WO2005080547A1

WO2005080547A1 - Patterning substrate for cell culture

Info

Publication number: WO2005080547A1
Application number: PCT/JP2005/002608
Authority: WO
Inventors: Hideyuki Miyake; Hideshi Hattori; Hironori Kobayashi; Hidetsugu Tazawa
Original assignee: Dai Nippon Printing Co., Ltd.
Priority date: 2004-02-19
Filing date: 2005-02-18
Publication date: 2005-09-01
Also published as: JP4589012B2; KR20070006781A; KR100886291B1; JP2005229914A; KR20080096714A

Abstract

A patterning substrate for cell culture that can realize efficiently arranging of cells in a regular fashion over a large area on a base material so as to attain forming of a tissue, etc. There is provided a patterning substrate for cell culture, comprising a base material and, superimposed thereon, a cell culture region being a region for cell incubation and including a cell adhesive layer having adherence to cells, characterized in that the cell culture region has cell adhesive parts wherein the cell adhesive layer is provided and a cell adhesion auxiliary part that is provided in patterned form and inhibits cell adhesion, and that the cell adhesion auxiliary part is provided so that at the time of sticking of cells to the cell adhesive parts, the cells on two cell adhesive parts adjacent to the cell adhesion auxiliary part can bind each other on the cell adhesion auxiliary part.

Description

Specification

Patterning substrate for cell culture

Technical field

TECHNICAL FIELD [0001] The present invention relates to a pattern culturing substrate used for culturing cells such as blood vessels.

Background art

[0002] Currently, cell cultures of animals, plants, and the like are being performed, and new cell culture methods are being developed. Cell culture technology is used to elucidate the biochemical phenomena and properties of cells and to produce useful substances. Further, attempts have been made to examine the bioactivity and toxicity of artificially synthesized drugs using cultured cells.

[0003] Some cells, particularly many animal cells, have an adhesion dependency of growing by adhering to something, and cannot survive for a long period of time in a floating state outside a living body. Cultivation of cells having such adhesion dependence requires a carrier for the cells to adhere to the cells. Generally, a plastic-made cell on which a cell adhesion protein such as collagen fibronectin is uniformly applied is generally used. A culture dish is used. These cell adhesion proteins are known to act on cultured cells, facilitating cell adhesion and affecting cell morphology.

[0004] On the other hand, a technique has been reported in which cultured cells are adhered to only minute portions on a substrate and arranged. Such a technique makes it possible to apply cultured cells to artificial organs, biosensors, bioreactors, and the like. A method for arranging cultured cells is to use a substrate having a patterned surface with different ease of adhesion to the cells, cultivate the cells on this surface, and allow the cells to adhere. A method is used in which cells are arranged by adhering the cells only to the processed surface.

[0005] For example, in Patent Document 1, a charge holding medium having an electrostatic charge pattern formed thereon is applied to cell culture for the purpose of, for example, growing nerve cells in a circuit form. Further, Patent Document 2 attempts to arrange cultured cells on a surface obtained by patterning a non-cell-adhesive or cell-adhesive photosensitive hydrophilic polymer by a photolithography method. [0006] Further, Patent Document 3 describes a cell culture substrate on which a substance such as collagen which affects cell adhesion rate and morphology is patterned, and a method for producing the substrate by photolithography. Has been disclosed. By culturing the cells on such a base material, more cells can be adhered to the surface on which collagen or the like is put on, thereby realizing the cell patterning.

[0007] However, in such a cell culturing method, when the area of the cell culture pattern is large, cells can be regularly arranged at the end of the cell culture pattern. In the central part, there is a problem that the alignment of the cells may be poor or the cells may not adhere. In addition, general cells are those in which individual cells undergo morphological changes to form tissues, and when cells are cultured using the above-described cell culture pattern or the like so as to form such tissues, The boundary region between the cell adhesion portion having adhesiveness to the cell and the cell adhesion inhibition portion that inhibits adhesion to the cell stimulates the cell, thereby causing a morphological change of the cell, and this morphological change gradually occurs. It is shown in Non-Patent Document 1 and the like that it propagates to the central part of the cell culture pattern. However, if the area of this cell culture pattern is increased, the morphological change of the cells is difficult to be transmitted to the central part, and the central part is not susceptible to the morphological change of the cells. was there. Furthermore, when cells are seeded and adhered to a substrate in this way, there is a problem that it takes time to adhere the cells.

Patent Document 1: JP-A-2-245181

Patent Document 2: JP-A-3-7576

Patent Document 3: JP-A-5-176753

Non-patent literature 1: Spargo et al., Proceedings of tne National Academy of sciences of the United States of America (1994) p.11070—

Disclosure of the invention

Problems to be solved by the invention

[0009] Therefore, there is a demand for a cell culture puttering substrate capable of forming cells and the like efficiently and regularly in a large area on a base material and forming a tissue or the like. Means for solving the problem [0010] The present invention provides a cell culture butterfly comprising a base material, a cell culture region formed on the base material and culturing cells, and a cell culture region containing a cell adhesive layer having adhesive properties to cells. A substrate,

The cell culture region has a cell adhesion portion on which the cell adhesion layer is formed, and a cell adhesion auxiliary portion formed in a pattern and inhibiting adhesion to cells. When the cells are attached to the cell adhesion part, the cells on the two cell adhesion parts adjacent to the cell adhesion part are formed so that they can be bonded on the cell adhesion part. Provided is a puttering substrate for cell culture, which is characterized in that:

[0011] According to the present invention, since a cell adhesion auxiliary portion is formed in the cell culture region, when a cell adheres to the cell adhesion portion, the cell can be activated. Cells can be cultured efficiently and in a short time. In addition, since the cells are cultured in each region sandwiched between the cell adhesion assisting portions, compared with the case where the cells are cultured in the entire cell culture region without the cell adhesion assisting portions, Boundary region force The number of cells that can be stimulated can be increased. As a result, the arrangement of cells can be improved, and the morphology of the cells can be uniformly changed. Further, in the present invention, when the cells are attached to the cell adhesion layer, the cell adhesion assisting portion is formed so as not to inhibit the binding between the cells attached to the adjacent cell adhesion portions, As a result, cells in the entire cell culture region can be finally combined, and the obtained tissue and the like can be made large.

[0012] In the above invention, the cell adhesion auxiliary portion may be formed in a line in the cell culture region. In this case, there is an advantage that the design when forming the cell culture region is facilitated, and the cells are easily arranged regularly when the cells are attached.

[0013] In the above invention, the boundary between the cell adhesion auxiliary part and the cell adhesion part may be formed in a pattern having a concave and convex shape. When the cells are adhered along the pattern having irregularities in this manner, the stimulation that the cells sense from the boundary region increases, and the cells can be arranged in a more aligned manner. In addition, since the adhesion of the cells to the cell adhesion portion can be activated, the cells can be efficiently and quickly placed on the substrate. Can be used as a cell culture puttering substrate.

[0014] Further, the present invention provides a cell culture comprising a substrate, and a cell culture region formed on the substrate and culturing cells, the cell culture region comprising a cell adhesion layer having adhesiveness to cells. A patterning substrate for

The cell adhesion layer is provided with a patterning substrate for cell culture, wherein the end portion is formed in a pattern having irregularities.

[0015] According to the present invention, since the end of the cell adhesive layer is formed in a pattern having irregularities, when the cells are attached to the cell adhesive layer, the stimulus that the cells sense from the boundary region is not affected. As the number of cells increases, the cells can be more aligned and arranged along the edge of the cell adhesion layer. In addition, since the adhesiveness of the cells to the cell adhesion portion can be made active, a cell culture pattern-junging substrate capable of efficiently adhering cells to the substrate in a short time can be obtained.

[0016] In the above invention, the distance between the concave end force of the irregularities and the convex end is such that the cells are linearly aligned when the cells are adhered to the cell adhesion layer. Is preferred. This is because by setting the size of the irregularities to such a value, cells can be favorably arranged.

In the above invention, the average of the distance from the concave end to the convex end of the unevenness is preferably in the range of 0.5 μm to 30 μm. By setting the size of the irregularities in such a range, the cells can be arranged well and the cells can be activated. The invention's effect

According to the present invention, when a cell is adhered to a cell adhesion portion, the cell can be activated, and the cell can be efficiently cultured in a short time in a wide area. It can be used as a patterning substrate for culture. Also, at this time, there is an effect that the arrangement of the cells can be improved and the morphological change of the cells can be uniformly performed. Brief Description of Drawings

FIG. 1 is a schematic sectional view showing an example of a cell culture patterning substrate of the present invention.

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

FIG. 3 is a schematic sectional view showing another example of the cell culture patterning substrate of the present invention. FIG. 4 is a schematic sectional view showing another example of the cell culture patterning substrate of the present invention.

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

FIG. 6 is a process chart showing an example of a method for forming a cell adhesion assisting portion in a cell culture putter-jung substrate of the present invention.

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

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

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

FIG. 10 is a process chart showing another example of the method for forming a cell adhesion auxiliary portion in the putter substrate for cell culture of the present invention.

FIG. 11 is a process chart showing an example of a method for forming a cell adhesive layer on the cell culture patterning substrate of the present invention.

FIG. 12 is a schematic sectional view showing another example of the cell culture patterning substrate of the present invention. Explanation of symbols

[0020] 1 ... substrate

2… cell culture area

3… cell adhesion

4… cell adhesion assistant

5… Photo mask

6… energy

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to a patterning substrate for cell culture used for cell culture, and the puttering substrate for cell culture of the present invention has two embodiments. Hereinafter, each embodiment will be described.

A. First Embodiment

First, a first embodiment of the cell culture patterning substrate of the present invention will be described. A first embodiment of the putter substrate for cell culture according to the present invention comprises a base material, a cell-adhesion layer formed on the base material, for culturing cells, and having an adhesive property to cells. A cell culture putter substrate having a cell culture region, The cell culture region has a cell adhesion portion on which the cell adhesion layer is formed, and a cell adhesion auxiliary portion formed in a pattern and inhibiting adhesion to cells. When the cells are attached to the cell adhesion part, the cells on the two cell adhesion parts adjacent to the cell adhesion part are formed so that they can be bonded on the cell adhesion part. Things.

The putter substrate for cell culture according to the present embodiment has a substrate 1 and a cell culture region 2 formed on the substrate 1 as shown in FIG. 1, for example. The culturing region 2 has a cell adhesion portion 3 having an adhesive property to the cell on which the cell adhesion layer is formed, and a cell adhesion assisting portion 4 for inhibiting adhesion to the cell.

Here, in general, when cells are cultured by attaching cells to a cell culture region to form a tissue, the cells are gradually arranged from the outside to the inside of the cell culture region. In addition, during tissue formation, it is necessary that individual cells undergo morphological changes and be arranged, and the morphological changes of these cells are also made gradually from the end to the center of the cell culture region. is there.

[0025] Therefore, when cells are cultured using a general pattern jungle substrate for cell culture, if the area of the cell culture region for culturing the cells is large and large, the cell arrangement at the center is large. In some cases, poorly formed tissues are not formed, or cells do not adhere to the center of the cell culture area. In addition, there was a problem that a target tissue having poor cell morphological change in the central portion was not formed.

[0026] On the other hand, according to the present invention, a cell adhesion assisting portion is formed in the cell culture region. For example, as shown in FIG. Will be cultured. That is, the arrangement of the cells and the morphological change of the cells can be caused from the boundary between the cell adhesion auxiliary part 4 and the cell adhesion part 3, and the cell adhesion auxiliary part 4 is not formed as compared with the case where the cell adhesion auxiliary part 4 is not formed. A boundary region can also be provided inside the cell culture 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 auxiliary portion 4. This makes it possible to improve the cell arrangement and morphological change over the entire cell culture region 2. [0027] In the present embodiment, the cell adhesion auxiliary portion is formed so that cells adhering to two adjacent cell adhesion layers can be bonded to each other on the cell adhesion auxiliary portion. For example, as shown in FIG. 1, the cells adhered to the cell adhesion part 3 in the area a and the cells adhered to the cell adhesion part 3 in the area b can be bonded on the cell adhesion auxiliary part 4. In addition, a cell adhesion assistant 4 is formed. As a result, the cells can be finally cultured in the same area as when the cells are cultured on the entire surface of the cell culture region 2. This means that even if it is a region that inhibits adhesion to cells, if cells are present on both sides of them and they are close enough to affect each other, they will not adhere to cells. This is because cells can interact with each other even in the region where the inhibition occurs.

[0028] Further, it is known that when there is a defect or the like in the cell adhesion layer, the cells are activated and the cells easily adhere to the region. In the present embodiment, the cell adhesion assisting portion formed in the cell culture region exhibits the same effect as such a defect, and the cells are activated. Can be adhered to the substrate.

Hereinafter, each configuration of the pattern culturing substrate for cell culture according to the present embodiment will be described.

[0030] 1. Cell culture area

First, a cell culture region on the putter substrate for cell culture of the present embodiment will be described. The cell culture region according to the present embodiment is a region formed for culturing cells, and includes a cell adhesion portion having a cell adhesion layer having an adhesive property to cells, a cell adhesion portion formed in a pattern, and a cell adhesion region. And a cell adhesion assisting portion that inhibits adhesion.

In the present embodiment, the cell culture region may be formed on a part of the substrate 1 as shown in FIG. 1, for example, or the entire surface of the substrate may be a cell culture region. , You can. Here, for example, when the cell culture region 2 is formed on a part of the substrate 1 as shown in FIG. 1, regions other than the cell culture region on the substrate 1 inhibit adhesion to cells. This is a non-cultured area. In the present embodiment, the number of cell culture regions formed on one substrate is not limited to one.For example, as shown in FIG. May be formed plurally. Also in this case, the area other than each cell culture area on the substrate 1 is the above-mentioned cell non-culture area. [0032] In addition, the force usually is different also depending on the size and type of yarn且織of interest, the size of one cell culture area, 0. 05mm ^² - 8000mm ^2, among others 0. 1 mm ² - It is in the range of 10 mm ^2.

Here, in the above-described cell culture region, a cell adhesion auxiliary portion is formed in a pattern in the cell adhesion portion. In the present embodiment, the leverage cell adhesion auxiliary part is formed so that cells attached to two cell adhesion parts adjacent to the cell adhesion auxiliary part can be bonded on the cell adhesion auxiliary part, and There is no particular limitation as long as the cells adhered on the cell adhesion layer are regularly arranged and are formed so that the cell morphological change is uniformly generated. For example, as shown in FIG. 1, the cell adhesion auxiliary part 4 may be formed in a line in the cell culture region 2, or as shown in FIG. 3, for example, the cell adhesion auxiliary part 4 may be formed in the cell culture region 2. Is randomly formed.

[0034] The width of the cell adhesion assisting portion varies depending on the type and size of the cells to be cultured, etc., but is usually within a range of 0.5 m to 10 m, especially 1 μm to 5 μm. It is preferred that If the width is wider than the above range, it becomes difficult for the cells attached to the two cell adhesion parts adjacent to the cell adhesion part to interact with each other on the cell adhesion part. When the width is narrower than the range, the cell adhesion auxiliary part, which makes it difficult to obtain a pattern of such a size in a fine pattern by the Patterjung technique described below, has an influence on the arrangement property and morphological change as described above. It is difficult to exert.

[0035] At this time, the width of the cell-adhesive portion sandwiched between the cell-adhesion assisting portions (for example, the distance indicated by X in FIG. 1), or the width of the cell-adhesive portion sandwiched between the cell-adhesive assisting portion and the non-cell culture region The width (for example, the distance represented by y in Fig. 1) is appropriately selected depending on the size and type of cells to be cultured, the type of target tissue, and the like. Normally, 1 μm to 200 m, especially 40 / zm-80 / zm is preferable. Thereby, the cells adhered to the cell adhesion portion can be regularly arranged, and a morphological change can be satisfactorily formed to form a tissue.

[0036] In the present embodiment, it is particularly preferable that the cell adhesion auxiliary portion is formed in a line shape. This facilitates the design when forming the cell culture region, and improves the arrangement of the cells to be cultured. Line shape means cell adhesion This means that the auxiliary part is formed in a straight line.For example, as shown in FIG. 1, only when the cell adhesion auxiliary part 4 is formed continuously, for example, the cell adhesion auxiliary part is formed in a broken line. This includes cases in which it is performed. Further, in this embodiment, it is not only the case where the cell adhesion assisting portion is formed in a line in one direction. For example, as shown in FIG. 4, the cell adhesion assisting portion 4 is formed in a line in a plurality of directions. Shall be included.

Furthermore, in the present embodiment, the boundary between the cell adhesion part and the cell adhesion auxiliary part may be formed in a pattern having irregularities. This is because, by arranging the cells along the pattern having such irregularities, the cells can be arranged more regularly. In this case, there is also an advantage that the attached cells are activated more and the cells can be efficiently cultured. Here, the pattern having irregularities is not particularly limited as long as the pattern allows cells to be regularly arranged.For example, as shown in FIG. The boundary with the adhesion auxiliary portion 4 may have a rectangular irregularity, or may have a corrugated irregularity. In addition, for example, even when the cell adhesion assisting portion is formed in a broken line or when the cell adhesion assisting portion is formed in a random pattern, the cell adhesion assisting portion and the cell adhesion portion are not separated. The boundary may be formed in a pattern having irregularities. Even in such a case, it is a force that can achieve the same effect.

Here, it is preferable that the distance from the concave end to the convex end of the unevenness is such that the cells are linearly aligned when the cells are attached to the cell adhesive layer. Specifically, the average size of the distance from the concave end to the convex end of the irregularities is 0.5 / ζπι-30 /, which is appropriately selected depending on the shape of the cells to be cultured. ππι, particularly preferably in the range of 1 m−5 / zm. Thereby, when the cells are cultured, it becomes possible to form the tissue by culturing the cells into a target shape without chipping at the end of the cell culture region. Here, the average measurement of the distance from the concave end to the convex end of the pattern having irregularities is determined by measuring the distance between the cell adhesion part and the cell adhesion auxiliary part in the range of 200 μm at the bottom force of each irregularity to the top part. Is measured and the average is taken as the calculated value.

Hereinafter, the cell adhesion part and the cell adhesion auxiliary part constituting such a cell culture region will be described. And each will be described.

[0039] (Cell adhesion part)

First, the cell adhesion portion used in the present embodiment will be described. The cell adhesion portion in the present embodiment is an area where a cell adhesion layer having adhesiveness to cells is formed on a substrate in a cell culture area. The cell adhesive layer is not particularly limited as long as it has an adhesive property to cells, and a layer having an adhesive property to cells used for a general patterning substrate for cell culture can be used. In this embodiment, by forming such a cell adhesive layer in a pattern, a cell adhesive portion can be formed. For example, a cell adhesive layer-forming coating containing a material having an adhesive property to cells can be obtained. By applying the solution in a pattern or the like, a cell adhesion portion can be formed. Alternatively, the coating solution for forming a cell adhesion layer may be formed on the entire surface of the cell culture region, and the cell adhesion portion may be formed by a photolithography method or the like.

[0040] In the present embodiment, the cell adhesive layer contains a cell adhesive material that has adhesiveness to cells and is decomposed or denatured by the action of a photocatalyst accompanying irradiation with energy. By irradiating the cell adhesion layer with energy, the pattern adhesion can be performed to form a cell adhesion portion. In this case, for example, after the cell adhesion layer is formed on the entire surface of the cell culture area, the cell adhesion material is decomposed or degraded by the action of the photocatalyst by irradiating energy in a pattern forming the cell adhesion auxiliary part. Thus, it is possible to form a cell adhesion assisting portion that inhibits adhesion to cells and a cell adhesion portion having adhesion to cells. The cell adhesive layer containing such a cell adhesive material and the method for forming the cell adhesion auxiliary portion at that time will be described in detail later.

[0041] The cell adhesion layer used in the present embodiment has a cell adhesion inhibitory property and has a cell adhesion inhibitory material that is degraded by the action of a photocatalyst accompanying energy irradiation. By coating the entire surface of the cell culture area, and then irradiating energy to an area other than the cell adhesion auxiliary part, the above-mentioned cell adhesion inhibiting material is decomposed or denatured and formed to have adhesiveness to cells.たIn this case, adhesion to cells is inhibited except for the area that has been irradiated with energy and becomes the cell adhesion part. Since it is an area to be used, it can be used as a cell adhesion auxiliary part. The cell adhesion-inhibiting layer containing such a cell adhesion-inhibiting material, the method for forming the cell adhesion layer at that time, and the like will be described later in detail.

(Auxiliary part for cell adhesion)

Next, the cell adhesion assisting portion in the cell culture region in the present embodiment will be described. The cell adhesion assisting portion in this embodiment is formed in a pattern in the cell culture region and inhibits adhesion to cells. When the cells are adhered to the cell adhesion portion, the cell adhesion assisting portion is There is no particular limitation as long as the cells on the two cell adhesion parts adjacent to the adhesion auxiliary part are formed so as to be bonded on the cell adhesion auxiliary part.

[0043] The cell adhesion assisting portion in this embodiment may be, for example, a region where the base material is exposed, which will be described later, or may be a commonly used cell adhesion inhibiting layer or the like that inhibits adhesion to cells. May be formed. Examples of the method for forming the cell adhesion inhibiting layer include a general printing method, a photolithography method, and a patterning method utilizing the action of a photocatalyst accompanying energy irradiation. The patterning method using the action of the photocatalyst accompanying the energy irradiation will be described later in the description of the cell adhesion layer using the cell adhesion inhibition layer having the cell adhesion inhibition material. The description here is omitted.

Further, as described above, when the cell adhesion layer is a layer containing a cell adhesion material that is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation, the cell adhesion auxiliary part is It may be a region where a decomposed or denatured material of the material remains or the like! / ヽ. The method of forming the cell adhesion assisting portion in this case will be described together with the description of the cell adhesion layer containing the cell adhesion material which is decomposed by the action of the photocatalyst accompanying the energy irradiation. Omitted.

[0045] 2. Base material

Next, the base material used in the present embodiment will be described. The substrate used in the present embodiment is not particularly limited as long as it can form the above-described cell culture region. Examples thereof include inorganic materials such as metal, glass, and silicon, and plastics. Organic materials and the like can be used. The flexibility and transparency of the substrate are appropriately selected depending on the type and use of the patterning substrate for cell culture.

Here, in this embodiment, since the region other than the cell culture region on the substrate is a cell non-culture region without culturing cells, adhesion to cells is inhibited. For example, a layer or the like that inhibits adhesion to cells may be formed in a non-cultured cell region other than the above-mentioned cell culture region.

[0047] 3. Patterning substrate for cell culture

Next, the putter substrate for cell culture according to the present embodiment will be described. The cell culture puttering substrate of the present embodiment is not particularly limited as long as the cell culture region is formed on the above-described base material. It may be formed, something.

[0048] 4. Other

As described above, the cell adhesion layer used for the cell culture region of the cell culture puttering substrate of the present embodiment contains (1) a cell adhesion material that is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation. In addition, (2) a cell adhesion-inhibiting material that has a cell adhesion-inhibiting property of inhibiting cell adhesion and is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation. It may be formed by decomposing or denaturing a cell adhesion inhibiting material by irradiating energy after forming a cell adhesion inhibiting layer to be contained.

Hereinafter, each will be described separately.

[0049] I. In the case of (1)

First, the case where the cell adhesive layer contains a cell adhesive material that is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation will be described. Examples of the cell adhesion layer containing such a cell adhesion material include the following three embodiments.

[0050] 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 photocatalyst-containing cell This is the case where the cell adhesive material is decomposed or denatured by the action of the photocatalyst contained in the adhesive layer itself. [0051] In a second embodiment, a cell adhesion layer containing at least a cell adhesion material is formed on a photocatalyst treatment layer containing at least a photocatalyst, and when the cell adhesion layer is irradiated with energy, This is the case when the cell adhesive material in the layer is decomposed or denatured by the action of the photocatalyst in the adjacent photocatalyst treatment layer.

[0052] In a third embodiment, at least a cell adhesion layer containing a cell adhesion material is formed on a base material, and at the time of energy irradiation, at least a photocatalyst-containing layer containing a photocatalyst is referred to as a cell adhesion layer. This is the case where the cell adhesive material is decomposed or denatured by the action of the photocatalyst in the opposing photocatalyst-containing layer by irradiating the energy with facing the energy. Hereinafter, each embodiment will be described.

[0053] (1) First aspect

First, 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 photocatalyst contained in the photocatalyst-containing cell adhesion layer itself is removed. The case where the cell adhesive material is decomposed or denatured by the action will be described.

[0054] According to this embodiment, since the photocatalyst-containing cell adhesive layer contains the photocatalyst and the above-mentioned cell adhesive material, by irradiating the photocatalyst-containing cell adhesive layer with energy, the action of the photocatalyst is improved. Thus, the region irradiated with energy can be used as a cell adhesion assisting portion to which cells do not adhere. In addition, since the cell adhesive material remains in the area where the energy has not been irradiated, a cell adhesive portion having good adhesion to cells can be formed. Therefore, by irradiating energy in a pattern without special devices or complicated steps, it is possible to easily form a cell adhesion auxiliary part in the cell adhesion part that inhibits adhesion to cells. Becomes possible.

[0055] In forming such a photocatalyst-containing cell adhesive layer, a coating solution for forming a photocatalyst-containing cell adhesive layer containing a photocatalyst and a cell adhesive material that is decomposed or modified by the action of the photocatalyst accompanying energy irradiation is applied. It can be performed by the following. The coating solution for forming the photocatalyst-containing cell adhesive layer can be applied by a general coating method, for example, a spin coating method, a spray coating method, a dip coating method, a roll coating method, a bead coating method, or the like. Can be used. At this time, the film thickness of the photocatalyst-containing cell adhesive layer is a force which is appropriately selected depending on the type of the cell culture patterning substrate and the like, and is usually about 0.1 Ol / zm—l.O / zm, Above all, a force of about 0.1 μm-0.3 μm can be achieved.

Hereinafter, a cell adhesive material and a photocatalyst contained in the photocatalyst-containing cell adhesive layer used in the present embodiment will be described, and further, a method for forming a cell adhesion auxiliary portion will be described.

[0058] a. Cell adhesive material

First, the cell adhesive material contained in the photocatalyst-containing cell adhesive layer of the present embodiment will be described. The type of the cell adhesive material contained in the photocatalyst-containing cell adhesive layer of this embodiment is not particularly limited as long as it has adhesiveness to cells and is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation. Not something. Here, having an adhesive property with a cell means that the cell adheres well to the cell, and when the adhesive property with the cell varies depending on the type of the cell, etc., it adheres well to a target cell. That means.

[0059] The cell adhesive material used in the present embodiment has such adhesion to cells, and is degraded or denatured by the action of a photocatalyst accompanying energy irradiation, and has adhesion to cells. Those that disappear or those that change to those having cell adhesion inhibitory properties that inhibit adhesion to cells are used.

[0060] Here, the material having an adhesive property to cells as described above includes a material having an adhesive property to cells due to physical properties and a material having an adhesive property to cells due to biochemical properties. There are two types; ^.

[0061] Physically determinant factors that determine the adhesiveness between cells and a material having adhesiveness to cells based on physicochemical properties include surface free energy and electrostatic interaction. For example, when the adhesiveness to cells is determined by the surface free energy of the material, if the material has a surface free energy within a predetermined range, the adhesiveness between the cells and the material becomes good, and if the material falls outside the range, the material has good surface free energy. Adhesion between the cells and the material will be reduced. As the change in cell adhesiveness due to such surface free energy, for example, the experimental results shown in the lower part of Yoshinobu Raft, supervised by CMC Publishing Noo Materials, p. 109, are known. Materials having adhesive properties to cells due to such factors include, for example, Examples include hydrophilized polystyrene and poly (N-isopropylacrylamide). When such a material is used, the surface free energy changes due to, for example, substitution or decomposition of a functional group on the surface of the material due to the action of a photocatalyst accompanying energy irradiation, and adhesion to cells. It may be one having no property, or one having cell adhesion inhibitory property.

[0062] Further, when the adhesiveness between the cell and the material is determined by the electrostatic interaction or the like, the adhesiveness to the cell is determined by, for example, the amount of the positive charge of the material. Examples of the material having an adhesive property to cells by such an electrostatic interaction include basic polymers such as polylysine, aminopropyltriethoxysilane, N- (2-aminoethyl) -3-aminopropyltrimethoxysilane. And condensates containing them. When such a material is used, the above-mentioned material is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation, for example, the amount of positive charges present on the surface can be changed, and the adhesion to cells can be improved. Having no, or having cell adhesion inhibitory properties.

[0063] Examples of the material having adhesive properties to cells due to its biological properties include those having good adhesive properties with specific cells, those having good adhesive properties with many cells, and the like. Examples include fibronectin, laminin, tenascin, vitronectin, a peptide containing an RGD (arginine-glycine-aspartate) sequence, a peptide containing a YIGSR (tyrosine isoleucine glycine-serine arginine) sequence, collagen, atelocollagen, gelatin and the like. When such a material is used, it does not have adhesiveness to cells by the action of a photocatalyst accompanying energy irradiation, for example, by destroying a part of the structure of the material or destroying the main chain. Or have cell adhesion inhibitory properties.

[0064] Such a cell adhesive material varies depending on the kind of the above-mentioned material and the like, but is usually 0.01% to 95% by weight, especially 1% to 10% by weight in the photocatalyst-containing cell adhesive layer. % Is preferably contained. Thereby, the region containing the cell adhesive material can be a region having good adhesion to cells.

[0065] b. Photocatalyst

Next, the photocatalyst contained in the photocatalyst-containing cell adhesion layer of the present embodiment will be described. The photocatalyst used in the present embodiment is not particularly limited as long as it can decompose or modify the above-mentioned cell adhesive material by the action of the photocatalyst accompanying energy irradiation.

Here, the action mechanism of a photocatalyst represented by titanium oxide as described later is not necessarily clear, but it is possible to directly react with a compound near the carrier force generated by light irradiation, or It is thought that active oxygen species generated in the presence of oxygen, water, and water change the chemical structure of organic matter. In this embodiment, it is considered that this carrier has an effect on the above-mentioned cell adhesion material.

As the photocatalyst used in the present embodiment, specifically, for example, titanium dioxide (TiO 2), zinc oxide (ZnO), tin oxide (SnO 2), strontium titanate (S

twenty two

rTiO), tungsten oxide (WO), bismuth oxide (Bi O), and iron oxide (Fe O)

3 3 2 3 2 3, and these forces can also be selected and used alone or in combination of two or more.

[0068] In this embodiment, titanium dioxide is particularly preferably used because it has a high band gap energy, is chemically stable, is toxic, and is easily available. Titanium dioxide has an anatase type and a rutile type, and any of them can be used in the present embodiment. However, an anatase type titanium dioxide is preferred.

380 or less.

Examples of such anatase-type titanium dioxide include, for example, anatase-type titania sol of peptized hydrochloric acid (STS-02 (average particle size: 7 nm) manufactured by Ishihara Sangyo Co., Ltd.) and ST-K01 manufactured by Ishihara Sangyo Co., Ltd. ), Nitrate peptized anatase-type titazole (TA-15 (average particle size: 12 nm) manufactured by Nissan Chemical Industries, Ltd.), and the like.

[0070] The smaller the particle size of the photocatalyst is, the more effectively the photocatalytic reaction takes place. Therefore, it is particularly preferable to use a photocatalyst having a preferred average particle size of 50 nm or less, preferably 20 nm or less.

[0071] The content of the photocatalyst in the photocatalyst-containing cell adhesion layer of this embodiment can be set in the range of 5 to 95% by weight, preferably 10 to 60% by weight, and more preferably 20 to 40% by weight. Wear.

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

Here, it is preferable that the photocatalyst used in the present embodiment has low adhesiveness to cells, for example, by having high hydrophilicity. This makes it possible to use the region where the photocatalyst is exposed due to the decomposition of the cell adhesive material or the like as the region having low adhesion to cells.

[0073] c Other

In this embodiment, the photocatalyst-containing cell adhesive layer may contain, for example, a binder or the like that improves the strength, resistance, or the like, if necessary, in addition to the cell adhesive material and the photocatalyst alone. In the present embodiment, it is preferable that a material having a cell adhesion-inhibiting property of inhibiting adhesion to cells at least after energy irradiation is used as the binder. Thereby, the adhesiveness of the cell adhesion assisting portion, which is the region irradiated with energy, to the cells can be reduced. As such a material, for example, a material having the above-described cell adhesion inhibitory property before the energy irradiation may be made to have the cell adhesion inhibitory property by the action of the photocatalyst accompanying the energy irradiation. You may.

[0074] In this embodiment, it is preferable to use, as a binder, a material that has cell adhesion inhibitory property particularly by the action of a photocatalyst accompanying energy irradiation. As a result, in the area before the energy irradiation, only the energy-irradiated area where the adhesion of the cell adhesive material to the cell is not hindered, Is a force that can be

As a material used as such a binder, for example, an organic substituent whose main skeleton has a high binding energy that is not decomposed by the photoexcitation of the photocatalyst and that is decomposed by the action of the photocatalyst is used. For example, (1) an organopolysiloxane which exerts large strength by hydrolyzing or polycondensing a black hole or alkoxysilane by a sol-gel reaction or the like, (2) water repellency, oil repellency And organopolysiloxanes obtained by cross-linking reactive silicones having excellent properties.

In the case of the above (1), the general formula:

Y SiX (Where Y is an alkyl group, fluoroalkyl group, butyl group, amino group, phenol group or epoxy group, or an organic group containing them, and X represents an alkoxyl group, an acetyl group or a halogen. Η is an integer from 0 to 3.)

It is preferable that the organopolysiloxane is one or more hydrolytic condensates or cohydrolytic condensates of the silicon compound represented by Here, the carbon number of the organic group represented by Υ is preferably in the range of 120.Alkoxy group represented by X is a methoxy group, an ethoxy group, a propoxy group, or a butoxy group. Preferably, there is.

[0077] Examples of the reactive silicone of the above (2) include compounds having a skeleton represented by the following general formula.

[0078] [Formula 1]

[0079] Here, n is an integer of 2 or more, and R ¹ and R ² are each a substituted or unsubstituted alkyl, alkaryl, aryl, or cyanoalkyl group having a carbon number of 120, and the whole is a molar ratio. Less than 40% of these are bulls, phenols and halogenated phenols. When RR ² is a methyl group, the surface energy is minimized, so that the methyl group is preferably 60% or more at a preferable molar ratio. Further, the chain terminal or the side chain has at least one or more reactive group such as a hydroxyl group in the molecular chain. By using such a material, the surface of the region irradiated with the energy can be made highly hydrophilic by the action of the photocatalyst accompanying the energy irradiation. Thereby, the adhesion to the cells is inhibited, and the cells do not adhere to the energy-irradiated region.

[0080] When the above material is used as a material having cell adhesion inhibiting properties, the contact angle with water before irradiation with energy is in the range of 15 ° to 120 °, especially 20 ° to 100 °. Is preferred. This makes it possible to improve the adhesion to cells. It is.

[0081] Further, when energy is applied to the material having the cell adhesion inhibiting property, the material preferably has a contact angle with water of 10 ° or less. By setting the content in the above range, it is possible to obtain a polymer having high hydrophilicity and low adhesiveness to cells.

[0082] The contact angle with water here is measured using a contact angle measuring instrument (CA-Z type manufactured by Kyowa Interface Science Co., Ltd.) with a contact angle with water or a liquid having an equivalent contact angle. Measurement (micro-syringe force 30 seconds after dropping the droplet), and obtained from the results or as a graph.

Further, a stable organosilicon conjugate, which does not undergo a crosslinking reaction like dimethylpolysiloxane, may be mixed with the above-mentioned organopolysiloxane in a binder.

Further, in the present embodiment, the adhesiveness to cells is reduced by causing a change in wettability of an area irradiated with energy, or a substance containing a decomposed substance or the like that assists such a change. It may be.

[0084] Examples of such a decomposed substance include, for example, a surfactant that is decomposed or the like by the action of a photocatalyst accompanying energy irradiation, becomes hydrophilic, and reduces the adhesiveness to cells. it can. Specific examples include hydrocarbons such as NIKKOL BL, BC, BO, and BB series from Nikko Chemicals, ZONYL FSN and FSO from DuPont, Surflon S-141, 145 from Asahi Glass, and Dainippon Japan. Megafac F-141, 144, manufactured by Ink Chemical Industry Co., Ltd., Futergent F-200, F251, manufactured by Neos Co., Ltd., Dudyne DS-401, 402, manufactured by Daikin Industries, Ltd., Florad manufactured by Threeem Co., Ltd. Examples thereof include silicone-based nonionic surfactants such as FC-170 and 176, and cationic surfactants, ion-based surfactants, and amphoteric surfactants can also be used.

[0085] 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, polychlorinated vinyl, polyamide, polyimide, styrene butadiene rubber, chloroprene rubber, polypropylene, polybutylene, polystyrene, polyvinyl acetate, nylon, polyester, polybutadiene, Examples thereof include oligomers and polymers such as polybenzimidazole, polyacryl-tolyl, epichlorohydrin, polysulfide, and polyisoprene.

[0086] In the present embodiment, such a binder is contained in the photocatalyst-containing cell adhesion layer in an amount of 5 wt% to 95 wt%, particularly 40 wt% to 90 wt%, particularly 60 wt% to 80 wt%. It is preferable to be contained within the range.

[0087] In this embodiment, a light-shielding portion may be formed on the cell culture region of the base material, if necessary. Accordingly, when the entire surface of the photocatalyst-containing cell adhesion 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 region other than the region where the light shielding portion is formed is not excited. It is a force capable of decomposing or denaturing the cell adhesion material contained in the cell adhesion layer.

[0088] Such a light-shielding portion is not particularly limited as long as it can block the energy applied when the cell adhesion assisting portion is formed. For example, a sputtering method, a vacuum deposition method, or the like can be used. It may be formed by forming a metal thin film of chrome or the like having a thickness of about 1000 to 2000 A by a method or the like, and patterning the thin film. As a method of this puttering, a normal puttering method such as sputtering can be used.

[0089] Further, a method in which a layer containing light-shielding particles such as carbon fine particles, metal oxides, inorganic pigments, and organic pigments in a resin binder may be formed in a pattern. Examples of the resin binder used include one or a mixture of two or more resins such as polyimide resin, acrylic resin, epoxy resin, polyacrylamide, polybutyl alcohol, gelatin, casein, and cellulose. A reactive resin, or an OZW emulsion type resin composition, for example, an emulsion obtained by emulsifying a reactive silicone can be used. The thickness of such a resin light-shielding portion can be set within a range of 0.5 to 10 m. As a method of patterning the resin light-shielding portion, a commonly used method such as a photolithography method and a printing method can be used.

[0090] d. Method of forming cell adhesion auxiliary part

Next, a method for forming the cell adhesion assisting portion in this embodiment will be described. In the present embodiment, for example, as shown in FIG. 6, an energy 6 is applied to the photocatalyst-containing cell adhesion layer 7 containing the cell adhesion material and the photocatalyst by using, for example, a photomask 5 or the like. By irradiating in a pattern to form the adhesion-assisting portion (FIG. 6 (a)), the cell-adhesive material is decomposed or denatured in the cell-adhesive layer 7 and inhibits adhesion to cells. The part 4 can be formed (FIG. 6 (b)). In this case, the photocatalyst and the decomposed or denatured product of the cell adhesion material are contained in the cell adhesion auxiliary part.

[0091] Here, in the present embodiment, "energy irradiation (exposure)" includes irradiation with a single energy beam capable of decomposing or denaturing a cell adhesion material by the action of a photocatalyst accompanying the energy irradiation. This is a concept and is not limited to light irradiation.

[0092] The wavelength of light used for such energy irradiation is generally set to a range of 400 nm or less, preferably 380 nm or less. This is because, as described above, a preferred photocatalyst used as a photocatalyst is titanium dioxide, and light having the above-mentioned wavelength is preferred as energy for activating photocatalysis by the titanium dioxide. is there.

[0093] Examples of the light source that can be used for such energy irradiation include a mercury lamp, a metal halide lamp, a xenon lamp, an excimer lamp, and various other light sources.

[0094] In addition to the method of performing pattern irradiation through a photomask using the above-described light source, a method of performing pattern drawing irradiation using a laser such as excimer or YAG can also be used. In addition, as described above, when the substrate has the light-shielding portion in the same pattern as the cell adhesion portion, the substrate-side power can also be applied by irradiating the entire surface with energy. In this case, there is an advantage that a step such as alignment that requires a photomask or the like is not required.

[0095] The amount of energy irradiation at the time of energy irradiation is an irradiation amount necessary for the cell adhesion material to be decomposed or denatured by the action of a photocatalyst.

[0096] At this time, by irradiating the layer containing the photocatalyst with energy while heating, the sensitivity can be increased, and the cell adhesion material can be efficiently decomposed or denatured, which is preferable. . Specifically, heating within the range of 30 ° C-80 ° C is preferred! / ヽ

[0097] In the present embodiment, the direction of the energy irradiation performed through the photomask is, when the above-mentioned base material is transparent, from the direction of displacement between the base material side and the photocatalyst-containing cell adhesive layer side. Energy irradiation may be performed. On the other hand, when the substrate is opaque, it is necessary to perform energy irradiation from the photocatalyst-containing cell-adhesive layer side.

[0098] (2) Second aspect

Next, a cell adhesive layer containing at least a cell adhesive material is formed on the photocatalyst treatment layer containing at least a photocatalyst, and when the cell adhesive layer is irradiated with energy, the cell adhesive material in the cell adhesive layer is formed. Is decomposed or denatured by the action of the photocatalyst in the adjacent photocatalyst treatment layer.

[0099] In the present embodiment, since the cell adhesion layer is formed on the photocatalyst treatment layer, by irradiating energy in a pattern for forming a cell adhesion auxiliary part, the cell adhesion layer is formed in the cell adhesion layer. Since the cell adhesive material is decomposed or denatured by the action of the photocatalyst in the adjacent photocatalyst treatment layer and the adhesiveness to cells in the region is reduced, it becomes possible to use the cell adhesive as a cell adhesion auxiliary part. is there. At this time, if the cell adhesion material is decomposed by the action of a photocatalyst accompanying energy irradiation, for example, the cell adhesion auxiliary portion contains a small amount of the cell adhesion material or the cell adhesion material. The photocatalyst-treated layer is exposed due to the presence of a decomposed product or the like, or the cell adhesion layer is completely decomposed and removed. When the above-mentioned cell adhesive material is modified by the action of a photocatalyst accompanying energy irradiation, the modified substance and the like are contained in the cell adhesion auxiliary part.

[0100] Hereinafter, the cell adhesion layer and the photocatalyst treatment layer used in the present embodiment will be described.

The method for forming the cell adhesion assisting portion in this embodiment is the same as that in the first embodiment described above, and thus description thereof will be omitted.

[0101] a. Cell adhesion layer

First, the cell adhesion layer used in this embodiment will be described. The cell adhesive layer used in this embodiment is a layer having at least a cell adhesive material having an adhesive property to cells, and a layer generally used as a layer having an adhesive property to cells can be used.

[0102] As a specific cell adhesive material, the same material as the cell adhesive material used for the photocatalyst-containing cell adhesive layer described in the first embodiment can be used. Omitted. Further, the photocatalyst-containing cell adhesion of the first embodiment is also provided in the cell adhesion layer of the present embodiment. It is preferable that a material having the cell adhesion inhibitory property described in the layer is contained. This makes it possible to lower the adhesiveness of the cell adhesion assisting portion, which is the region irradiated with energy, to the cells.

[0103] Further, the formation of such a cell adhesion layer can be carried out by applying a coating solution for forming a cell adhesion layer containing the above-mentioned cell adhesion material by a general application method or the like. Since the method can be the same as the method for forming the photocatalyst-containing cell adhesive layer of the embodiment, the description thereof is omitted here.

[0104] The thickness of such a cell adhesive layer is appropriately selected depending on the type of the cell culture puttering substrate and the like. Normally, about 0.1 OOl / zm-l.O / zm, Above all, it can be set to about 0.005 μm-0.3 μm.

[0105] b. Photocatalyst treatment layer

Next, the photocatalyst treatment layer used in the present embodiment will be described. The photocatalyst treatment layer used in the present embodiment is not particularly limited as long as it is a layer containing at least a photocatalyst. The photocatalyst treatment layer may be a layer having only the power of the photocatalyst or a layer containing other components such as a binder. It may be.

The photocatalyst used in this embodiment can be the same as that used for the photocatalyst-containing cell adhesion layer in the first embodiment, and in this embodiment, titanium oxide is particularly used. Is preferred.

[0107] Here, when a photocatalyst treatment layer that can only act as a photocatalyst is used, the efficiency with respect to the decomposition or denaturation of the cell adhesion material in the cell adhesion layer is improved, which is advantageous in terms of cost such as shortening of treatment time. It is. On the other hand, when a photocatalyst treatment layer including a photocatalyst and a binder is used, there is an advantage that the formation of the photocatalyst treatment layer is easy.

[0108] Examples of a method for forming a photocatalyst treatment layer in which only a photocatalyst is effective include a method using a vacuum film forming method such as a sputtering method, a CVD method, and a vacuum evaporation method. By forming the photocatalyst treatment layer by the vacuum film formation method, it is possible to form a uniform film and a photocatalyst treatment layer containing only the photocatalyst, which enables the cell adhesion material to be uniformly decomposed or denatured. In addition, since only the photocatalyst becomes strong, the cell adhesion material can be decomposed or denatured more efficiently than in the case where a binder is used. [0109] Further, as another example of a method for forming a photocatalyst treatment layer comprising only a photocatalyst, for example, when the photocatalyst is titanium dioxide, amorphous titania is formed on a base material, and then the crystalline titania is formed by firing. A method of changing the phase to titania may be used. The amorphous titanium used herein includes, for example, hydrolysis, dehydration condensation of inorganic salts of titanium such as titanium tetrachloride and titanium sulfate, tetraethoxytitanium, tetraisopropoxytitanium, tetra-n-propoxytitanium, and tetrabutoxytitanium. Organic titanium conjugates such as titanium and tetramethoxytitanium can be obtained by hydrolysis and dehydration condensation in the presence of an acid. Then, it can be modified to anatase type titania by baking at 400 ° C to 500 ° C, and modified to rutile type titania by baking at 600 ° C to 700 ° C.

[0110] When a binder is used, a binder having a high binding energy such that the main skeleton of the binder is not decomposed by the photoexcitation of the photocatalyst is preferable. And the organopolysiloxanes described in the above section.

[0111] When the organopolysiloxane is used as a binder as described above, the photocatalyst treatment layer is formed by dispersing the organocatalyst, which is a photocatalyst, and a binder together with other additives as necessary. It can be formed by preparing a coating solution and applying the coating solution onto a substrate. As the solvent to be used, alcohol-based organic solvents such as ethanol and isopropanol are preferable. The coating can be performed by a known coating method such as spin coating, spray coating, dip coating, roll coating, and bead coating. In the case of containing an ultraviolet curable component as a solder, the photocatalyst treatment layer can be formed by performing a curing treatment by irradiating ultraviolet rays.

[0112] An amorphous silica precursor can be used as a binder. This amorphous silica precursor is represented by the general formula SiX, where X is a halogen, methoxy, ethoxy, or acetyl group.

Four

Preferred are silicon compounds such as hydroxyl groups, silanols which are hydrolysates thereof, and polysiloxanes having an average molecular weight of 3000 or less!

[0113] Specific examples include tetraethoxysilane, tetraisopropoxysilane, tetra-n-propoxysilane, tetrabutoxysilane, tetramethoxysilane and the like. In this case, the precursor of the amorphous silica and the particles of the photocatalyst are uniformly dispersed in the non-aqueous solvent to obtain a transparent resin. The photocatalyst-treated layer can be formed by forming a silanol on a bright substrate by hydrolysis with moisture in the air, followed by dehydration-condensation polymerization at room temperature. If the dehydration polycondensation of silanol is carried out at 1 oo ° C or more, the degree of polymerization of silanol increases and the strength of the film surface can be improved. These binders can be used alone or in combination of two or more.

[0114] The content of the photocatalyst in the photocatalyst treatment layer can be set in the range of 5 to 60% by weight, preferably 20 to 40% by weight. The thickness of the photocatalyst treatment layer is preferably in the range of 0.05-10 / zm.

[0115] The photocatalyst-treated layer may contain, in addition to the above-mentioned photocatalyst and binder, a surfactant and the like used for the above-mentioned cell adhesion layer.

Here, in the present embodiment, it is preferable that the surface of the photocatalyst-treated layer has low adhesion to cells, for example, low adhesion due to hydrophilicity of the surface. Thereby, when the photocatalyst treatment layer is exposed due to the decomposition of the cell adhesion layer or the like, the region can be a region having low adhesion to cells.

[0117] Further, in the present embodiment, as described above, a light shielding portion may be formed on the photocatalyst treatment layer. Thus, when the entire surface of the cell adhesive layer is irradiated with energy, the photocatalyst on the region where the light-shielding portion is formed is not excited, and is contained in the cell adhesive layer other than the region where the light-shielding portion is formed. A force that can degrade or denature cell adhesion materials. Further, in this case, since the photocatalyst in the region where the light-shielding portion is formed is not excited, there is an advantage that the direction of energy irradiation is not particularly limited.

[0118] As such a light-shielding portion, the same light-shielding portion as that described in the first embodiment can be used, and thus the detailed description thereof is omitted here.

(3) Third Embodiment

Next, a cell adhesive layer containing at least a cell adhesive material is formed on the substrate, and at the time of energy irradiation, at least a photocatalyst containing layer containing a photocatalyst is opposed to the cell adhesive layer, and energy is irradiated. The case where the cell adhesive material is decomposed or denatured by the action of the photocatalyst in the opposing photocatalyst-containing layer will be described.

[0120] In this embodiment, the cell adhesion layer and the photocatalyst-containing layer are arranged so as to face each other, and energy is irradiated in a pattern for forming a cell adhesion auxiliary portion, whereby the photocatalyst-containing layer is formed. By the action of the photocatalyst in the layered layer, the cell adhesive material in the cell adhesive layer is decomposed or denatured, and it becomes possible to form a cell adhesion auxiliary part.

[0121] Hereinafter, a photocatalyst-containing layer-side substrate used in the present embodiment and a method for forming a cell adhesion auxiliary portion using the photocatalyst-containing layer-side substrate will be described. The cell adhesion layer used in the present embodiment is the same as the cell adhesion layer used in the above-described second embodiment, and thus the description thereof will be omitted.

[0122] a. Photocatalyst containing layer side substrate

First, a photocatalyst-containing layer-side substrate having a photocatalyst-containing layer containing a photocatalyst used in this 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 usually has a substrate and a photocatalyst-containing layer formed on the substrate. The photocatalyst-containing layer-side substrate may include, for example, a photocatalyst-containing layer-side light-shielding portion or a primer layer formed in a pattern.

. Hereinafter, each configuration of the photocatalyst-containing layer-side substrate used in the present embodiment will be described.

(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 adhesion material in the adjacent cell adhesion layer. The film may be composed of a binder and a photocatalyst alone. In addition, the characteristics of the surface may be lyophilic or lyophobic.

[0124] The photocatalyst-containing layer used in this embodiment may be formed on the entire surface of the substrate, but, for example, as shown in Fig. 7, a photocatalyst-containing layer 12 is formed on the substrate 11 on a pattern. It may have been done.

[0125] By forming the photocatalyst-containing layer in a pattern in this manner, when irradiating energy to form the cell adhesion assisting portion, it is necessary to perform pattern irradiation using a photomask or the like over the entire surface. By irradiating, the cell adhesion material contained in the cell adhesion layer can be decomposed or denatured to form a cell adhesion auxiliary part.

[0126] The method for patterning the photocatalyst-containing layer is not particularly limited. This can be performed by a photolithography method or the like.

In addition, since only the portion of the cell adhesive layer that actually faces the photocatalyst-containing layer is decomposed or denatured, the direction of energy irradiation is such that the photocatalyst-containing layer and the cell adhesion layer face each other. Irradiation can be performed from any direction as long as the part is irradiated with energy.Furthermore, the irradiation energy is not particularly limited to parallel light such as parallel light. Become.

Here, the photocatalyst-containing layer used in the present embodiment can be the same as the photocatalyst-treated layer described in the second embodiment, and a detailed description thereof will be omitted. .

(Ii) Substrate

Next, the substrate used for the photocatalyst-containing layer-side substrate will be described. 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 base to be used is appropriately selected depending on the direction of energy irradiation described later, whether the obtained pattern formed body needs transparency, and the like.

[0129] The substrate used in the present embodiment may be a flexible substrate, for example, a resin film, or a non-flexible substrate, for example, a glass substrate. This is appropriately selected depending on the energy irradiation method.

[0130] An anchor layer may be formed on the substrate in order to improve the adhesion between the substrate surface and the photocatalyst-containing layer. Examples of such an anchor layer include silane-based and titanium-based coupling agents.

(Iii) Photocatalyst-containing layer-side light-shielding portion

The photocatalyst-containing layer-side substrate used in the present embodiment may be one having a photocatalyst-containing layer-side light-shielding portion formed in a pattern. By using the photocatalyst-containing layer-side substrate having the photocatalyst-containing layer-side light-shielding portion in this way, it is not necessary to use a photomask or perform drawing irradiation with a laser beam during energy irradiation. Therefore, there is no need to align the photocatalyst-containing layer-side substrate with the photomask, so that it is possible to simplify the process, and it is not necessary to use an expensive apparatus required for drawing irradiation, thereby reducing costs. It is advantageous when it is advantageous. [0132] The photocatalyst-containing layer-side substrate having such a photocatalyst-containing layer-side light-shielding portion can be in the following two modes depending on the position where the photocatalyst-containing layer-side light-shielding portion is formed.

For example, as shown in FIG. 8, a photocatalyst-containing layer-side light-shielding portion 14 is formed on a substrate 11, and a photocatalyst-containing layer 12 is formed on the photocatalyst-containing layer-side light-shielding portion 14. This is an embodiment in which a layer-side substrate is used. The other is a mode in which a photocatalyst-containing layer 12 is formed on a substrate 11 and a photocatalyst-containing layer-side light-shielding portion 14 is formed thereon to form a photocatalyst-containing layer-side substrate, as shown in FIG. 9, for example.

In any of the embodiments, the light-shielding portion on the photocatalyst-containing layer side is disposed near the position where the photocatalyst-containing layer and the cell adhesion layer are disposed, as compared with the case where a photomask is used. Since the influence of energy scattering in the body or the like can be reduced, it is possible to perform energy pattern irradiation extremely accurately.

Here, in the present embodiment, when the photocatalyst-containing layer-side light-shielding portion 14 is formed on the photocatalyst-containing layer 12 as shown in FIG. 9, the photocatalyst-containing layer and the cell adhesion layer are When the photocatalyst-containing layer-side light-shielding portion is arranged at the position indicated by the numeral, the film thickness of the photocatalyst-containing layer-side light-shielding portion is made equal to the width of the gap, so that the photocatalyst-containing layer-side light-shielding portion has a constant gap. If it can be used as a spacer, it has the following advantages.

That is, when the photocatalyst-containing layer and the cell adhesive layer are arranged facing each other with a predetermined gap, the photocatalyst-containing layer-side light-shielding portion and the cell adhesive layer are in close contact with each other. By arranging, the predetermined gap can be made accurate, and by irradiating energy in this state, the portion of the cell adhesive layer where the cell adhesive layer and the light shielding portion are in contact with each other is Since the adhesive material is not decomposed or denatured, it is possible to accurately form the cell adhesion auxiliary part.

[0137] The method of forming the light-blocking portion on the photocatalyst-containing layer side is not particularly limited, and may be appropriately determined according to the characteristics of the surface on which the light-blocking portion on the photocatalyst-containing layer side is formed, the shielding property against required energy, and the like. Since it can be selected and used and can be the same as the light-shielding portion provided on the base material described in the first embodiment, detailed description is omitted here.

[0138] In the above description, the two cases where the photocatalyst-containing layer-side light-shielding portion is formed are described between the substrate and the photocatalyst-containing layer and on the surface of the photocatalyst-containing layer. In addition, it is also possible to adopt a mode in which a photocatalyst-containing layer-side light-shielding portion is formed on the surface of the substrate on which the photocatalyst-containing layer is not formed. In this embodiment, for example, a case where a photomask is brought into close contact with the surface to such an extent that the photomask can be detached is considered, and it can be suitably used when the turn of the cell adhesion auxiliary portion is changed in a small lot.

(Iv) Primer layer

Next, the primer layer used in the photocatalyst-containing layer-side substrate of the present embodiment will be described. In this embodiment, as described above, in the case where the photocatalyst-containing layer-side substrate is formed by forming the photocatalyst-containing layer-side light-shielding portion on the substrate in a pattern and forming the photocatalyst-containing layer thereon. A primer layer may be formed between the light-shielding portion on the containing layer side and the photocatalyst containing layer.

[0140] The function and function of this primer layer are not always clear, but by forming the primer layer between the light-shielding portion and the photocatalyst-containing layer on the photocatalyst-containing layer side, the primer layer becomes a cell by the action of the photocatalyst. Impurities from the photocatalyst-containing layer-side light-shielding portion and the openings existing between the photocatalyst-containing layer-side light-shielding portions that cause degradation or denaturation of the adhesive material, particularly residues generated when patterning the photocatalyst-containing layer-side light-shielding portion It is considered to have a function of preventing diffusion of impurities such as metal and metal ions. Therefore, by forming the primer layer, the process of decomposing or denaturing the cell adhesion material proceeds with high sensitivity, and as a result, it is possible to obtain a cell adhesion auxiliary portion formed with high definition. You.

[0141] In this embodiment, the primer layer is intended to prevent impurities existing not only in the light-shielding portion on the photocatalyst-containing layer side but also in the openings formed between the light-shielding portions on the photocatalyst-containing layer from affecting the action of the photocatalyst. Therefore, it is preferable that the primer layer is formed over the entire light-shielding portion on the photocatalyst-containing layer side including the opening.

[0142] The primer layer in this embodiment is not particularly limited as long as the primer layer is formed so that the light-blocking portion on the photocatalyst-containing layer side of the substrate on the photocatalyst-containing layer side does not contact the photocatalyst-containing layer.

[0143] The material constituting the primer layer is not particularly limited, but an inorganic material that is not easily decomposed by the action of a photocatalyst is preferable. Specific examples include amorphous silica Can. When such an amorphous silica is used, the precursor of the amorphous silica is represented by the general formula SiX, wherein X is a halogen, a methoxy group, an ethoxy group, or an acetyl group.

Four

Silanols, which are silicon compounds that are groups, and hydrolysates thereof, or polysiloxanes having an average molecular weight of 3000 or less are preferable.

The thickness of the primer layer is preferably in the range of 0.001 μm to 1 μm, particularly preferably in the range of 0.001 μm to 0.1 μm.

[0144] b. Method of forming cell adhesion auxiliary part

Next, a method for forming the cell adhesion assisting portion in this embodiment will be described. In this embodiment, for example, as shown in FIG. 10, the cell adhesion layer 7 and the photocatalyst-containing layer 12 of the photocatalyst-containing layer-side substrate 13 are arranged at a predetermined gap, for example, a photomask 5 or the like. Is used to irradiate energy 6 from a predetermined direction. As a result, the cell adhesive material in the area irradiated with the energy is decomposed or denatured, and the cell adhesion auxiliary part 4 that inhibits adhesion to cells is formed in the cell adhesion layer 7. At this time, if the cell adhesive material is decomposed by the action of a photocatalyst accompanying energy irradiation, the cell adhesive material contains a small amount of the cell adhesive material. Or the cell adhesion material is decomposed, or the cell adhesion layer is completely released and the substrate is exposed. When the above-mentioned cell adhesive material is modified by the action of a photocatalyst accompanying energy irradiation, the modified substance or the like is contained in the cell adhesion auxiliary part.

[0145] The above-mentioned arrangement refers to a state in which the photocatalyst is substantially placed on the surface of the cell adhesive layer, and in addition to a state in which it is in actual physical contact, The photocatalyst-containing layer and the cell adhesion layer are arranged at an interval. This gap is preferably 200 μm or less.

[0146] In the present embodiment, the gap is particularly excellent in consideration of the fact that the pattern accuracy is extremely good, the sensitivity of the photocatalyst is high, and the efficiency of decomposition or denaturation of the cell adhesive material in the cell adhesive layer is good. It is preferable to be within the range of 2 μη−10 μm, preferably within the range of 1 μm−5 μm. Such a range of the gap is particularly effective for a small-area cell adhesion layer capable of controlling the gap with high accuracy. [0147] On the other hand, when processing is performed on a cell adhesion layer having a large area of, for example, 300 mm x 300 mm or more, a fine gap as described above is formed between the photocatalyst-containing layer-side substrate and the cell adhesion layer without contact. It is extremely difficult to form between them. Therefore, when the cell adhesion layer has a relatively large area, the gap is preferably in the range of 10 to 100 / zm, particularly preferably in the range of 50 to 75 m. By setting the gap within such a range, problems such as a decrease in pattern accuracy such as blurring of the pattern and a decrease in the efficiency of decomposing or denaturing the cell adhesive material due to a decrease in the sensitivity of the photocatalyst are caused. It is also a force that has the effect of preventing unevenness in the decomposition or denaturation of the cell adhesive material without causing any problem.

[0148] When irradiating a relatively large area of the cell adhesive layer with energy as described above, the gap in the positioning device between the photocatalyst-containing layer-side substrate and the cell adhesive layer in the energy irradiation device is set to 10 µm- It is preferable to set within the range of 200 μm, especially within the range of 25 μm-75 μm. By setting the set value within such a range, it is possible to prevent the cell-adhering layer from being in contact with the photocatalyst-containing layer-side substrate without causing a significant decrease in pattern accuracy or a significant deterioration in photocatalytic sensitivity. This is because it is possible to dispose them.

[0149] By arranging the photocatalyst-containing layer and the cell adhesion layer surface at a predetermined distance in this manner, oxygen and water and active oxygen species generated by the photocatalysis can be easily desorbed. That is, if the distance between the photocatalyst-containing layer and the cell adhesive layer is narrower than the above range, it becomes difficult to desorb the active oxygen species, and as a result, the rate at which the cell adhesive material is decomposed or denatured is reduced. It is not preferable because it may be delayed. In addition, if the distance is larger than the above range, the generated reactive oxygen species may not easily reach the cell adhesive layer, and in this case, the rate of decomposition or denaturation of the cell adhesive material may be reduced. Not preferred from

[0150] An example of a method of forming such an extremely narrow gap uniformly and arranging the photocatalyst-containing layer and the cell adhesion layer includes a method using a spacer. By using the spacer in this manner, a uniform gap can be formed, and the portion where the spacer comes into contact is not affected by the action of the photocatalyst on the surface of the cell adhesive layer. By making the spacer have the same pattern as the above-mentioned cell adhesion portion, The cell-adhesive material of only the portion where the psa is formed can be decomposed or denatured, and the cell-adhesion assisting portion can be formed with high definition. In addition, by using such a spacer, the active oxygen species generated by the action of the photocatalyst reach the surface of the cell adhesive layer at a high concentration without being diffused. Can be formed.

[0151] In the present embodiment, such an arrangement state of the photocatalyst-containing layer-side substrate should be maintained at least only during irradiation with one energy.

[0152] Here, in the present embodiment, "energy irradiation (exposure)" also includes irradiation with a single energy beam capable of decomposing or denaturing a cell adhesive material by the action of a photocatalyst accompanying the energy irradiation. This is a concept and is not limited to light irradiation.

[0153] Here, the type of energy applied in this embodiment is the same as that described in the above-described first embodiment, and thus the details and description thereof will be omitted.

[0154] In the present embodiment, the direction of energy irradiation performed through the photomask is such that, when the above-described base material is transparent, the energy irradiation is performed from the direction of displacement between the base material side and the photocatalyst containing layer side substrate. May be. On the other hand, when the substrate is opaque, it is necessary to perform energy irradiation from the photocatalyst-containing layer side substrate side.

[0155] II. Case (2)

Next, a cell adhesion inhibitory layer containing a cell adhesion inhibitory material which has a cell adhesion inhibitory property of inhibiting adhesion to cells and which is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation. A description will be given of a case where the cell adhesion inhibiting material is formed by decomposing or denaturing the cell adhesion inhibiting material by irradiating energy after the formation. In this case, the following three embodiments can be mentioned.

[0156] In a first embodiment, the cell adhesion-inhibiting layer is a photocatalyst-containing cell adhesion-inhibiting layer containing a cell adhesion-inhibiting material that inhibits adhesion to cells and a photocatalyst. By irradiating energy in a pattern that forms the cell adhesion layer, the cell adhesion inhibition material is decomposed or modified by the action of the photocatalyst contained in the photocatalyst-containing cell adhesion inhibition layer itself to form a cell adhesion layer. .

[0157] As a second embodiment, a cell adhesion-inhibiting layer containing at least a cell adhesion-inhibiting material, It is formed on a photocatalyst-treated layer containing at least a photocatalyst. By irradiating this cell adhesion-inhibiting layer with energy in a pattern for forming a cell-adhesive layer, cell adhesion is caused by the action of the photocatalyst contained in the photocatalyst-treated layer This is the case where the inhibitory material is a degraded or denatured cell adhesion layer.

[0158] In a third embodiment, a cell adhesion-inhibiting layer containing at least a cell adhesion-inhibiting material is formed on a substrate, and the cell adhesion-inhibiting layer and at least a photocatalyst-containing layer containing a photocatalyst are combined with a cell. In this case, the cell adhesion-inhibiting material is decomposed or denatured to form a cell-adhesion layer by irradiating energy in a pattern that forms the cell-adhesion layer in opposition to the adhesion-inhibition layer.

Hereinafter, each of the aspects will be described separately.

(1) First Embodiment

First, the cell adhesion-inhibiting layer is a cell adhesion-inhibiting layer that inhibits adhesion to cells and a photocatalyst-containing cell adhesion-inhibiting layer containing a photocatalyst. A case in which the cell adhesion inhibiting material is decomposed or denatured by the action of the photocatalyst contained in the photocatalyst treatment layer by irradiating energy in a manner to form the cell adhesion layer will be described.

[0160] According to this embodiment, since the photocatalyst-containing cell adhesion-inhibiting layer contains the photocatalyst and the above-mentioned cell adhesion-inhibiting material, the photocatalyst-containing cell adhesion-inhibiting layer is irradiated with energy, whereby the cell adhesion-inhibiting material is obtained. Can be decomposed or denatured by the action of a photocatalyst, and the area irradiated with energy can be used as a cell adhesion portion having adhesiveness to cells, that is, a cell adhesion layer. At this time, a region not irradiated with energy can be used as a cell adhesion assisting portion.

[0161] Such formation of the photocatalyst-containing cell adhesion inhibitory layer is performed by using a cell adhesive material that is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation and a coating solution for forming a photocatalyst-containing cell adhesion inhibitory layer containing a photocatalyst. It can be performed by, for example, applying to a cell culture region. The application of the coating solution for forming the photocatalyst-containing cell adhesion inhibiting layer can be performed by a general coating method, for example, a spin coating method, a spray coating method, a dip coating method, a roll coating method, a bead coating method. Etc. can be used. [0162] At this time, the thickness of the photocatalyst-containing cell adhesion-inhibiting layer is selected appropriately depending on the type of the cell culture patterning substrate and the like, and is generally about 0.1 Ol / zm-l. Above all, a force of about 0.1 μm-0.3 μm can be achieved.

[0163] Hereinafter, the cell adhesion inhibiting material will be described, and further, a method for forming the cell adhesion layer will be described. Note that the photocatalyst used in the present embodiment can be the same as the photocatalyst used in the first embodiment of “1. (1)” described above, and a detailed description thereof will be omitted.

a.Cell adhesion inhibitor

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

[0164] The cell adhesion-inhibiting material used in this embodiment has a cell adhesion-inhibiting property of inhibiting adhesion to cells and is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation. The type and the like are not particularly limited.

[0165] Here, having the cell adhesion inhibitory property means that the cell has a property of inhibiting the cell from adhering to the cell adhesion inhibitory material, that is, when the adhesiveness to the cell differs depending on the type of the cell. And the like means having the property of inhibiting adhesion to a target cell.

[0166] The cell adhesion-inhibiting material used in this embodiment has such cell adhesion-inhibiting properties, and is degraded or denatured by the action of a photocatalyst accompanying energy irradiation, and has no cell adhesion-inhibiting properties. And those having good adhesion to cells are used.

[0167] As such a cell adhesion inhibiting material, for example, a material having a high hydration ability can be used. A material with high hydration ability forms a hydration layer in which water molecules are gathered around it. Generally, a substance with such high hydration ability has a higher adhesiveness to water molecules than an adhesiveness to cells. Therefore, the cells cannot adhere to the material having a high hydration ability and have low adhesion to the cells. Here, the above-mentioned hydration ability means a property of hydration with water molecules, and a high hydration ability means that it is easy to hydrate with water molecules.

[0168] Examples of the material having a high hydration ability and used as a cell adhesion inhibiting material include polyethylene glycol, zwitterionic materials having a betaine structure and the like, and phospholipid-containing materials. When such a material is used as the cell adhesion inhibiting material, it will be described later. In the energy irradiation step, when the cell is irradiated with energy, the cell adhesion inhibiting material is decomposed or deteriorated by the action of a photocatalyst, and the hydration layer on the surface is separated, so that the cell adhesion inhibiting property is not obtained. , It can be.

In the present embodiment, a surfactant having a water-repellent or oil-repellent organic substituent which can be decomposed by the action of a photocatalyst can also be used as the above-mentioned cell adhesion-inhibiting material. Examples of such a surfactant include hydrocarbons such as NIKKOL BL, BC, BO, and BB series manufactured by Nikko Chemicals Co., Ltd., ZONYL FSN and FS0 manufactured by DuPont, and Surflon S manufactured by Asahi Glass Co., Ltd. — 141, 145, MegaFac F-141, 144, manufactured by Dainippon Ink & Chemicals, Inc., Futagent F-200, F251, manufactured by Neos Corporation, Dudyne DS-401, 402, manufactured by Daikin Industries, Ltd. Examples include fluorine-based or silicone-based nonionic surfactants such as Florad FC-170 and 176 manufactured by K.K., and cationic surfactants, anionic surfactants, and amphoteric surfactants. It can also 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 unevenly distributed on the surface. As a result, the water repellency and oil repellency of the surface can be increased, and the interaction with the cell, which has a small interaction with the cell, can be reduced. When the layer is irradiated with energy in the energy irradiation step, the layer is easily decomposed by the action of the photocatalyst to expose the photocatalyst and not have the cell adhesion inhibitory property. You can.

[0171] In the present embodiment, it is particularly preferable to use, as the above-mentioned cell adhesion-inhibiting material, a material having good adhesion to cells by the action of a photocatalyst accompanying energy irradiation. Examples thereof include materials having oil repellency and water repellency.

[0172] When the above-mentioned water-repellent or oil-repellent material is used as the cell adhesion-inhibiting material, the water-repellent or oil-repellent properties of the cell-adhesion-inhibiting material, such as hydrophobicity, Adhesion with cells with small interaction such as sexual interaction can be reduced.

[0173] As such a material having water repellency or oil repellency, for example, the skeleton has a photocatalytic action. Examples thereof include those having a high binding energy that are not decomposed by water and those having a water-repellent or oil-repellent organic substituent that is decomposed by the action of a photocatalyst.

[0174] Examples of those having a high binding energy such that the skeleton is not decomposed by the action of a photocatalyst and having a water-repellent or oil-repellent organic substituent capable of being decomposed by the action of a photocatalyst include those described above. (1) An organopolysiloxane that exerts high strength by hydrolyzing and polycondensing the lip or alkoxysilane etc. by sol-gel reaction, etc. (2) Reaction And organopolysiloxanes obtained by crosslinking functional silicone.

[0175] When such a substance is used as a binder in "1. (1)", the side chain of the above-mentioned organopolysiloxane or the like is acted upon by a photocatalyst accompanying energy irradiation to a high rate. It is used as a material having cell adhesion inhibitory property by decomposing or denaturing to make it super-hydrophilic.

[0176] On the other hand, in the present embodiment, when used as a cell adhesion-inhibiting material, the side chains of the above-mentioned organopolysiloxane and the like are not decomposed or denatured completely by the action of a photocatalyst accompanying energy irradiation. By irradiating the energy, the region irradiated with the energy can have adhesiveness to cells.

[0177] Here, when the above-mentioned material is used as a cell adhesion-inhibiting material, a material having a contact angle with water of 80 ° or more, particularly in the range of 100 ° to 130 °, is usually used as the cell adhesion-inhibiting material. Is preferred. Thereby, the adhesiveness to cells can be reduced. Note that the upper limit of the angle is the upper limit of the contact angle of the cell adhesion-inhibiting material with water on a flat substrate, for example, with the water of the cell adhesion-inhibiting material on a substrate having irregularities. When the contact angle is measured, the upper limit is about 160 ° as shown in, for example, Material Japanese 'Journal of Applied' Physics, Part 2, Volume 32, L614-L615, 1993 Ogawa et al. In some cases.

[0178] When the material for inhibiting cell adhesion is irradiated with energy so as to have adhesiveness to cells, the contact angle with water is in the range of 10 ° to 40 °, particularly 15 ° to 30 °. It is preferable to irradiate the energy so as to be within. As a result, adhesion to cells is high. It is because it can be. The contact angle with water can be obtained by the method described above.

[0179] A stable organosilicon conjugate, which does not undergo a crosslinking reaction like dimethylpolysiloxane, may be separately mixed with the above-mentioned organopolysiloxane and the like.

[0180] By using the reactive silicone as described above, water repellency and oil repellency can be increased, and interaction with cells is small and adhesion to cells is low. it can. In addition, when energy is irradiated to the above-mentioned materials, the substituents can be easily removed to introduce OH groups and the like to the surface, and interaction with cells can be increased. Thus, the adhesiveness to cells can be improved.

[0181] Such a cell adhesion-inhibiting material is preferably contained in the photocatalyst-containing cell adhesion-inhibiting layer in the range of 0.01% by weight to 95% by weight, particularly preferably 1% by weight to 10% by weight. This is a force that enables the region containing the cell adhesion inhibiting material to be a region having low adhesion to cells.

[0182] The cell adhesion-inhibiting material preferably has surface activity. For example, when a coating solution for forming a photocatalyst-containing cell adhesion inhibitor layer containing the above-mentioned cell adhesion inhibitor material is applied and then dried, the rate of uneven distribution on the surface of the coating film increases, resulting in good cell adhesion. This is because inhibitory properties can be obtained.

[0183] b. Other

In addition, the photocatalyst-containing cell adhesion-inhibiting layer of the present embodiment contains a binder or the like in accordance with required properties such as coatability when forming the layer, strength and resistance when forming the layer, and the like. May be. Further, the cell adhesion-inhibiting material may function as the binder.

As such a binder, for example, a binder having such a high binding energy that the main skeleton is not decomposed by the action of the photocatalyst can be used. Specifically, polysiloxane having no organic substituent or having a small amount of organic substituent that does not affect the adhesion can be mentioned, such as tetramethoxysilane and tetraethoxysilane. Can be obtained by hydrolysis and polycondensation.

[0185] In the present embodiment, such a binder is contained in the photocatalyst-containing cell adhesion-inhibiting layer in an amount of 5% by weight. % -95% by weight, especially 40% -90% by weight, particularly 60% -80% by weight. This makes it possible to exhibit properties such as facilitating formation of the photocatalyst-containing cell adhesion inhibition layer and imparting strength to the photocatalyst-containing cell adhesion inhibition layer.

[0186] In this embodiment, it is particularly preferable that the photocatalyst-containing cell adhesion-inhibiting layer contains a cell-adhesive material having adhesive properties to cells after at least energy irradiation. Thus, the photocatalyst-containing cell adhesion-inhibiting layer is a force capable of improving the adhesion of the cell adhesion portion, which is the area irradiated with energy, that is, the cell adhesion layer to the cells. Such a cell adhesive material may be used as the above-mentioned binder, or may be used separately from the binder. Further, for example, the force before irradiation with energy may be one having good adhesion to cells. Even one having good adhesion to cells by the action of a photocatalyst accompanying energy irradiation may be used. Good. Here, having the above-mentioned adhesive property with the cell means that the cell adheres well to the cell, and when the adhesive property with the cell differs depending on the type of the cell, etc., it should adhere well to the target cell. ,

[0187] In the present embodiment, if the above-mentioned cell adhesive material has good adhesiveness to cells at least after being irradiated with energy, the adhesion to cells may be, for example, hydrophobic interaction or electrostatic. It may be good due to physical interaction such as interaction, hydrogen bonding, van der Waals force, etc. .

[0188] In the present embodiment, such a cell adhesion material is contained in the photocatalyst-containing cell adhesion inhibition layer.

It is preferable that the content is 0.01% by weight to 95% by weight, particularly 1% by weight to 10% by weight. Accordingly, the photocatalyst-containing cell adhesion-inhibiting layer is also capable of improving the adhesiveness of the cell adhesion layer, which is a region irradiated with energy, to cells. In addition, when a material having good adhesiveness to the capillar cells before the energy irradiation is used as the cell adhesive material, the cell adhesion inhibition in the region not irradiated with the energy, that is, the region serving as the cell adhesion auxiliary part. It is preferably contained to such an extent that the cell adhesion inhibitory property of the material is not inhibited. [0189] c. Method for forming cell adhesion layer

Next, a method for forming the cell adhesion layer will be described. In this embodiment, for example, as shown in FIG. 11, a photomask containing a photocatalyst-containing cell adhesion-inhibiting layer 8 containing the cell adhesion-inhibiting material and the photocatalyst formed on the cell culture region on the substrate 1 Using 5 or the like, irradiate energy 6 in a pattern to form a cell adhesion layer (cell adhesion part) (Fig. 11 (a)). As a result, the region irradiated with energy can be made into a cell adhesion layer (cell adhesion portion) 7 having a cell adhesion-inhibiting material degraded or denatured and having adhesiveness to cells, and is not irradiated with energy. It can be a cell adhesion assistant 4 that inhibits the region from adhering to cells. At this time, the cell adhesion portion contains a photocatalyst, a decomposed product or a modified product of the cell adhesion inhibiting material, and the like.

[0190] Here, in the present embodiment, "energy irradiation (exposure)" refers to irradiation of a line of energy capable of decomposing or denaturing a cell adhesion-inhibiting material by the action of a photocatalyst accompanying the energy irradiation. And is not limited to light irradiation.

[0191] Note that such an energy irradiation method and the like are the same as those described in the first embodiment of the above-mentioned "1. (1) case", and thus the details and description thereof are omitted here. I do.

[0192] (2) Second aspect

Next, a cell adhesion-inhibiting layer containing at least a cell adhesion-inhibiting material is formed on the photocatalyst-treated layer containing at least a photocatalyst, and the cells are irradiated with energy in a pattern to form the cell adhesion layer. The case where the adhesion-inhibiting material is a decomposed or denatured cell adhesion layer will be described.

[0193] In the present embodiment, since the cell adhesion inhibiting layer is formed on the photocatalyst treatment layer, the photocatalyst contained in the photocatalyst treatment layer is excited by irradiating the cell adhesion inhibition layer with energy. In addition, the cell adhesion-inhibiting material in the cell adhesion-inhibiting layer can be decomposed or denatured, and a cell adhesion portion (cell adhesion layer) can be formed. Further, at this time, a region where the energy is not irradiated and the cell adhesion inhibiting material remains can be used as a cell adhesion auxiliary part.

[0194] Here, "the cell adhesion-inhibiting material is degraded or denatured!" Means that the cell adhesion-inhibiting material is contained, or that the cell adhesion-inhibiting material is contained in the cell adhesion-assisting layer. Compared to the amount of the inhibitory material, the cell adhesion inhibitory material contained a smaller amount. For example, when the cell adhesion inhibiting material is decomposed by the action of a photocatalyst accompanying energy irradiation, the cell adhesion portion contains a small amount of the cell adhesion inhibiting material, or the cell adhesion inhibiting material It means that decomposed products and the like are contained. Further, when the above-mentioned cell adhesion inhibiting material is modified by the action of a photocatalyst accompanying energy irradiation, the modified substance or the like is contained in the cell adhesion portion. In this embodiment, it is preferable that the cell adhesion portion contains a cell adhesion substance having an adhesive property to cells after at least energy irradiation. Thereby, the adhesiveness between the cell adhesion portion and the cell can be further enhanced, and the cell can be adhered to only the cell adhesion portion with high definition.

[0195] Hereinafter, the cell adhesion inhibiting layer used in the present embodiment will be described. The photocatalyst treatment layer used in the present embodiment can be the same as that described in the second embodiment of the above-mentioned “1. The forming method can be the same as in the first embodiment described above, and the description is omitted here.

[0196] (Cell adhesion inhibition layer)

The cell adhesion-inhibiting layer used in this embodiment is formed on the photocatalyst-treated layer, has a cell adhesion-inhibiting property of inhibiting adhesion to cells, and is decomposed or degraded by the action of a photocatalyst accompanying energy irradiation. It is not particularly limited as long as it contains a cell adhesion inhibiting material to be denatured.

In the present embodiment, the formation method and the like are not particularly limited as long as such a layer can be formed. For example, a coating for forming a cell adhesion inhibition layer containing the above-mentioned cell adhesion inhibition material The solution can be formed by applying the solution to the cell culture area by a general application method. Further, the thickness of such a cell adhesion-inhibiting layer is appropriately selected depending on the kind of the cell culturing pattern substrate, etc. Usually, the force is about 0.001 m-1.0 μm, and especially 0.005 μm. m— 0.3 μm.

[0198] Here, as a specific cell adhesion-inhibiting material used for the cell adhesion-inhibiting layer formed according to this embodiment, the photocatalyst-containing cell adhesion-inhibiting layer described in the first embodiment is used. The same material as the cell adhesion inhibitor can be used. Omitted. Further, it is preferable that the cell adhesion-inhibiting layer of the present embodiment also contains the material having the cell adhesion described in the photocatalyst-containing cell adhesion-inhibiting layer described in the first embodiment. Thereby, the adhesiveness with the cells of the cell adhesion portion (cell adhesion layer), which is the region irradiated with energy, can be enhanced.

(3) Third Mode

Next, a cell adhesion-inhibiting layer containing at least a cell adhesion-inhibiting material is formed on the substrate, and the cell adhesion-inhibiting layer and at least a photocatalyst-containing layer containing a photocatalyst are opposed to the cell adhesion-inhibiting layer. The case where the cell adhesion-inhibiting material is decomposed or denatured to form a cell adhesion layer by irradiating energy in a pattern for forming the cell adhesion layer will be described.

[0200] In the present embodiment, since the cell adhesion inhibiting layer decomposed or denatured by the action of a photocatalyst accompanying energy irradiation is contained in the cell adhesion inhibiting layer, the cell adhesion inhibiting layer And the photocatalyst-containing layer are arranged to face each other, and energy is irradiated in a pattern that forms the cell adhesion layer (cell adhesion part), thereby inhibiting cell adhesion by the action of the photocatalyst in the cell photocatalyst-containing layer. The cell adhesion-inhibiting material in the layer is decomposed or denatured to form a cell adhesion layer (cell adhesion part). At this time, since the cell adhesion-inhibiting material remains in the region where the energy is not irradiated, it can be prevented from adhering to the cells, and can be used as a cell adhesion auxiliary part. is there.

[0201] Here, "the cell adhesion-inhibiting material is degraded or denatured!" Means that the cell adhesion-inhibiting material is contained, or that the cell adhesion-inhibiting material is contained in the cell adhesion auxiliary layer. That the cell adhesion-inhibiting material is contained in a smaller amount than the amount of the cell adhesion inhibitor. For example, when the above-mentioned cell adhesion inhibiting material is decomposed by the action of a photocatalyst accompanying energy irradiation, the cell adhesion portion (cell adhesion layer) contains a small amount of the cell adhesion inhibiting material, or That is, a decomposition product of the cell adhesion inhibiting material or the like is contained. When the above-mentioned cell adhesion-inhibiting material is modified by the action of a photocatalyst accompanying energy irradiation, the cell adhesion portion contains a modified product thereof. In this embodiment, at least energy irradiation is performed on the cell adhesion portion. After that, it is preferable to contain a cell adhesive substance having an adhesive property to cells. Thereby, the adhesiveness between the cell adhesion portion and the cell can be further improved, and the cell can be adhered to only the cell adhesion portion with high definition.

[0202] The cell adhesion-inhibiting layer used in this embodiment is the same as the cell adhesion-inhibiting layer described in the second embodiment. For the substrate on the photocatalyst-containing layer side and its arrangement, see "1. )) ", The detailed description is omitted here. Further, the method of irradiating energy and the like are the same as those in the first embodiment, and thus description thereof will be omitted.

[0203] B. Second embodiment

Next, a second embodiment of the puttering substrate for cell culture of the present invention will be described. A second embodiment of the puttering substrate for cell culture of the present invention comprises a substrate, a cell adhesion region formed on the substrate and culturing cells, and having a cell adhesion property to the cells. A cell culture putter substrate having a cell culture region,

The cell adhesive layer is formed in a pattern having edges with irregularities. The puttering substrate for cell culture according to the present embodiment has a substrate 1 and a cell culture region 2 formed on the substrate 1 as shown in FIG. The end part a of the cell adhesive layer 7 formed in this manner is formed in a pattern having irregularities.

[0204] As described above, when cells are attached to the cell culture region, the cells are regularly arranged from the end with a morphological change, and a tissue is formed. Here, when culturing cells, when cells are adhered along a straight line, and when cells are adhered along an uneven line, cells are adhered along an irregular line. In this case, the morphological changes of the cells are activated and the cells can be arranged regularly. According to this embodiment, since the end of the cell adhesive layer where cells start to be arranged is formed in a pattern having irregularities, cells attached to the end can be activated. It is possible to arrange the cells regularly and well.

[0205] Hereinafter, each configuration of the pattern culturing substrate for cell culture according to the present embodiment will be described. The substrate used in this embodiment is the same as that used in the first embodiment described above. V, which is the same as that described above, and a detailed description thereof will be omitted here.

[0206] 1. Cell culture area

First, a cell culture region on the putter substrate for cell culture of the present embodiment will be described. The cell culture region in the patterning substrate for cell culture according to the present embodiment is a region for culturing cells and is not particularly limited as long as it has a cell adhesive layer formed in a pattern having irregularities at the ends. What is not done.

[0207] Here, the cell culture region is formed on the base material, and the portion of the base material other than the cell culture region is a cell non-culture region that inhibits adhesion to cells. In addition, the edge of the cell adhesion layer is usually a boundary between the cell culture region and the cell non-culture region (indicated by a in FIG. 12).

[0208] In the present embodiment, the entire edge of the cell adhesion layer formed in the cell culture region may be provided with irregularities. For example, as shown in FIG. Only the one with irregularities may be used! / ,.

[0209] The unevenness formed at the end of the cell adhesive layer is preferably such that the cells adhered to the cell adhesive layer can be regularly arranged. It is preferable that the distance between the concave end force and the convex end is formed so that the cells are linearly aligned when the cells are attached to the cell adhesive layer.

[0210] The specific size of the unevenness is appropriately selected depending on the shape of the cells to be cultured and the like, but usually, the average of the distance from the concave end to the short end of the unevenness is 0.5 m— It is preferably within a range of 30 m, especially 1 μm-5 μm. Accordingly, when the cells are cultured, the cells can be cultured in a desired shape without lacking the cells at the end of the cell culture region and a tissue can be formed. Here, the average measurement of the distance from the concave end to the convex end of the unevenness was measured by measuring the distance from the bottom of each unevenness to the top of each unevenness in the range of 200 μm between the cell adhesion part and the cell adhesion auxiliary part. This is the calculated value of the average.

[0211] The cell adhesive layer having such an end is not particularly limited as long as it is a layer having an adhesive property to cells. The method for forming the cell adhesion layer is not particularly limited as long as the method is capable of forming the above-mentioned end portion. A method of applying a coating solution for forming a cell adhesive layer containing a material having adhesive properties by a general printing method, or a method of applying the coating solution for forming a cell adhesive layer, followed by a pattern lithography method or the like. And the like. In the present embodiment, as described in the first embodiment, a cell adhesive layer containing a cell adhesive material that is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation is formed. The cell adhesion layer may be putterung. Further, as described in the first embodiment, a cell adhesion-inhibiting layer containing a cell adhesion-inhibiting material having cell-adhesion-inhibiting properties is formed, and this cell adhesion-inhibiting layer is formed by the action of a photocatalyst accompanying energy irradiation. The cell adhesion layer may be formed by decomposing or denaturing the inhibitor.

[0212] Since these methods, materials, and the like are the same as those in the first embodiment, detailed description thereof will be omitted.

[0213] Here, also in the present embodiment, it is preferable that the cell adhesion auxiliary portion described in the first embodiment is formed in the cell culture region. Thereby, cells can be cultured efficiently and a large-area tissue or the like can be formed.

[0214] 2. Patterning substrate for cell culture

Next, the putter substrate for cell culture according to the present embodiment will be described. The pattern culturing substrate for cell culture of the present embodiment is not particularly limited as long as the cell adhesion layer having the above-mentioned end is formed on the above-mentioned base material. A member such as a light shielding portion may be formed.

[0215] The present invention is not limited to the above embodiment. The above embodiment is an exemplification, and has substantially the same configuration as the technical idea described in the claims of the present invention. Included in the technical scope of the invention.

Example

Hereinafter, the present invention will be described more specifically with reference to Examples.

[Example 1]

Open B 咅 width 60/60 πι, shading 咅 width S 300m, 60m / 300m line & switch A photomask having a pace and formed so that the boundary between the opening and the light-shielding portion had irregularities of 1 μm square was formed.

Next, 5 g of trimethoxymethylsilane TSL8114 (manufactured by GE Toshiba Silicone Co., Ltd.) and 2.5 g of 0.5 N hydrochloric acid were mixed and stirred for 8 hours. This was diluted 10-fold with isopropyl alcohol to obtain a primer layer composition. This primer layer composition was applied onto the pattern surface of a photomask by a spin coating method, and the substrate was dried at a temperature of 150 ° C. for 10 minutes to form a primer layer.

Next, 30 g of isopropyl alcohol, 3 g of trimethoxymethylsilane TSL8114 (manufactured by GE Toshiba Silicone Co., Ltd.) and 20 g of a photocatalytic inorganic coating agent ST-K03 (Ishihara Sangyo) were mixed and stirred at 100 ° C. for 20 minutes. This was diluted 3-fold with isopropyl alcohol to obtain a photocatalyst-containing layer composition. The composition for a photocatalyst-containing layer is applied to a photomask substrate on which a primer layer is formed by a spin coater, and dried at 150 ° C. for 10 minutes to obtain a photomask having a transparent photocatalyst-containing layer. A mask was formed.

[0217] <Method of Making Patterning Substrate for Cell Culture>

(Formation of cell adhesion inhibition layer)

Organosilane TSL-8114 (GE Toshiba Silicone Co., Ltd.) 5. Og, fluoroalkylsilane TSL-8233 (1.5 g, GE Toshiba Silicone Co., Ltd.) 1.5 g, and 0.005N hydrochloric acid 2.36 g were mixed and stirred for 24 hours. . This solution was diluted 100-fold with isopropyl alcohol, applied to a quartz substrate that had been alkali-treated in advance by spin coating, and dried at 150 ° C for 10 minutes to conduct hydrolysis and polycondensation reactions. By proceeding, a substrate having a cell adhesion inhibiting layer having a thickness of 0.2; zm was obtained.

[0218] (Putter Jung on Putter Jung Substrate)

The cell adhesion layer of the substrate and to face the photocatalyst-containing organic layer of photomask having a photocatalyst-containing layer described above, performs ultraviolet exposure energy amount of 6JZcm ² by a mercury lamp through a photomask, unexposed portions A cell culture patterning substrate having a cell adhesion inhibitory property and having a cell adhesive surface patterned so that the exposed portion has the cell adhesive property was obtained.

[0219] (Culture of cells) The above-mentioned putter substrate for cell culture was immersed in DMEM medium supplemented with 10% fetal bovine serum, and primary human umbilical vein cells (HUVEC) were seeded. The cells were cultured at 37 ° C in a 5% carbon dioxide environment for 16 hours, and the cells were 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 in the cell culture region and further exhibited an extended shape.

Furthermore, the DMEM medium was exchanged for one that had been mashed at a concentration of bFGF (Sigma) lOngZml, and cultivation was continued at 37 ° C in a 5% diacid carbon environment for 24 hours to form a continuous capillary tissue. I confirmed that I did.

[0220] [Comparative Example 1]

Cell culture was performed in the same manner as in Example 1, except that the photomask was only 60 μm / 300 μm lines and spaces and had no irregularities at the boundary between the opening and the light-shielding portion. It was confirmed that the adhesion of the cells to the substrate 16 hours after seeding of the cells was smaller than that in Example 1. When the culture was performed for up to 24 hours, it was confirmed that the number of cells adhered to the substrate increased, but the degree of cell orientation and growth shape was inferior to Example 1.

Furthermore, when bFGF was added to DMEM medium as in Example 1 and the cells were organized, the cells formed kyaryari, but the length of the kyaryri was shorter than that in Example 1. It was confirmed that the tissue formation was incomplete.

[0221] [Example 2]

It has 190 m / 500 / ζm line & space with opening width 190 111, light shielding width 500 / ζ πι, and 5 m every 60 m in the above opening. Cells were cultured in the same manner as in Example 1 except that a photomask having a light-shielding portion was prepared and used. The cell morphology was observed 16 hours after the seeding of the cells, and the orientation and extension of the cells were observed for all cells in the cell culture area.

[0222] [Comparative Example 2]

The cells were cultured in the same manner as in Example 2 except that a 190 μm / 500 μm line & space photomask was used. In this case, even after 24 hours of seeding of the cells, it was confirmed that the cells near the center of the cell adhesion portion adhered to the substrate, but did not undergo orientation or extension.

[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), 0.04 g of N- (2-aminoethyl) -3-aminopropyltrimethoxysilane (manufactured by Huels America), and photocatalytic inorganic coating 1.5 g of ST-KOI (Ishihara Sangyo Co., Ltd.) was mixed and heated at 100 ° C. for 20 minutes with stirring.

This solution is applied to a quartz glass substrate that has been previously alkali-treated by spin coating, and the substrate is dried at 150 ° C for 10 minutes to allow hydrolysis and polycondensation reactions to proceed. A patterning substrate having a photocatalyst-containing cell-adhesive layer having a thickness of 0.2 m and a cell-adhesive layer that changes from cell-adhesive to cell-adhesive-inhibiting by the action of photocatalyst accompanying energy irradiation was formed.

[0224] (Patterning of patterning substrate)

This patterning substrate has a line and space of 60 μm / 300 μm with an opening width of 60 μm and a light shielding portion of 300 μm. Using a photomask formed so that the boundaries have irregularities of 1 μm square, UV exposure was performed with a mercury lamp (wavelength 365 nm) at an illuminance of 300 mWZcm ² for 900 seconds, and the unexposed areas showed cell adhesion. Thus, a puttering substrate for cell culture having a cell-adhesive surface patterned so that exposed portions have cell-adhesion inhibiting properties was obtained.

[0225] (Culture of cells)

The cells were cultured in the same manner as in Example 1, and the morphology of the cells was observed 16 hours after seeding of the cells. As a result, 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 inhibition layer)

Isopropyl alcohol 3g, organosilane TSL8114 (GE Toshiba Silicone) 0.4g, fluoroalkylsilane TSL8233 (GE Toshiba Silicone) 0.04g, photocatalyst organic coating agent ST-K01 (Ishihara Sangyo) 1 .5 g were mixed and heated at 100 ° C. for 20 minutes with stirring.

[0227] This solution was applied to a quartz glass substrate which had been previously subjected to an alkali treatment by a spin coating method, and the substrate was dried at a temperature of 150 ° C for 10 minutes to perform a hydrolysis and polycondensation reaction. A 0.2 m-thick photocatalyst-containing cell-adhesion-inhibiting layer, which changes from cell-adhesion-inhibiting to cell-adhesive by the action of photocatalyst accompanying energy irradiation, has a photocatalyst firmly fixed in the organopolysiloxane. Having a patterning substrate.

[0228] (Patterning of Patterning Substrate)

The above-mentioned substrate for puttering was irradiated with ultraviolet rays in the same manner as in Example 3 to obtain a patterned substrate for cell culture having a pattern in which the unexposed portion was a cell adhesion inhibiting portion and the exposed portion was a cell adhesion portion.

[0229] (Culture of cells)

[0230] [Example 5]

(Formation of photocatalyst containing layer)

3 g of isopropyl alcohol, 0.4 g of organosilane TSL8114 (manufactured by GE Toshiba Silicones) and 1.5 g of photocatalytic inorganic coating agent ST-KOI (Ishihara Sangyo) were mixed and heated at 100 ° C for 20 minutes with stirring. .

This solution is applied to a quartz glass substrate that has been preliminarily alkali-treated by spin coating, and the substrate is dried at a temperature of 150 ° C for 10 minutes to allow hydrolysis and polycondensation reactions to proceed. A photocatalyst-containing layer having a thickness of 0.2 IX m firmly fixed in siloxane was formed on the substrate.

[0231] (Formation of Cell Adhesive Layer)

Fibronectin F- 4759 and (Sigma) 0. 2 mg, was mixed with purified water 200 ml, the solution to the photocatalyst-containing layer of the substrate provided with the photocatalyst-containing layer, dropwise at a ratio of substrate area lcm ² per ³ 00 1 Then, this was allowed to stand at 4 ° C. for 24 hours. Further, the substrate was washed twice with PBS, exposed to nitrogen gas and dried to obtain a substrate for a pattern having a photocatalyst-containing layer and a cell adhesion layer on the substrate.

[0232] (Patterning of patterning substrate)

The above-mentioned patterning substrate is irradiated with ultraviolet rays in the same manner as in Example 3, and the unexposed portion has a pattern in which a cell adhesion portion and the exposed portion has a pattern in which a cell adhesion inhibition portion is formed. A substrate was obtained.

[0233] (Culture of cells)

[Example 6]

(Formation of photocatalyst containing layer)

Mix 3 g of isopropyl alcohol, 0.4 g of organosilane TSL8114 (GE Toshiba Silicone), and 1.5 g of photocatalytic inorganic coating agent ST-KOI (Ishihara Sangyo), and stir without force. Heated.

(Formation of Cell Adhesion Inhibiting Layer)

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

[0236] (Patterning of Patterning Substrate)

[0237] (Culture of cells)

[Example 7]

(Formation of cell adhesion layer)

3 g of isopropyl alcohol, organosilane TSL8114 (GE Toshiba Silicone) 0. 4 g and 0.4 g of aminopropyltriethoxysilane were mixed and heated at 100 ° C. for 20 minutes with stirring. This solution is applied to a pre-alkali glass substrate by a spin coating method, and the substrate is dried at a temperature of 150 ° C for 10 minutes. An amino group-containing organopolysiloxane layer having a thickness of about 80 nm was formed on the substrate to provide a substrate for notching.

[0239] (Patterning of Patterning Substrate)

The substrate for puttering was irradiated with ultraviolet rays in the same manner as in Example 1 to obtain a patterned substrate for cell culture having a pattern in which the unexposed portion became a cell adhesion portion and the exposed portion became a cell adhesion inhibition portion.

[0240] (Culture of cells)

Claims

The scope of the claims

[1] A cell culture patterning substrate having a substrate and a cell culture region formed on the substrate and for culturing cells and containing a cell adhesion layer having adhesive properties to cells. hand,

The cell culture region includes a cell adhesion portion on which the cell adhesion layer is formed, and a cell adhesion auxiliary portion formed in a pattern and inhibiting adhesion to cells, wherein the cell adhesion auxiliary portion is When a cell is attached to the cell adhesion part, the cells on the two cell adhesion parts adjacent to the cell adhesion part are formed so as to be able to be bonded on the cell adhesion part. A putter-jung substrate for cell culture, comprising:

[2] The puttering substrate for cell culture according to claim 1, wherein the cell adhesion auxiliary part is formed in a line shape in the cell culture region.

[3] The cell culture according to [1] or [2], wherein a boundary between the cell adhesion auxiliary part and the cell adhesion part is formed in a pattern having irregularities. For putter jung board.

[4] A cell culture patterning substrate having a substrate and a cell culture region formed on the substrate and culturing cells and containing a cell adhesion layer having adhesive properties to cells. hand,

A patterning substrate for cell culture, wherein the cell adhesion layer is formed in a pattern having an end with irregularities.

[5] The distance between the concave end and the convex end of the unevenness is such that the cells are linearly aligned when the cells are adhered on the cell adhesive layer. The putter-jung substrate for cell culture according to claim 1.

[6] The pattern jungle for cell culture according to claim 4 or 5, wherein the concave-to-concave edge force of the irregularities has an average distance to the convex portion end in a range of 0.5 m to 30 m. Substrate