JP5477423B2 - Cell culture substrate - Google Patents

Description

  The present invention relates to a cell culture substrate for use in cell processing, regenerative medicine and the like, which can transfer cells cultured in adhesion to a cell sheet, organ, protein sheet or the like alive. .

  Currently, various animal and plant cell cultures are being performed, and new cell culture methods have been developed. Cell culture techniques are used for the purpose of elucidating biochemical phenomena and properties of cells and producing useful substances. In addition, attempts have been made to examine the physiological activity and toxicity of artificially synthesized drugs using cultured cells. Furthermore, in recent years, research on regenerative medicine in which cultured cells and cultured tissues are transplanted into a patient through a process of culturing cells in vitro has been actively conducted.

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

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

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

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

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

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

  Recently, after culturing cells in a pattern, a technique for transferring the cells to a tissue or an organ alive and a tissue body using the technique have been reported (Patent Document 8 and Patent Document 9). However, this technique has a problem that, since it takes time to transfer the cells, the operation for keeping the activity of the cells is complicated.

JP-A-2-245181 Japanese Patent Laid-Open No. 3-7576 Japanese Patent Laid-Open No. 5-176653 US Pat. No. 5,512,131 US Pat. No. 5,900,160 JP-A-9-240125 Japanese Patent Laid-Open No. 10-12545 JP-A-2005-342112 International Publication WO2005 / 038011 Pamphlet

  The present invention provides a means for enabling cell sheets, sparse adherent cells, cell colonies, cell networks, cell patterns, and the like to be transferred at high speed to cell sheets, tissues, organs, organs, protein sheets, and the like. With the goal.

The present invention includes the following inventions.
(1) A cell culture substrate comprising a base material and a cell adhesive region formed on the surface of the base material,
The cell adhesion region is formed of a cell adhesion-inhibiting hydrophilic film containing an organic compound having a carbon-oxygen bond, which is subjected to oxidation treatment and / or decomposition treatment to make it cell adhesion. Cell culture substrate.
(2) A cell culture substrate comprising a substrate, a cell adhesion region and a cell adhesion inhibitory region formed on the surface of the substrate,
The cell adhesion region is formed of a cell adhesion-inhibiting hydrophilic film containing an organic compound having a carbon-oxygen bond, which has been subjected to oxidation treatment and / or decomposition treatment to make it cell adhesive.
A cell culture substrate, wherein the cell adhesion-inhibiting region is formed of a hydrophilic film containing an organic compound having a carbon-oxygen bond.
(3) The cell culture substrate according to (1) or (2), wherein the decomposition treatment is a decomposition treatment by oxidation or ultraviolet irradiation.

(4) A cell culture substrate comprising a base material and a cell adhesive region formed on the surface of the base material,
The cell adhesion region is formed of a hydrophilic film containing an organic compound having a carbon-oxygen bond,
A cell culture substrate, wherein the density of the organic compound in the cell adhesion region is low enough to allow cells to adhere.
(5) A substrate for cell culture comprising a substrate, a cell adhesion region and a cell adhesion inhibitory region formed on the surface of the substrate,
The cell adhesion region and the cell adhesion inhibitory region are formed of a hydrophilic film containing an organic compound having a carbon-oxygen bond,
A cell culture substrate, wherein the density of the organic compound in the cell adhesion region is lower than the density of the organic compound in the cell adhesion inhibition region.
(6) The cell culture according to any one of (2) to (5), wherein the amount of carbon in the cell adhesion region is 20 to 99% of the amount of carbon in the cell adhesion inhibition region substrate.

(7) The ratio of the percentage of carbon bonded to oxygen among the carbon in the cell adhesion region is the percentage of the carbon bonded to oxygen among the carbon in the cell adhesion inhibitory region (%) The cell culture substrate according to any one of (2) to (6), which is 35 to 99% with respect to the value of.
(8) The cell culture substrate according to any one of (1) to (7), wherein the organic compound having a carbon-oxygen bond is an alkylene glycol-based material.
(9) A cell adhesion substrate comprising the cell culture substrate according to any one of (1) to (8) and cells adhered to the cell adhesion region of the cell culture substrate.

(10) A method for producing a tissue body, comprising a step of transferring cells on the cell adhesion substrate according to (9) to a target material.
(11) The method according to (10), wherein the step is performed a plurality of times.
(12) A tissue produced by the method according to (10) or (11).

  According to the present invention, cell sheets, sparse adherent cells, cell colonies, cell networks, cell patterns, and the like can be transferred at high speed to cell sheets, tissues, organs, organs, protein sheets, and the like.

FIG. 1 shows a procedure for producing a cell adhesion substrate. FIG. 2 shows a procedure for transferring and culturing cells from a cell adhesion substrate to a target material.

  The cell culture substrate of the present invention comprises a base material and a cell adhesive region formed on the surface of the base material.

  It is preferable that not only a cell adhesion region but also a cell adhesion inhibition region is formed on the substrate surface. And it is preferable that the cell adhesion region and the cell adhesion inhibition region are arranged in a pattern.

  In the present invention, “cell adhesion” means strength for cell adhesion, that is, ease of cell adhesion. The cell adhesion region means a region with good cell adhesion, and the cell adhesion inhibitory region means a region with poor cell adhesion. Therefore, when cells are seeded on a substrate on which a cell adhesion region and a cell adhesion inhibition region are patterned, cells adhere to the cell adhesion region, but cells do not adhere to the cell adhesion inhibition region. The cells are arranged in a pattern on the surface of the cell culture substrate.

  Since cell adhesion may vary depending on the cells to be adhered, good cell adhesion means good cell adhesion to certain types of cells. Therefore, on the cell culture substrate, there may be a plurality of cell adhesion regions for a plurality of types of cells, that is, there may be two or more levels of cell adhesion regions having different cell adhesion properties.

  The cell culture substrate of the present invention is characterized by the structure of the cell adhesion region. In the present invention, the structure of the cell adhesion region can be roughly divided into two forms.

  In the first form, the cell adhesion region is formed of a membrane that is made cell-adhesive by subjecting a cell adhesion-inhibiting hydrophilic membrane containing an organic compound having a carbon-oxygen bond to oxidation treatment and / or degradation treatment. It is a form. In this form, a cell adhesion-inhibiting hydrophilic film containing an organic compound having a carbon-oxygen bond is formed on the entire surface of the substrate, and then an oxidation treatment and / or a degradation treatment are performed on a region where cell adhesion is desired. To give cell adhesion to the region to modify the region. The part not subjected to the treatment is a cell adhesion-inhibiting region. When the entire surface of the cell adhesion-inhibiting hydrophilic membrane is subjected to oxidation treatment and / or decomposition treatment, a cell culture substrate having only a cell adhesion region on the surface is obtained.

  In the second form, the cell adhesive region is formed of a hydrophilic film containing an organic compound having a carbon-oxygen bond at a low density. This form utilizes the fact that a hydrophilic film containing an organic compound having a carbon-oxygen bond at a high density has cell adhesion inhibitory properties, whereas a hydrophilic film containing the compound at a low density has a cell adhesion property. It is a thing. When a first region where the compound is likely to bind to the substrate surface and a second region where the compound is difficult to bind are provided on the substrate surface, and a film of the compound is formed on the substrate surface, the first region becomes a cell adhesion inhibitory region, The region becomes a cell adhesive region.

The following description applies to both of the above two forms unless otherwise specified.
(Base material)
The base material used for the cell culture substrate of the present invention is not particularly limited as long as it is formed of a material capable of forming a film of an organic compound having a carbon-oxygen bond on the surface thereof. . Specifically, inorganic materials such as metals, glass, ceramics, silicon, elastomers, plastics (for example, polyester resins, polyethylene resins, polypropylene resins, ABS resins, nylons, acrylic resins, fluororesins, polycarbonate resins, polyurethane resins, methyl resins) Organic materials represented by pentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin). The shape is not limited, and examples thereof include a flat shape such as a flat plate, a flat membrane, a film, and a porous membrane, and a three-dimensional shape such as a cylinder, a stamp, a multiwell plate, and a microchannel. When using a film, the thickness in particular is not restrict | limited, However, Usually, it is 0.1-1000 micrometers, Preferably it is 1-500 micrometers, More preferably, it is 10-200 micrometers.

(Cell adhesion inhibitory area)
The cell adhesion-inhibiting region is formed by a hydrophilic film formed by an organic compound having a carbon-oxygen bond. The hydrophilic film is a thin film mainly composed of an organic compound having a carbon-oxygen bond, which has water solubility and water swellability, and has a cell adhesion inhibitory property before being oxidized and is oxidized and / or decomposed. There is no particular limitation as long as it has cell adhesion.

  In the present invention, the carbon-oxygen bond means a bond formed between carbon and oxygen, and is not limited to a single bond but may be a double bond. Examples of the carbon-oxygen bond include a C—O bond, a C (═O) —O bond, and a C═O bond.

  Examples of main raw materials include water-soluble polymers, water-soluble oligomers, water-soluble organic compounds, surfactants, amphiphiles, etc., which are physically or chemically cross-linked with each other to form a physical bond with the substrate. Or it becomes a hydrophilic thin film by combining chemically.

  Specific water-soluble polymer materials include polyalkylene glycol and derivatives thereof, polyacrylic acid and derivatives thereof, polymethacrylic acid and derivatives thereof, polyacrylamide and derivatives thereof, polyvinyl alcohol and derivatives thereof, and zwitterionic polymers. , Polysaccharides, and the like. Examples of the molecular shape include a straight chain, a branched one, and a dendrimer. More specifically, polyethylene glycol, a copolymer of polyethylene glycol and polypropylene glycol, such as Pluronic F108, Pluronic F127, poly (N-isopropylacrylamide), poly (N-vinyl-2-pyrrolidone), poly (2- Hydroxyethyl methacrylate), poly (methacryloyloxyethylphosphorylcholine), copolymers of methacryloyloxyethylphosphorylcholine and acrylic monomers, dextran, and heparin, but are not limited thereto.

  Specific water-soluble oligomer materials and water-soluble low molecular weight compounds include alkylene glycol oligomers and derivatives thereof, acrylic acid oligomers and derivatives thereof, methacrylic acid oligomers and derivatives thereof, acrylamide oligomers and derivatives thereof, saponified vinyl acetate oligomers and Derivatives thereof, oligomers composed of zwitterionic monomers and derivatives thereof, acrylic acid and derivatives thereof, methacrylic acid and derivatives thereof, acrylamide and derivatives thereof, zwitterionic compounds, water-soluble silane coupling agents, water-soluble thiol compounds, etc. be able to. More specifically, ethylene glycol oligomer, (N-isopropylacrylamide) oligomer, methacryloyloxyethylphosphorylcholine oligomer, low molecular weight dextran, low molecular weight heparin, oligoethylene glycol thiol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene Glycols, 2- [methoxy (polyethyleneoxy) -propyltrimethoxysilane, and triethylene glycol-terminated-thiols, but are not limited to these.

  It is desirable that the hydrophilic film has a high cell adhesion inhibitory property before the treatment and shows a weak cell adhesion property after the oxidation treatment and / or the degradation treatment.

  The average thickness of the hydrophilic film is preferably 0.8 nm to 500 μm, more preferably 0.8 nm to 100 μm, more preferably 1 nm to 10 μm, and most preferably 1.5 nm to 1 μm. If the average thickness is 0.8 nm or more, it is preferable because protein adsorption and cell adhesion are not easily affected by the region not covered with the hydrophilic thin film on the substrate surface. Moreover, if the average thickness is 500 μm or less, coating is relatively easy.

  The hydrophilic film can be formed on the substrate surface by directly adsorbing the hydrophilic organic compound on the substrate, coating the hydrophilic organic compound directly on the substrate, or coating the hydrophilic organic compound on the substrate. A method of performing a crosslinking treatment later, a method of forming a hydrophilic thin film in a multi-stage manner in order to increase the adhesion to the substrate, a base layer is formed on the substrate in order to increase the adhesion to the substrate, Examples thereof include a method of coating a hydrophilic organic compound, a method of forming a polymerization initiation point on the substrate surface, and then polymerizing a hydrophilic polymer brush.

  Among the above film forming methods, particularly preferable methods include a method of forming a hydrophilic thin film in a multistage manner, and a base layer is formed on the base material in order to improve adhesion to the base material, and then hydrophilic organic Mention may be made of a method of coating a compound. This is because using these methods makes it easy to enhance the adhesion of the hydrophilic organic compound to the substrate. In this specification, the term “bonding layer” is used. The bonding layer means a layer existing between the outermost hydrophilic thin film layer and the substrate in the case of forming a hydrophilic organic compound thin film in a multistage manner. When a hydrophilic thin film layer is formed on an underlayer, the underlayer is meant. The binding layer is preferably a layer containing a material having a binding portion (linker). Examples of combinations of the linker and the functional group at the end of the material to be bonded to the linker include epoxy group and hydroxyl group, phthalic anhydride and hydroxyl group, carboxyl group and N-hydroxysuccinimide, carboxyl group and carbodiimide, amino group and glutaraldehyde, etc. Is mentioned. In each combination, any of them may be a linker. In these methods, before coating with a hydrophilic material, a bonding layer is formed of a material having a linker on a substrate. The density of the material in the bonding layer is an important factor that defines the bonding force. The density can be easily evaluated using the contact angle of water on the surface of the bonding layer as an index. For example, taking a silane coupling agent having an epoxy group at the end (epoxysilane) as an example, the water contact angle of the substrate surface to which epoxysilane is added is typically 45 ° or more, preferably 47 ° or more. Then, a substrate having sufficient cell adhesion inhibitory property can be made by adding an ethylene glycol-based material or the like in the presence of an acid catalyst.

(Formation of cell adhesion region by oxidizing and / or decomposing hydrophilic membrane)
In the first embodiment of the present invention, the cell adhesion region is formed by a membrane that is made cell-adhesive by subjecting a cell adhesion-inhibiting hydrophilic membrane containing an organic compound having a carbon-oxygen bond to oxidation treatment and / or degradation treatment. It is formed. Since the cell adhesion region formed in this manner has a relatively weak ability to adhere cells, the material adhered on the region has a relatively strong interaction with cells such as protein sheets, cell sheets, tissues and organs. It is possible to transfer at a high speed.

  In the present invention, “oxidation” has a narrow meaning, and means a reaction in which an organic compound reacts with oxygen and the content of oxygen is higher than before the reaction.

  In the present invention, “decomposition” refers to a change in which a bond of an organic compound is broken to produce two or more organic compounds from one organic compound. “Decomposition treatment” typically includes, but is not limited to, decomposition by oxidation treatment and decomposition by ultraviolet irradiation. When the “decomposition process” is a decomposition accompanied by oxidation (that is, oxidative decomposition), the “decomposition process” and the “oxidation process” indicate the same process.

  Decomposition by ultraviolet irradiation means that an organic compound absorbs ultraviolet rays and decomposes through an excited state. In addition, when an organic compound is irradiated with ultraviolet rays in a system that contains molecular species containing oxygen (oxygen, water, etc.), the molecular species are activated in addition to being absorbed by the compound and causing decomposition. May react with organic compounds. The latter reaction can be classified as “oxidation”. A reaction in which an organic compound is decomposed by oxidation by an activated molecular species can be classified as “decomposition by oxidation” rather than “decomposition by ultraviolet irradiation”.

  As described above, “oxidation treatment” and “decomposition treatment” may overlap as operations, and the two cannot be clearly distinguished. Therefore, in this specification, the term “oxidation treatment and / or decomposition treatment” is used.

  Examples of the oxidation treatment and / or decomposition treatment include a method of subjecting the hydrophilic film to ultraviolet irradiation treatment, a method of photocatalytic treatment, and a method of treatment with an oxidizing agent. The entire hydrophilic film may be oxidized and / or decomposed, or partially oxidized and / or decomposed. In the case of partial oxidation treatment and / or decomposition treatment, a mask such as a photomask or a stencil mask or a stamp may be used. Further, the oxidation treatment and / or the decomposition treatment may be performed by a direct drawing method such as a method using a laser such as an ultraviolet laser.

  In the case of the ultraviolet irradiation treatment, it is preferable to use a lamp that emits ultraviolet rays in the UV-C region from the VUV region, such as a mercury lamp that emits ultraviolet rays having a wavelength of 185 nm or 254 nm or an excimer lamp that emits ultraviolet rays having a wavelength of 172 nm. When the photocatalytic treatment is performed, it is preferable to use a light source that emits ultraviolet light having a wavelength of 365 nm or less, and it is more preferable to use a light source that emits ultraviolet light having a wavelength of 254 nm or less. As the photocatalyst, it is preferable to use a titanium oxide photocatalyst, a titanium oxide photocatalyst activated with a metal ion or a metal colloid. As the oxidizing agent, an organic acid or an inorganic acid can be used without any particular limitation. However, since a high concentration acid is difficult to handle, it is preferable to dilute it to a concentration of 10% or less. The optimum ultraviolet treatment time, photocatalyst treatment time, and oxidant treatment time can be appropriately determined according to various conditions such as the ultraviolet light intensity of the light source used, the activity of the photocatalyst, the oxidizing power and concentration of the oxidant.

(Formation of cell adhesion region by reducing the density of hydrophilic membrane)
In the second embodiment of the present invention, the cell adhesive region is formed by a hydrophilic film containing an organic compound having a carbon-oxygen bond at a low density. Since the cell adhesion region thus formed also has a relatively weak ability to adhere cells, the cells adhered on the region have a relatively strong interaction with cells such as protein sheets, cell sheets, tissues and organs. It is possible to transfer to the material at high speed.

  In this embodiment of the present invention, both the cell adhesion region and the cell adhesion inhibition region are formed of a hydrophilic film containing an organic compound having a carbon-oxygen bond. The two regions differ in the density of the organic compound. The higher the density, the more difficult the cells adhere. In the cell adhesion region, the density of the organic compound is low enough to allow cells to adhere. On the other hand, in the cell adhesion-inhibiting region, the density of the organic compound is so high that cells cannot adhere.

  Examples of the method for controlling the density of the hydrophilic organic compound include a method in which a bonding layer is provided between the hydrophilic organic compound thin film and the substrate surface, and the bonding force of the bonding layer with the hydrophilic organic compound is adjusted. It is done. Here, the “binding layer” is as defined above, and may be composed of the preferred materials described above. The bonding force of the bonding layer increases as the density of the material having the linker in the bonding layer increases, and decreases as the density decreases. As described above, the density of the material having a linker in the bonding layer can be easily evaluated using the water contact angle on the surface of the bonding layer as an index.

  In this embodiment of the invention, the density of the material with the linker in the tie layer of the cell adhesion region is low. The water contact angle on the surface of the bonding layer before forming the hydrophilic organic compound thin film in the cell adhesive region is when a silane coupling agent (epoxy silane) having an epoxy group at the end is used as a material having a linker. Is typically 10 ° to 43 °, desirably 15 ° to 40 °. As a method for forming such a bonding layer, a method of forming a coating (bonding layer) of a material having a linker on the surface of the substrate and then oxidizing and / or decomposing the surface of the bonding layer can be mentioned. Examples of the method for oxidizing and / or decomposing the bonding layer surface include a method of treating the bonding layer surface with ultraviolet irradiation, a method of treating with a photocatalyst, and a method of treating with an oxidizing agent. The entire surface of the bonding layer surface may be oxidized and / or decomposed or partially processed. The partial processing can be performed by using a mask such as a photomask or a stencil mask, or by using a stamp. Further, the oxidation treatment and / or the decomposition treatment may be performed by a direct drawing method such as a method using a laser such as an ultraviolet laser. As for various conditions, the same conditions as in the method of forming a cell adhesive region by oxidizing and / or decomposing a hydrophilic film can be applied. A cell adhesion region can be formed by forming a thin film of a hydrophilic organic compound on the binding layer thus formed.

  In this embodiment of the present invention, the density of the material having a linker in the binding layer of the cell adhesion-inhibiting region is high. The water contact angle on the surface of the bonding layer before forming a thin film of hydrophilic organic compound in the cell adhesion-inhibiting region uses a silane coupling agent (epoxysilane) having an epoxy group as a terminal as a material having a linker. Taking the case as an example, it is typically 45 ° or more, preferably 47 ° or more. Such a bonding layer can be obtained by forming a coating of a material having a linker on the substrate surface. When the bonding layer surface is partially oxidized and / or decomposed, the remaining portion not subjected to the treatment becomes the bonding layer having the water contact angle. A cell adhesion-inhibiting region can be formed by forming a hydrophilic organic compound thin film on the binding layer thus formed.

(Comparison between cell adhesion region and cell adhesion inhibitory region)
The following description applies to both of the above two forms.
The amount of carbon in the cell adhesion region (including the bonding layer when a bonding layer is present) is lower than the amount of carbon in the cell adhesion inhibiting region (including the bonding layer when the bonding layer is present). It is preferable. Specifically, the amount of carbon in the cell adhesion region is preferably 20 to 99% with respect to the amount of carbon in the cell adhesion inhibition region. Corresponding to this range is particularly suitable when the thickness of the hydrophilic film (the total of the thickness of the bonding layer and the hydrophilic film when a bonding layer is present) is 10 μm or less. “Carbon content (atomic concentration,%)” is as defined below.

  In addition, the value of the ratio (%) of the carbon bonded to oxygen in the cell adhesive region (including the bonded layer when the bonded layer is present) is the cell adhesion inhibitory region (the bonded layer is It is preferable that the value be smaller than the value of the ratio (%) of carbon bonded to oxygen among the carbon in the carbon (including the bonded layer if present). Specifically, the value of the ratio (%) of carbon bonded to oxygen in the cell adhesion region is equal to the ratio of carbon bonded to oxygen among the carbon in the cell adhesion inhibitory region ( %) Is preferably 35 to 99%. Corresponding to this range is particularly suitable when the thickness of the hydrophilic film (the total of the thickness of the bonding layer and the hydrophilic film when a bonding layer is present) is 10 μm or less. “The ratio of carbon bonded to oxygen (atomic concentration,%)” is as defined below.

(Evaluation method of hydrophilic thin film)
Evaluation methods for the hydrophilic thin film of the present invention (including a bonding layer when a bonding layer is present) include contact angle measurement, ellipsometry, atomic force microscope observation, electron microscope observation, Auger electron spectroscopy measurement, X-ray measurement Photoelectron spectroscopy, various mass spectrometry, etc. can be used. Among these methods, X-ray photoelectron spectroscopy (XPS / ESCA) is most excellent in quantification. What is obtained by this measurement method is a relative quantitative value, and is generally calculated by an element concentration (atomic concentration,%). Hereinafter, the X-ray photoelectron spectroscopic analysis method in the present invention will be described in detail.

(Calculation method for carbon content of hydrophilic thin film and calculation method for the proportion of carbon bonded to oxygen)
In the present invention, the “carbon amount” of the hydrophilic thin film is defined as “the carbon amount obtained from the analytical value of the C1s peak obtained using an X-ray photoelectron spectrometer”. In the present invention, the “ratio of carbon bonded to oxygen” of the hydrophilic thin film is “the ratio of carbon bonded to oxygen determined from the analytical value of the C1s peak obtained using an X-ray photoelectron spectrometer. Is defined. Two specific measurement methods are described below. Note that the present invention is not limited to these measurement methods.

(Measurement method 1)
X-ray photoelectron spectrometer: VG_Theta Probe manufactured by Thermo Electron.
X-ray source: Monochromated aluminum Kα ray (15 kV-6.67 mA = 100 W).
Measurement area: 400μmφ
Positional relationship between sample and detector: A photoelectron capture lens exists at 53 ° from the sample normal.
Carbon content: Determine the set of photoelectrons to be measured, assuming the elements that make up the substrate and hydrophilic thin film. The atomic concentration derived from each photoelectron when the measured total photoelectron is 100% is calculated. The elemental concentration (atomic concentration%) of the C1s peak is defined as the carbon content.
C1s peak fitting method: Fitting is performed using a C—O bond, a C (═O) —O bond, a C═O bond, or a C—C bond.
Formula for calculating the proportion of carbon bonded to oxygen: {[ratio of carbon in C—O bond] + [ratio of carbon in C (═O) —O bond] + [ratio of carbon in C = O bond] } ÷ {[C-O bond carbon ratio] + [C (= O) -O bond carbon ratio] + [C = O bond carbon ratio] + [C—C bond carbon ratio] + [(If necessary) Carbon ratio of other bonds]} × 100 (%).
If necessary, fit with other bonds. Based on this data, the carbon concentration (atomic concentration%) of each bonded state of the C1s peak is calculated.

(Measurement method 2)
X-ray photoelectron spectrometer: ESCA-3400 (Amicus) manufactured by KRATOS Analytical.
X-ray source: non-monochromated magnesium Kα ray (10 kV-20 mA = 200 W).
Measurement area: 6mmφ
Positional relationship between sample and detector: A photoelectron capture lens exists on the sample normal.
Carbon content: Determine the set of photoelectrons to be measured, assuming the elements that make up the substrate and hydrophilic thin film. The atomic concentration derived from each photoelectron when the measured total photoelectron is 100% is calculated. The C1s peak element concentration (atomic concentration%) is defined as the carbon content.
C1s peak fitting method: Fitting is performed using a C—O bond, a C (═O) —O bond, a C═O bond, or a C—C bond.
Formula for calculating the proportion of carbon bonded to oxygen: {[ratio of carbon in C—O bond] + [ratio of carbon in C (═O) —O bond] + [ratio of carbon in C = O bond] } ÷ {[C-O bond carbon ratio] + [C (= O) -O bond carbon ratio] + [C = O bond carbon ratio] + [C—C bond carbon ratio] + [(If necessary) Carbon ratio of other bonds]} × 100 (%).

  If necessary, fit with other bonds. Based on this data, the carbon concentration (atomic concentration%) of each bonded state of the C1s peak is calculated.

(Pattern shape)
In the cell culture substrate of the present invention, it is preferable that the cell adhesion region and the cell adhesion inhibition region are arranged in a pattern. The shape of the pattern is not particularly limited as long as it is a two-dimensional pattern, and can be selected depending on the type of cell, the tissue to be formed, and the like. For example, a line shape, a tree shape (dendritic shape), a mesh shape, a lattice shape, a circular shape, a quadrangular pattern, a pattern in which the inside of a graphic shape such as a circular shape and a quadrangular shape is a cell adhesive region or a cell adhesion inhibitory region. Can be formed. When a tissue is formed by culturing vascular endothelial cells or nerve cells, it is preferable to form a pattern in which cells adhere in a line shape, a tree shape (dendritic shape), a mesh shape, or a lattice shape. When this is transferred to a material to be transferred and cultured, in the case of vascular endothelial cells, it is arranged in a line, tree (dendritic), mesh, or lattice, which promotes organization and promotes the formation of blood vessels. Prompted. When forming a line-like, tree-like (dendritic), mesh-like or lattice-like pattern, the line width in the pattern is usually 20 to 200 μm, preferably 30 to 100 μm. In particular, when capillary vessels are formed by arranging and culturing vascular endothelial cells in a line, the cell adhesion change pattern in which the spaces between the line-shaped cell adhesion region and the cell adhesion inhibition region are alternately arranged It is preferable that vascular endothelial cells are adhered in a line shape. In such an embodiment, it is preferable to form a pattern in which cells are adhered with a line width of 1 to 10 cells, preferably 1 to 6 cells. Specifically, the line width of the cell adhesion region is usually 20 to 200 μm, preferably 30 to 80 μm, and the space width of the cell adhesion inhibitory region between the lines is usually 80 to 1000 μm, preferably 100 ˜800 μm. By setting the line width within the above numerical range, vascular endothelial cells can be efficiently organized into a tube. By forming such a cell adhesion change pattern, the vascular endothelial cells adhered and transferred in a line form are organized to efficiently form a line capillary. If you want to form a cell pattern in which multiple lines are lined up without crossing, make sure that the space width between the lines to which the cells are attached is greater than a certain value as described above. In addition, it is possible to prevent the lines from being distorted due to the extension of pseudo feet from the cells between the lines.

(cell)
The cells to be seeded on the cell culture substrate may be floating cells such as blood cells and lymphoid cells, or may be adhesive cells, but the present invention is preferably used for cells having adhesiveness. Examples of such cells include liver cells that are liver parenchymal cells, Kupffer cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, Epidermal cells such as epidermal keratinocytes, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells such as corneal epithelial cells, mammary cells, pericytes, muscle cells such as smooth muscle cells and cardiomyocytes, kidneys Examples include cells, pancreatic islets of Langerhans, nerve cells such as peripheral nerve cells and optic nerve cells, chondrocytes, and bone cells. These cells may be primary cells collected directly from tissues or organs, or may be passaged from one generation to another. Furthermore, these cells are undifferentiated embryonic stem cells, pluripotent stem cells such as pluripotent mesenchymal stem cells, unipotent stem cells such as vascular endothelial progenitor cells that have unipotency, and differentiation has ended. Any of the cells may be used. In addition, the cells may be cultivated as a single species, or two or more types of cells may be co-cultured.

(Cell adhesion substrate)
The present invention provides a cell adhesion substrate comprising a cell culture substrate and cells adhered to the cell adhesion region of the cell culture substrate. The cell adhesion substrate can itself be distributed as a product.

  A method for producing a cell adhesion substrate will be described with reference to FIG. In FIG. 1, a case where a cell culture substrate having a cell adhesion region and a cell adhesion inhibition region on a substrate is used as an example will be described. It is also within the scope of the present invention to use a cell culture substrate that is entirely a cell adhesion region.

  As shown in FIG. 1 (a), cells are uniformly seeded on the surface of the cell culture substrate, and as shown in FIG. 1 (b), the cells are cultured for a certain period of time, and extra cells in the cell adhesion-inhibiting region. As shown in FIG. 1C, a cell pattern was formed in which cells were adhered to the cell adhesion region but cells were not adhered to the cell adhesion inhibitory region. A cell adhesion substrate is obtained.

  The culture sample containing the target cells is preliminarily subjected to a dispersion process in which the biological tissue is finely dispersed in a liquid, a separation process in which impurities other than the target cells in the biological tissue and other cell debris are removed. It is preferable to go.

  Prior to seeding of the cells on the cell culture substrate, it is preferable to increase the number of target cells by pre-culturing a culture sample containing the target cells in advance by various culture methods. For the preliminary culture, a normal culture method such as monolayer culture, coat dish culture, or on-gel culture can be employed. In the preculture, one of the methods of culturing with cells attached to the surface of the support is already known as a so-called monolayer culture method. Specifically, for example, by storing a culture sample and a culture solution in a culture container and maintaining the environment at a certain level, only specific living cells are adhered to the surface of a support such as a culture container. Multiply. The apparatus to be used, the treatment conditions, etc. are performed according to the usual monolayer culture method. As materials for the surface of the support on which cells adhere and grow, materials such as polylysine, polyethyleneimine, collagen, and gelatin that can adhere and proliferate well can be selected, glass dishes, plastic dishes, slide glasses, covers A so-called cell adhesion factor, which is a chemical substance that favors cell adhesion and proliferation, is also applied to the surface of a support such as glass, plastic sheet and plastic film.

  After preliminary culture, the culture solution in the culture vessel is removed, so that unnecessary components such as clumps and fibrous impurities that do not adhere to the support surface in the culture sample are removed, and only living cells that adhere to the support surface are removed. Can be recovered. Means such as EDTA-trypsin treatment can be applied to recovering living cells adhered to the support surface.

Cells preliminarily cultured as described above are seeded on a cell culture substrate in a culture solution as shown in FIG. The seeding method and the seeding amount of the cell are not particularly limited, and for example, the method described in “A tissue culture technique (1999)” published by Asakura Shoten, pages 266 to 270 can be used. It is preferable to seed the cells so that the cells adhere in a single layer in an amount sufficient to prevent the cells from growing on the cell culture substrate. Usually, it is preferable to seed so that cells are contained in an order of 10 ⁴ to 10 ⁶ per ml of culture medium, and seeding so that cells are contained in an order of 10 ⁴ to 10 ⁶ per 1 cm ^{2 of} the substrate. Is preferred. This is because when the cells aggregate, the organization of the cells is inhibited, and the function decreases even if the cells are transferred to the target material and cultured. Specifically, seeding is performed at about 2 × 10 ⁵ per 400 mm ² .

  It is preferable to adhere the cells to the cell adhesion region by culturing the cell culture substrate seeded with the cells in a culture solution. Any culture medium can be used without particular limitation as long as it is a cell culture medium usually used in the art. For example, depending on the type of cell used, MEM medium, BME medium, DME medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI1640 medium, etc. A basal medium as described in “Tissue culture technology third edition”, page 581, edited by the Japanese Society for Tissue Culture can be used. Furthermore, serum (such as fetal bovine serum), various growth factors, antibiotics, amino acids and the like may be added to the basal medium. A commercially available serum-free medium such as Gibco serum-free medium (Invitrogen) can also be used. Considering the clinical application of the finally obtained cell tissue, it is preferable to use a medium that does not contain animal-derived components.

  As shown in FIG. 1B, the step of culturing the cells aims to adhere the cells to the cell adhesive region of the cell culture substrate. The culture time depends on the presence or absence of cell manipulation during the culture, but is usually 6 to 96 hours, preferably 12 to 72 hours. By culturing for an appropriate time, the cells in the cell adhesion-inhibiting region of the cell culture substrate are washed away when washed, and the cells in the cell adhesion region remain on the cell culture substrate with an appropriate adhesive force. The remaining cells can be easily transferred to the target material.

The temperature for culturing is usually 37 ° C. It is preferable to culture in a CO ₂ concentration atmosphere of about 5% using a CO ₂ cell culture device or the like. After culturing, the cell culture substrate is washed to wash away non-adherent cells, and the cell adhesion substrate of the present invention in which cells adhere to the cell adhesion region can be prepared.

(How to use cell adhesion substrate)
A method for transferring cells to the target material using the cell adhesion substrate prepared by the above procedure and culturing the cells after the transfer will be described. In FIG. 2, a case where a substrate having a cell culture layer provided on the surface is used as a material to be transferred and a vascular endothelial cell is used as a cell is shown for illustrative purposes. The embodiment is not limited.

  As shown in FIG. 2A, a cell adhesion substrate having cells adhered to the cell adhesion region of the cell culture substrate is brought into close contact with the cell culture layer of the target material. Next, as shown in FIG. 2B, the cells are cultured to adhere the cells to the cell culture layer of the target material. Furthermore, since the adhesive force of the cells to the cell adhesive region is smaller than the adhesive force to the cell culture layer, as shown in FIG. 2 (c), when the cell culture substrate is peeled from the target material, Cells are transferred to the material. When the transferred cells are further cultured, the cells are functionalized as shown in FIG. 2D, and the cyclic structure is reproduced if the cells are vascular endothelial cells.

  The material to which the cells are transferred is not particularly limited as long as the cells can be adhered and cultured, but those having a surface that has a stronger adhesion to the cells than the cell adhesion region of the cell culture substrate are preferable. . For example, the following reference shows that it is desirable that the surface of the target material is soft and the cell adherence is not too high in order to stably organize cells. Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: Role of extracellular matrix. Donald E. Ingber et al., J. of cell biol. (1989) p.317. Particularly preferably, as detailed below, cell sheets, tissues, organs, organs, protein sheets, biological gels, protein coatings, and the like can be used as target materials.

  As a suitable target material, a collagen sheet, a fibrin sheet, an elastin sheet, an amniotic membrane and the like can be used. When a cell culture layer described later is provided on the substrate, the material of the substrate may be any material that does not inhibit the cell culture on the cell culture layer. For example, glass, polystyrene, polyethylene terephthalate, polycarbonate Polyimide, polylactic acid, polyglycolic acid, poly (lactic acid-glycolic acid) copolymer, polycaprolactone, poly (glycerol-sebacate) and the like can be used. What was illustrated about the base material used for the substrate for cell culture can also be used.

  The cell culture layer preferably contains a chemical substance or a cell adhesion factor that can favorably adhere and proliferate cells on its surface. Specific examples include extracellular substrates such as various types of collagen, fibronectin, laminin, vitronectin, cadherin, gelatin, peptide and integrin. These may be used alone or in combination of two or more. Also good. In view of high cell adhesiveness, it is more preferable to use various collagens, and among various collagens, it is particularly preferable to use type I collagen or type IV collagen. A cell culture layer can also be formed by culturing extracellular matrix-producing cells such as osteoblasts to generate an extracellular matrix.

  The shape of the target material is not particularly limited as long as it has a surface on which cells can be transferred. For example, a culture dish such as a petri dish or a multi-dish can be used. Moreover, the culture plate which consists of glass or the above-mentioned plastics can also be used.

Transfer of cells from the cell adhesion substrate to the target material can be performed by bringing the cell adhesion surface of the cell adhesion substrate into contact with the surface of the target material, for example, a cell culture layer. As described above, the cells can be transferred by culturing the cell adhesion substrate and the target material in contact with each other. Culturing can be carried out under suitable conditions depending on the substrate and cells to be used, and is usually performed at a CO ₂ concentration of 5% and 37 ° C. for 15 minutes to 96 hours, preferably 30 minutes to 36 hours.

  Thereafter, cell culture is performed in a culture solution, but cell culture may be performed in a state where the cell adhesion substrate and the target material are in contact with each other, or the cell culture substrate may be removed and the cell culture may be performed only with the target material. Good. It is preferable that cell culture is performed for a certain time in a state where the cell adhesion substrate and the target material are in contact with each other, and then the cell adhesion substrate is removed and further cell culture is performed. The culture conditions are not particularly limited and can be selected depending on the cell type to be cultured. As the culture solution, the same ones as described above can be used.

  The cell adhesion substrate of the present invention can be easily transferred in a state where cells adhered in a pattern are patterned on a target material, and after the transfer, the cells are washed and seeded with cells again. Since it can be used as a substrate, a cell pattern can be formed again, and this can be transferred and cultured. Therefore, a cell pattern can be created inexpensively and efficiently. In addition, since it is not necessary to form a special pattern for the target material, materials that are usually used for cell culture can be used, and the range of material selection is widened. I will not receive it.

  The target material includes biomaterials. The biological material means a biological material, and includes, for example, biological tissue or organ. Specific examples include organs such as lung, heart, liver, kidney, brain, stomach, small intestine, and large intestine, and tissues such as bone, cartilage, skin, muscle, eyeball, tongue, and peritoneum. In addition, cell aggregates such as cell sheets and spheroids can also be used as target materials. Examples of the cells constituting the cell aggregate include cells that produce extracellular matrix, such as stromal cells, epithelial cells, and parenchymal cells. Specifically, aggregates such as osteoblasts, fibroblasts, hepatocytes, feeder cells and the like can be preferably used.

  By directly transferring the cells on the cell culture substrate to these biomaterials, the cells can be directly cultured in a pattern on the tissue or organ. Examples of combinations of biomaterials to which cells are transferred and cells that are transferred to and cultured on the biomaterial include liver and vascular endothelial cells, dermis and vascular endothelial cells, osteoblast layer and vascular endothelial cells, and fibroblasts. Examples include cell layers and hepatocytes, endothelial cell layers and hepatocytes, feeder cell layers and corneal epithelial cells.

  In such an embodiment, since the cells can be directly transferred to a biological material such as an organ and cultured, it is not necessary to remove the cells from the cell culture carrier by enzymatic treatment or the like, thereby preventing cell damage. it can.

  In organ transplantation, capillaries are formed on the surface of the organ after transplantation, and there is known a technique for performing transplantation effectively by forming the capillaries in advance on the surface of the organ to be transplanted. It has been. However, in the prior art method in which capillaries are formed before transplantation and adhered to the organ surface, it takes time to create capillaries and rapid transplantation is impossible. Furthermore, the tissue is damaged when a blood vessel previously formed with a culture substrate or the like is peeled off from the substrate and transferred to the organ surface. In the method of the present invention, cells transferred in a pattern on the cell adhesion substrate can be transferred to the organ surface, and then transplantation can be performed without waiting for the complete formation of capillaries. It becomes possible. Since vascular endothelial cells transcribed linearly or on the surface of an organ are easily organized, the formation of capillaries in the body is promoted. Furthermore, in the present invention, when the cells are transferred, a treatment for peeling the cells from the cell culture substrate is not required, so that tissue damage does not become a problem. Furthermore, in the present invention, transcription is performed quickly, so there is little adverse effect on tissues or organs.

  In the culture of the transferred cells, a cell stimulating factor can be added as necessary to enhance cell activity or to promote the organization by expressing the functions inherent in the cells. As the cell stimulating factor, any substance having an activity of promoting cell organization can be used. For example, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), nerve growth factor (NGF) ), Epidermal growth factor (EGF), insulin-like growth factor (IGF) and the like.

  The process of transferring the cells on the cell adhesion substrate to the target material can be performed a plurality of times to create a multi-layered structure.

  The present invention also relates to a tissue body produced by a method including a step of transferring cells on a cell adhesion substrate to a target material by the above-described procedure. When the transfer process is performed a plurality of times, the created tissue has a multilayer structure. When the material to be transferred is a living tissue or organ, a tissue formed on the tissue or organ is also included in the scope of the present invention.

  The present invention also provides a method in which cells derived from a subject are adhered to the cell culture substrate of the present invention in a pattern and then patterned on the surface of the subject's biological tissue, such as the organ, skin, and bone. The present invention relates to a method for regenerating a tissue of a subject by transferring the cells in a state in which the cells are transferred and proliferating the cells. Although it does not specifically limit as a test subject, For example, a mammal is mentioned, Preferably a human is mentioned. For example, skin tissue can be regenerated in a subject by directly transferring dermal fibroblasts or epithelial cells to a skin damaged part in a living body and proliferating the cells by the method of the present invention. It is also possible to promote the regeneration of the skin by forming capillary blood vessels by transferring and proliferating the vascular endothelial cells in a pattern in the damaged part of the subject. Furthermore, by arranging and culturing nerve cells in a pattern, it becomes possible to create a neural circuit and a nerve cell computer.

Example 1
(Preparation of cell culture substrate)
(First stage reaction)
39.0 g of toluene and 13.5 g of epoxysilane TSL8350 (manufactured by GE Toshiba Silicone) were mixed, and a catalytic amount of triethylamine was added while stirring the mixed solution, followed by further stirring at room temperature for several minutes. A 10 cm square glass substrate that had been UV-cleaned in advance was immersed in the above epoxysilane solution and allowed to stand at room temperature for 12 hours. Thereafter, the glass substrate subjected to the ground treatment was washed with ethanol, then washed with water and dried. The average contact angle of water on the substrate surface after film formation was 50.2 °.

(Second stage reaction)
While stirring 50 g of tetraethylene glycol, a catalytic amount of concentrated sulfuric acid was slowly added and further stirred at room temperature for several minutes. The epoxy-treated substrate was immersed in the tetraethylene glycol and reacted at 80 ° C. for 20 minutes. After the reaction, the substrate was washed thoroughly with water and then dried. Thus, a glass substrate on which a hydrophilic thin film was formed could be made.

(Oxidation treatment)
A quartz plate coated with a titanium oxide photocatalyst was prepared. The illuminance of the exposure machine was measured in advance at a wavelength of 350 nm and used as a guide for setting the exposure time. The illuminance was 20 mW / cm ² . The glass substrate with the hydrophilic thin film and the quartz plate with the photocatalyst are placed so that the hydrophilic thin film and the photocatalyst layer of the quartz plate face each other, and placed in the exposure machine so that light is irradiated from the back side of the quartz plate. did. Exposure was performed for 25 seconds to perform oxidation treatment. Then, it cut | disconnected to the magnitude | size of 24 mm x 15 mm so that it might be easy to use for culture | cultivation.

(Surface analysis)
Using a VG_Theta Probe manufactured by Thermo Electron, the hydrophilic thin film before the oxidation treatment and the hydrophilic thin film after the oxidation treatment were measured. The amount of carbon in the hydrophilic thin film after the oxidation treatment in which the C1s peak is fitted with carbon of C—C bond, carbon of C—O bond, carbon of C (═O) —O bond, and C═O bond is as follows. It was 92.2% of the carbon content of the hydrophilic thin film. The ratio of carbon bonded to oxygen in the hydrophilic thin film before the oxidation treatment was 60.8%, and the ratio of carbon bonded to oxygen in the hydrophilic thin film after the oxidation treatment was 59.2%.

(Cell culture)
The substrate cut as described above was sterilized by high-pressure steam in an autoclave. This substrate was placed in a culture vessel, and 2.6 × 10 ⁵ bovine vascular endothelial cells (bEC) were seeded per substrate. Using MEM medium containing 5% serum, the cells were cultured for 46 hours in an incubator at 37 ° C. and 5% CO ₂ concentration. When observed with a phase contrast microscope, bEC adhered in a confluent state.

(Cell transcription)
A culture vessel was placed on ice, 200 μl of growth factor-reduced Matrigel (Becton Dickinson) was added to the culture vessel, and the Matrigel was spread to the same size as the cell adhesion substrate with a cell scraper cooled to 4 ° C. Thereafter, the culture vessel was allowed to stand at room temperature for about 5 minutes to gel the matrigel. The cell adhesion substrate was gently placed on the gelled matrigel so that the cell adhesion surface opposed to the matrigel. The bEC was kept in contact with Matrigel for several minutes. Thereafter, MEM medium containing 0.3% serum was added to the culture vessel and kept in an incubator at 37 ° C. and 5% CO ₂ concentration. After 2 hours, only the cell culture substrate could be easily removed. It was confirmed by phase contrast microscopy that bEC was transferred to almost 100% Matrigel.

(Comparative Example 1)
(Preparation of cell culture substrate)
The glass used in Example 1 was UV cleaned and cut into a size of 24 mm × 15 mm.
(Cell culture)
The substrate cut as described above was sterilized by high-pressure steam in an autoclave. This substrate was placed in a culture vessel and cultured for 46 hours under the same conditions as in Example 1. When observed with a phase contrast microscope, bEC adhered in a confluent state.

(Cell transcription)
As in Example 1, keep bEC in contact with Matrigel for a few minutes, then add MEM medium containing 0.3% serum to the culture vessel in an incubator at 37 ° C., 5% CO ₂ concentration. Retained. After 2 hours, bEC was not transferred when the cell culture substrate was peeled off. When another sample was further cultured for 2.5 hours for a total of 4.5 hours and then the cell culture substrate was peeled off, the cell transfer rate was 20%.

(Surface analysis)
The glass substrate after UV cleaning and high-pressure steam sterilization was measured using VG_Theta Probe manufactured by Thermo Electron. The C1s peak was fitted with C—C bond carbon, C—O bond carbon, C (═O) —O bond, and C═O bond carbon. The proportion of carbon bonded to oxygen was 32.0%.

(Example 2)
(Preparation of cell culture substrate)
(First stage reaction)
19.5 g of toluene and 6.8 g of epoxysilane TSL8350 (manufactured by GE Toshiba Silicone) were mixed, and a catalytic amount of triethylamine was added while stirring the mixed solution, followed by further stirring at room temperature for several minutes. A 10 cm square glass substrate that had been UV-cleaned in advance was immersed in the above epoxysilane solution and left at room temperature for 14 hours. The epoxidized glass substrate was then washed with ethanol, then washed with water and dried. The average value of the water contact angle on the substrate surface after film formation was 51.4 °.

(Second stage reaction)
While stirring 25 g of tetraethylene glycol (TEG), a catalytic amount of concentrated sulfuric acid was slowly added and further stirred at room temperature for several minutes. The above epoxidized substrate was immersed in TEG and reacted at 80 ° C. for 15 minutes. After the reaction, the substrate was washed thoroughly with water and then dried. Thus, a glass substrate on which a hydrophilic thin film was formed could be made.

(Oxidation treatment)
A photomask having a titanium oxide photocatalyst applied to the entire surface was prepared. In the photomask, line-shaped openings having a width of 60 μm perpendicular to the left line pattern are formed at intervals of 2.5 cm in a line pattern in which openings having a width of 60 μm are formed at a pitch of 300 μm, and a width of about 1. A 5-inch size having a 5 cm opening was used. The illuminance of the exposure machine was measured in advance at a wavelength of 350 nm and used as a guide for setting the exposure time. The illuminance was 20 mW / cm ² . Place the glass substrate on which the hydrophilic thin film is formed and the photomask with the above catalyst so that the hydrophilic thin film and the catalyst layer of the photomask face each other, and place it in the exposure machine so that light is irradiated from the back side of the photomask. did. The film was exposed for 75 seconds to be oxidized. Then, it cut | disconnected to the magnitude | size of 24 mm x 15 mm so that it might be easy to use for culture | cultivation.

(Surface analysis)
Using Thermo Electron's VG_Theta Probe, base glass substrate, hydrophilic thin film before oxidation treatment, hydrophilic thin film region after oxidation treatment (opening region with a width of about 1.5 cm around the photomask) Was measured. The C1s peak was fitted with C—C bond carbon, C—O bond carbon, C (═O) —O bond, and C═O bond carbon. The carbon content of the hydrophilic thin film in the oxidized region was 89.9% of the carbon content of the hydrophilic thin film in the non-oxidized region. Further, the ratio of carbon bonded to oxygen was 71.9% in the non-oxidized region of the hydrophilic thin film, and 47.6% in the oxidized region of the hydrophilic thin film. In addition, when the glass used as a base material was measured, the ratio of the carbon couple | bonded with oxygen was 30.2%.

(Cell culture)
The substrate cut as described above was sterilized by high-pressure steam in an autoclave. This substrate was placed in a culture vessel, and 1.5 × 10 ⁵ human umbilical vein endothelial cells (HUVEC, Kurabo Industries) were seeded per substrate. HuMedia-EG2 (Kurabo), which is a low serum medium for normal human vascular endothelial cell growth, was used so that the final cell concentration was 1.2 × 10 ⁵ cells / ml. The cells were cultured for 46 hours in an incubator at 37 ° C. and 5% CO ₂ concentration. When observed with a phase contrast microscope, HUVEC adhered in a confluent state only to the oxidized portion.

(Cell transcription)
A culture vessel was placed on ice, 200 μl of growth factor-reduced Matrigel (Becton Dickinson) was added to the culture vessel, and the Matrigel was spread to the same size as the cell adhesion substrate with a cell scraper cooled to 4 ° C. Thereafter, the culture vessel was allowed to stand at room temperature for about 5 minutes to gel the matrigel. The cell adhesion substrate was gently placed on the gelled matrigel so that the cell adhesion surface opposed to the matrigel. The cell pattern was kept for several minutes in contact with Matrigel. Subsequently, HuMedia-EG2 was added to the culture vessel. It was kept in an incubator at 37 ° C. and 5% CO ₂ concentration. After 30 minutes, it was confirmed by observation with a phase-contrast microscope that the HUVEC changed in shape and became a tube. Only the cell culture substrate could be easily removed. It was confirmed by observation with a phase contrast microscope that HUVEC was transferred to almost 100% Matrigel, and its tubular shape was hardly disturbed.

(Example 3)
(Preparation of cell culture substrate)
(First stage reaction)
39.0 g of toluene and 13.5 g of TSL8350 (manufactured by GE Toshiba Silicone) were mixed, and 450 μl of triethylamine was added while stirring. After stirring at room temperature for several minutes, the whole amount was transferred to a glass dish. A 10 cm square glass substrate that had been cleaned with UV was immersed therein and allowed to stand at room temperature for 16 hours. Thereafter, the glass substrate was washed with ethanol and water and dried by nitrogen blowing. Thereby, the thin film containing an epoxy group was formed in the glass substrate surface. The average value of the water contact angle on the substrate surface after film formation was 48.9 °.

(Second stage reaction)
While stirring 50 g of TEG, 250 μl of concentrated sulfuric acid was added dropwise. After stirring for several minutes, the whole amount was transferred to a glass dish. The above substrate was immersed therein and reacted at 80 ° C. for 15 minutes. After the reaction, the substrate was thoroughly washed with water and dried by nitrogen blowing. As a result, a uniform hydrophilic thin film was formed on the glass surface.

(Oxidation treatment)
The photocatalyst-equipped photomask used in Example 2 was used. The photocatalyst layer of the photomask and the hydrophilic thin film on the substrate surface were brought into contact with each other, and the photomask was placed in the exposure machine so that light was irradiated from the photomask side. The film was exposed for 50 seconds with a mercury lamp having a wavelength of 350 nm and an illuminance of 20 mW / cm ² to partially oxidatively decompose the hydrophilic thin film on the substrate surface. This substrate was cut into a size of 24 mm × 15 mm and used as a cell adhesion substrate.

(Surface analysis)
Using ESCA-3400 (Amicus) manufactured by KRATOS Analytical, elemental analysis was performed on regions that were oxidized and not decomposed by photocatalytic lithography. The carbon content of the hydrophilic thin film in the oxidized region was 63.8% of the carbon content of the hydrophilic thin film in the non-oxidized region. The proportion of carbon bonded to oxygen was 71.9% in the non-oxidized region of the hydrophilic thin film and 62.6% in the oxidized region of the hydrophilic thin film.

(Cell culture)
The autoclave-sterilized substrate was placed in a culture container, and an appropriate amount of 5% bovine serum-containing MEM medium was added. Here, 2.0 × 10 ⁵ bovine vascular endothelial cells (bEC) were seeded per substrate. This was cultured in an incubator (37 ° C., 5% CO ₂ ) for 40 hours and observed with a phase contrast microscope. As a result, bEC adhered only to a region having a relatively low carbon concentration and formed a cell pattern.

(Cell transcription)
200 μl of ice-cooled growth factor-reduced Matrigel (Becton Dickinson) was spread on a culture vessel and allowed to stand at room temperature for about 1 minute, and then the substrate was gently placed on the Matrigel with the cell adhesion surface down. In this state, the substrate was left for about 5 minutes to completely gelate, and then the MEM medium was added to immerse the substrate. The culture vessel was transferred into an incubator (37 ° C., 5% CO ₂ ), and bEC was cultured while being sandwiched between the substrate and Matrigel. When bEC was observed with a phase-contrast microscope after 2 hours, the width of the cell pattern was reduced by several tens of percent. At this time, when the substrate was peeled off, all of the bEC was moved to the gel side. When returned to the incubator and further cultured for 1 hour, bEC differentiated into structures on the tube in the gel.

Example 4
(Preparation of cell culture substrate)
(First stage reaction)
39.0 g of toluene, 0.48 g of TSL8350 (manufactured by GE Toshiba Silicone) and 0.97 g of triethylamine were mixed and stirred at room temperature for 10 minutes. A UV-cleaned 10 cm square glass substrate was immersed in this silane solution so that the cleaning surface was facing upward. After standing at room temperature for 16 hours, the substrate was washed with ethanol and water and dried by nitrogen blowing. Thereby, the thin film containing an epoxy group was formed in the glass substrate surface. The average value of the water contact angle on the substrate surface after film formation was 47.2 °.

(Oxidation treatment)
The photocatalyst-equipped photomask used in Example 2 was used. The photocatalyst layer of the photomask and the organic thin film on the substrate surface were brought into contact with each other, and the photomask was placed in the exposure machine so that light was irradiated from the photomask side. Exposure was performed with a mercury lamp having a wavelength of 350 nm and an illuminance of 20 mW / cm ² for 25 seconds, and the organic thin film on the substrate surface was partially oxidized and decomposed. After the oxidation treatment, the value of the water contact angle on the substrate surface in the region facing the 1.5 cm wide opening around the mask was 39.7 °.

(Second stage reaction)
While stirring 50 g of tetraethylene glycol, 25 μl of concentrated sulfuric acid was added dropwise. After stirring for several minutes, the whole amount was transferred to a glass dish. The above substrate was immersed therein and reacted at 80 ° C. for 15 minutes. After the reaction, the substrate was thoroughly washed with water and dried by nitrogen blowing. Thereby, the hydrophilic thin film which consists of two types of area | regions where carbon concentrations differ relatively was formed in the glass substrate surface. This substrate was cut into a size of 25 mm × 15 mm and used as a cell adhesion substrate.

(Surface analysis)
Using ESCA-3400 (Amicus) manufactured by KRATOS Analytical, elemental analysis of the hydrophilic thin film formed in the region not oxidized and decomposed and the hydrophilic thin film formed in the region oxidized and decomposed was performed. The amount of carbon in the hydrophilic thin film in the oxidized region, that is, the region having a low hydrophilic material density, is 91.2% of the amount of carbon in the hydrophilic thin film in the region that has not been oxidized, that is, the region having a high hydrophilic material density. Met. The ratio of carbon bonded to oxygen is 75.1% in the region of the hydrophilic thin film that has not been oxidized, that is, the region having a high hydrophilic material density, and the region that has been oxidized, that is, the region having a low hydrophilic material density. It was 70.1%.

(Cell culture)
The autoclave-sterilized substrate was placed in a culture container, and an appropriate amount of 5% bovine serum-containing MEM medium was added. Here, 2.0 × 10 ⁵ bovine vascular endothelial cells (bEC) were seeded per substrate. This was cultured for 40 hours in an incubator (37 ° C., 5% CO ₂ ) and then observed with a phase-contrast microscope. As a result, bEC adhered only to a region having a relatively low carbon concentration and formed a cell pattern. .

(Cell transcription)
200 μl of ice-cooled growth factor-reduced Matrigel (Becton Dickinson) was spread on a culture vessel and allowed to stand at room temperature for about 1 minute, and then the substrate was gently placed on the Matrigel with the cell adhesion surface down. In this state, the substrate was left for about 5 minutes to completely gelate, and then the MEM medium was added to immerse the substrate. The culture vessel was transferred into an incubator (37 ° C., 5% CO ₂ ), and bEC was cultured while being sandwiched between the substrate and Matrigel. When bEC was observed with a phase contrast microscope after 1 hour, the width of the cell pattern was reduced by several tens of percent. At this time, when the substrate was peeled off, all of the bEC was moved to the gel side. When returned to the incubator and further cultured for 1 hour, bEC differentiated into structures on the tube in the gel.

(Example 5)
(Preparation of cell culture substrate)
(First stage reaction)
39.0 g of toluene, 0.48 g of epoxysilane TSL8350 (manufactured by GE Toshiba Silicone) and 0.97 g of triethylamine were mixed and stirred at room temperature for 10 minutes. A UV-cleaned 10 cm square glass substrate was immersed in this silane solution so that the cleaning surface was facing upward. After standing at room temperature for 16 hours, the substrate was washed with ethanol and water and dried by nitrogen blowing. Thereby, the thin film containing an epoxy group was formed in the glass substrate surface. The average value of the water contact angle on the substrate surface after film formation was 49.8 °.

(Second stage reaction)
While stirring 50 g of polyethylene glycol (PEG 400) having an average molecular weight of 400, 25 μl of concentrated sulfuric acid was added dropwise. After stirring for several minutes, the whole amount was transferred to a glass dish. The above substrate was immersed therein and reacted at 80 ° C. for 20 minutes. After the reaction, the substrate was thoroughly washed with water and dried by nitrogen blowing. As a result, a uniform hydrophilic thin film was formed on the glass surface.

(Oxidation treatment)
The photocatalyst-equipped photomask used in Example 2 was used. The photocatalyst layer of the photomask and the hydrophilic thin film on the substrate surface were brought into contact with each other, and the photomask was placed in the exposure machine so that light was irradiated from the photomask side. The film was exposed for 50 seconds with a mercury lamp having a wavelength of 350 nm and an illuminance of 20 mW / cm ² to partially oxidatively decompose the hydrophilic thin film on the substrate surface. This substrate was cut into a size of 25 mm × 15 mm and used as a cell adhesion substrate.

(Surface analysis)
Using VG_Theta Probe manufactured by Thermo Electron Co., Ltd., elemental analysis was performed on areas that were not oxidized and decomposed by photocatalytic lithography. The carbon content of the hydrophilic thin film in the oxidized region was 84.8% of the carbon content of the hydrophilic thin film in the non-oxidized region. The ratio of carbon bonded to oxygen was 72.5% in the region of the hydrophilic thin film that was not oxidized and 77.1% in the region that was oxidized.

(Cell culture)
The autoclave-sterilized substrate was placed in a culture container, and an appropriate amount of 5% bovine serum-containing MEM medium was added. Here, 2.0 × 10 ⁵ bovine vascular endothelial cells (bEC) were seeded per substrate. This was cultured in an incubator (37 ° C., 5% CO ₂ ) for 24 hours and observed with a phase contrast microscope. As a result, bEC adhered only to a region having a relatively low carbon concentration and formed a cell pattern.

(Example 6)
(Preparation of cell culture substrate)
(First stage reaction)
39.0 g of toluene and 13.5 g of TSL8350 (manufactured by GE Toshiba Silicone) were mixed, and 450 μl of triethylamine was added while stirring. After stirring as it was for several minutes at room temperature, the whole amount was transferred to a glass dish. A 10 cm square glass substrate that had been cleaned with UV was immersed therein and allowed to stand at room temperature for 16 hours. Thereafter, the glass substrate was washed with ethanol and water and dried by nitrogen blowing. As a result, a thin film containing an epoxy group was formed on the surface of the glass substrate. The average value of the water contact angle on the substrate surface after film formation was 52.0 °.

(Second stage reaction)
While stirring 50 g of TEG, 250 μl of concentrated sulfuric acid was added dropwise. Stirring was continued for several minutes, and the whole amount was transferred to a glass dish. The silane-treated substrate was immersed therein and reacted at 80 ° C. for 15 minutes. After the reaction, the substrate was washed thoroughly with water and dried. As a result, a uniform hydrophilic thin film was formed on the glass surface.

(Oxidation treatment)
The photocatalyst-equipped photomask used in Example 2 was used. The photocatalyst layer of the photomask and the hydrophilic thin film on the substrate surface were brought into contact with each other, and the photomask was placed in the exposure machine so that light was irradiated from the photomask side. Exposure was performed with a mercury lamp having a wavelength of 350 nm and an illuminance of 20 mW / cm ² for 100 seconds, and the hydrophilic thin film on the substrate surface was partially oxidized and decomposed. This substrate was cut into a size of 24 mm × 15 mm.

(Surface analysis)
Using VG_Theta Probe manufactured by Thermo Electron Co., Ltd., elemental analysis was performed on areas that were not oxidized and decomposed by photocatalytic lithography. The carbon content of the hydrophilic thin film in the oxidized region was 61.8% of the carbon content of the hydrophilic thin film in the non-oxidized region. The proportion of carbon bonded to oxygen was 71.9% in the region of the hydrophilic thin film that was not oxidized and 44.5% in the region that was oxidized.

(BSA-FITC pattern adsorption)
2 ml of 20 μg / ml BSA-FITC was prepared using PBS buffer and transferred to a 35 mm dish. The above substrate was immersed in such a manner that the hydrophilic thin film faced up, and left overnight in an incubator (37 ° C., 5% CO ₂ ). After the substrate was washed twice with PBS, the surface of the substrate was observed with a fluorescence microscope, and a clear fluorescence pattern was observed. This is because BSA-FITC was adsorbed only in the oxidized region.

(Example 7)
(Preparation of cell culture substrate)
(First stage reaction)
39.0 g of toluene and 13.5 g of methacryloylsilane TSL8370 (GE Toshiba Silicone) were mixed, and 450 μl of triethylamine was added while stirring. After stirring as it was for several minutes at room temperature, the whole amount was transferred to a glass dish. A 5 cm square glass substrate that had been cleaned with UV was immersed therein and allowed to stand at room temperature for 16 hours. Thereafter, the glass substrate was washed with ethanol and water and dried by nitrogen blowing. As a result, a thin film containing a methacryloyl group was formed on the glass substrate surface.

(Second stage reaction)
0.1 g of a polymerization initiator 2,2′-dimethoxy-2-phenyl-acetophenone (DMPA, manufactured by Aldrich) was dissolved in 10 g of polyethylene glycol diacrylate (PEGdA, manufactured by Aldrich) at room temperature. This was spin coated on the methacryloylated substrate at 1500 rpm for 5 seconds. Thereafter, the entire surface of the substrate was immediately irradiated with ultraviolet rays for 3 seconds in a nitrogen atmosphere. Subsequently, it was post-baked at 160 ° C. for 10 minutes. The PEGdA substrate was immersed in water overnight, washed with water, and then dried. The average dry film thickness was 0.33 μm.

(Oxidation treatment)
A general photomask that was not photocatalyzed and had the same pattern as the photomask used in Example 2 was used. The mask was gently placed on the PEGdA surface of the PEGdA substrate, and irradiated with vacuum ultraviolet rays using a xenon excimer lamp (172 nm, 10 mW / cm ² ) as a light source from the back side of the mask for 1 minute. Thereby, the region corresponding to the opening of the photomask on the surface of the PEGdA film was oxidized. The substrate was cut into 2.5 cm square and used for cell culture.

(Cell culture)
A substrate sterilized with 70% ethanol was placed in a culture vessel, and an appropriate amount of MEM medium containing 5% bovine serum was added. Here, 8.3 × 10 ⁴ cells / cm ² of bovine vascular endothelial cells (bEC) were seeded. This was cultured in an incubator (37 ° C., 5% CO ₂ ) for 43 hours and then observed with a phase-contrast microscope. As a result, bEC adhered only to the oxidized region of the PEGdA film and formed a cell pattern. .

(Cell transcription)
Spread 400 μl of ice-cooled growth factor-reduced Matrigel (Becton Dickinson) on a culture vessel using a cell scraper and let stand at room temperature for about 3 minutes, and then gently place the above substrate on the Matrigel with the cell adhesion surface down. Put it on. After leaving in this state for about 5 minutes, MEM medium was added to immerse the substrate. The culture vessel was transferred into an incubator (37 ° C., 5% CO ₂ ), and bEC was cultured while being sandwiched between the substrate and Matrigel. When bEC was observed with a phase-contrast microscope after 5 hours, the cell pattern was morphologically changed into a luminal shape. At this time, when the substrate was peeled off, all of the bEC was moved to the gel side.

(Example 8)
(Preparation of cell culture substrate)
(First stage reaction)
39.0 g of toluene and 0.96 g of methacryloylsilane TSL8370 (manufactured by GE Toshiba Silicone) were mixed, and 450 μl of triethylamine was added while stirring. After stirring as it was for several minutes at room temperature, the whole amount was transferred to a glass dish. A 5 cm square glass substrate that had been cleaned with UV was immersed therein and allowed to stand at room temperature for 16 hours. Thereafter, the glass substrate was washed with ethanol and water and dried by nitrogen blowing. As a result, a thin film containing a methacryloyl group was formed on the glass substrate surface.

(Second stage reaction)
0.1 g of polymerization initiator DMPA (manufactured by Aldrich) was dissolved in a mixture of 8 g of PEGdA (manufactured by Aldrich) and 2 g of 1-vinyl 2-pyrrolidone (VP, manufactured by Tokyo Chemical Industry) at room temperature. This was spin coated on the methacryloylated substrate at 1500 rpm for 5 seconds. Thereafter, the entire surface of the substrate was immediately irradiated with ultraviolet rays for 3 seconds in a nitrogen atmosphere. Subsequently, it was post-baked at 160 ° C. for 10 minutes. This PEGdA-VP substrate was immersed in water overnight, washed with water, and then dried. The average dry film thickness was 0.28 μm.

(Oxidation treatment)
A general photomask that was not photocatalyzed and had the same pattern as the photomask used in Example 2 was used. The mask was gently placed on the PEGdA-VP surface of the PEGdA-VP substrate, and irradiated with vacuum ultraviolet rays using a xenon excimer lamp (172 nm, 10 mW / cm ² ) as a light source from the back side of the mask for 1 minute. Thereby, the region corresponding to the opening of the photomask on the surface of the PEGdA-VP film was oxidized. The substrate was cut into 2.5 cm square and used for cell culture.

(Cell culture)
A substrate sterilized with 70% ethanol was placed in a culture vessel, and an appropriate amount of MEM medium containing 5% bovine serum was added. Here, 8.3 × 10 ⁴ cells / cm ² of bovine vascular endothelial cells (bEC) were seeded. When this was cultured in an incubator (37 ° C., 5% CO ₂ ) for 43 hours and observed with a phase-contrast microscope, bEC adhered only to the oxidized region of the PEGdA-VP film and formed a cell pattern. It was.

(Cell transcription)
Spread 400 μl of ice-cooled growth factor-reduced Matrigel (Becton Dickinson) on a culture vessel using a cell scraper and let stand at room temperature for about 3 minutes, and then gently place the above substrate on the Matrigel with the cell adhesion surface down. Put it on. After leaving in this state for about 5 minutes, MEM medium was added to immerse the substrate. The culture vessel was transferred into an incubator (37 ° C., 5% CO ₂ ), and bEC was cultured while being sandwiched between the substrate and Matrigel. When bEC was observed with a phase-contrast microscope after 5 hours, the cell pattern was morphologically changed into a luminal shape. At this time, when the substrate was peeled off, all of the bEC was moved to the gel side.

Example 9
(Production of dish for cell culture)
(First stage reaction)
39.0 g of toluene and 0.48 g of epoxysilane TSL8350 (manufactured by GE Toshiba Silicone) were mixed, and 225 μl of triethylamine was added while stirring. After stirring as it was for several minutes at room temperature, the whole amount was transferred to a glass dish. A 10 cm square UV-washed glass substrate having a thickness of 0.1 mm was immersed therein and left at room temperature for 16 hours. Thereafter, the glass substrate was washed with ethanol and water and dried by nitrogen blowing. As a result, a thin film containing an epoxy group was formed on the surface of the glass substrate. The average value of the water contact angle on the substrate surface after film formation was 49.8 °.

(Second stage reaction)
While stirring 25 g of tetraethylene glycol, a catalytic amount of concentrated sulfuric acid was slowly added and further stirred at room temperature for several minutes. The epoxy-treated substrate was immersed in the tetraethylene glycol and reacted at 80 ° C. for 60 minutes. After the reaction, the substrate was washed thoroughly with water and then dried. Thus, a glass substrate on which a hydrophilic thin film was formed could be made.

(Oxidation treatment)
The photocatalyst-equipped photomask used in Example 2 was used. The photocatalyst layer of the photomask and the hydrophilic thin film on the substrate surface were brought into contact with each other, and the photomask was placed in the exposure machine so that light was irradiated from the photomask side. Exposure was performed for 188 seconds with a mercury lamp having an illuminance of 350 nm and an illuminance of 18.6 mW / cm ² to partially oxidatively decompose the hydrophilic thin film on the substrate surface. This substrate was cut into a size of 21 mm × 21 mm.

(Dish for pattern culture)
A plastic dish (manufactured by Corning) with a diameter of 35 mm having a hole with a diameter of 14 mm in the center of the bottom was used. The cell culture substrate having a thickness of 0.1 mm was attached using an adhesive KE45T (manufactured by Shin-Etsu Silicone) so as to close the hole at the bottom of the dish. It was dried at 35 ° C. for 4 hours and further dried at room temperature for 2 days. Sterilized with 70% ethanol and washed with phosphate buffer.

(Cell culture)
2 × 10 ⁵ bECs were suspended in 2.5 ml of 5% bovine serum-containing MEM medium and seeded on the pattern culture dish. This was cultured for 68 hours in an incubator (37 ° C., 5% CO ₂ ). When observed with a phase contrast microscope, bEC adhered only to the oxidized portion of the substrate.

(Cell transcription)
200 μl of ice-cooled growth factor-reduced Matrigel (Becton Dickinson) was spread on a tissue culture plastic sheet (manufactured by Wako Pure Chemical Industries) having a diameter of 23 mm to a diameter of about 15 mm and left at room temperature for about 10 minutes. The medium in the dish was removed by suction, and the plastic sheet on which the matrigel was placed was gently placed so that the matrigel faced the patterned cells on the bottom of the dish. The dish was covered and allowed to stand at room temperature for about 5 minutes, and then 2.5 ml of MEM medium containing 0.3% bovine serum was added and cultured in an incubator (37 ° C., 5% CO ₂ ) for 3 hours. When observed with a phase contrast microscope, the cell pattern was morphologically changed into a luminal shape. At this time, when the plastic sheet was peeled off, all bEC was transferred to Matrigel on the plastic sheet.

(Example 10)
(Fibroblast culture)
Using a 6 cm dish, mouse embryo fibroblast BALB / 3T3 clone A31 was cultured in DMEM medium containing 10% fetal bovine serum. After becoming 100% confluent, the culture was further continued for 24 hours.

(Fluorescent staining of bovine vascular endothelial cells)
Bovine vascular endothelial cells (bEC) were stained with the fluorescent dye PKH26 (Aldrich). The staining method was performed according to the manufacturer's protocol sheet.

(Pattern culture of fluorescently stained bEC)
Using the 0.1 mm-thick cell culture substrate prepared in Example 9, the fluorescently stained bEC was 48 in an incubator (37 ° C., 5% CO ₂ ) using MEM medium containing 5% fetal calf serum. Time pattern culture was performed. The cell pattern was confirmed to be good by observation with a phase contrast microscope. Moreover, it was confirmed that bEC was well dyed by observation with a fluorescence microscope.

(Cell transcription)
The medium is removed by suction from the dish in which the fibroblasts are cultured, the substrate on which the fluorescence-stained bEC is subjected to pattern culture is disposed so that the fibroblast sheet and the pattern-cultured bEC face each other, and the lid of the dish is disposed. And kept for 10 minutes in the incubator. Thereafter, MEM medium containing 0.3% bovine serum was gently added to the dish, taking care not to move the substrate, and further cultured in an incubator for 6 hours. It was confirmed by observation with a fluorescence microscope that the width of the bEC pattern was reduced by about 50%. Further, when the cell culture substrate was carefully peeled off, it was confirmed by microscopic observation that bEC did not remain on the cell culture substrate and that the bEC pattern was transferred onto the fibroblast sheet.

(Example 11)
(Substrate on which a hydrophilic thin film is formed)
The substrate produced in Example 2 was used.
(Oxidation treatment)
A photomask having a titanium oxide photocatalyst applied to the entire surface was prepared. As the photomask, a 5-inch photomask having a pattern in which openings with a width of 60 μm are arranged in lines at intervals of 140 μm and an opening with a width of about 1.5 cm is used. The illuminance of the exposure machine was measured in advance at a wavelength of 350 nm and used as a guide for setting the exposure time. The illuminance was 20 mW / cm ² . Place the glass substrate on which the hydrophilic thin film is formed and the photomask with the above catalyst so that the hydrophilic thin film and the catalyst layer of the photomask face each other, and place it in the exposure machine so that light is irradiated from the back side of the photomask. did. The film was exposed for 75 seconds to be oxidized. Then, it cut | disconnected to the magnitude | size of 24 mm x 15 mm so that it might be easy to use for culture | cultivation.

(Cell culture and cell transcription)
Experiments were performed in the same manner as in Example 2 using HUVEC. Even if the cell pattern density was high, it was confirmed that HUVEC was transferred to Matrigel in 30 minutes as in Example 2.

(Example 12)
(Substrate on which a hydrophilic thin film is formed)
The substrate manufactured in Example 1 was used.
(Oxidation treatment)
A photomask having a titanium oxide photocatalyst applied to the entire surface was prepared. As the photomask, a 5-inch photomask having a pattern in which 100 μm square openings are arranged at intervals of 100 μm and having an opening having a width of about 1.5 cm is used. The illuminance of the exposure machine was measured in advance at a wavelength of 350 nm and used as a guide for setting the exposure time. The illuminance was 20 mW / cm ² . Place the glass substrate on which the hydrophilic thin film is formed and the photomask with the above catalyst so that the hydrophilic thin film and the catalyst layer of the photomask face each other, and place it in the exposure machine so that light is irradiated from the back side of the photomask. did. Exposure was performed for 25 seconds to perform oxidation treatment. Then, it cut | disconnected to the magnitude | size of 24 mm x 15 mm so that it might be easy to use for culture | cultivation.

(Cell culture and cell transcription)
The experiment was performed in the same manner as in Example 1 using bEC. It was confirmed that bEC was transferred to Matrigel in 2 hours as in Example 1 even in a 100 μm square cell pattern.

Claims (6)

A method for producing a substrate for cell culture comprising a substrate and a cell adhesive region formed on the substrate surface,
A cell adhesion-inhibiting hydrophilic film containing an organic compound having a carbon-oxygen bond is formed on the surface of the substrate, and the cell adhesion-inhibiting area of the hydrophilic film is subjected to a decomposition treatment by ultraviolet irradiation to thereby apply the area to the area. A production method comprising modifying cell adhesion regions by imparting cell adhesion properties. A method for producing a substrate for cell culture comprising a substrate, a cell adhesion region formed on the substrate surface and a cell adhesion inhibitory region,
By forming a cell adhesion-inhibiting hydrophilic film containing an organic compound having a carbon-oxygen bond on the surface of the substrate, and subjecting a part of the cell adhesion-inhibiting area of the hydrophilic film to a decomposition treatment by ultraviolet irradiation. A method for producing a cell, wherein the partial region is imparted with cell adhesion and modified into a cell adhesion region.   The production method according to claim 1 or 2, wherein the ultraviolet rays are ultraviolet rays in a VUV region to a UV-C region.   The production method according to claim 2 or 3, wherein the amount of carbon in the cell adhesion region is 20 to 99% of the amount of carbon in the cell adhesion inhibitory region.   The value of the percentage of carbon bonded to oxygen in the cell adhesion region is changed to the value of the percentage of carbon bonded to oxygen in the cell adhesion inhibitory region. It is 35 to 99% with respect to it, The manufacturing method of any one of Claims 2-4 characterized by the above-mentioned. A method for producing a cell adhesion substrate , comprising culturing cells by adhering to a cell adhesion region of the cell culture substrate produced by the production method according to any one of claims 1 to 5.

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