substrate for cell culture having Patterned substrate for cell culture, characterized in that the shape of the end of the cell adhesive layer is planar, concave-convex shape. The method according to claim 4, wherein the distance from the concave end of the concave to the convex end is such that the cell linearly aligns along the uppermost part of the concave and convex portion of the cell adhesive layer when attaching the cell onto the cell adhesive layer. Patterned substrate for cell culture, characterized by. The patterned substrate for cell culture according to claim 4 or 5, wherein an average of the distances from the concave end to the convex end of the unevenness is in the range of 0.5 µm to 30 µm.

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