WO2005093039A1 - 細胞培養基板の製造方法および細胞培養基板製造装置 - Google Patents
細胞培養基板の製造方法および細胞培養基板製造装置 Download PDFInfo
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- 229920003213 poly(N-isopropyl acrylamide) Polymers 0.000 description 1
- 229920001084 poly(chloroprene) Polymers 0.000 description 1
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 229920002480 polybenzimidazole Polymers 0.000 description 1
- 229920002857 polybutadiene Polymers 0.000 description 1
- 229920001748 polybutylene Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 238000006068 polycondensation reaction Methods 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 239000009719 polyimide resin Substances 0.000 description 1
- 229920001195 polyisoprene Polymers 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 239000005077 polysulfide Substances 0.000 description 1
- 229920001021 polysulfide Polymers 0.000 description 1
- 150000008117 polysulfides Polymers 0.000 description 1
- 229920002635 polyurethane Polymers 0.000 description 1
- 239000004814 polyurethane Substances 0.000 description 1
- 229920002689 polyvinyl acetate Polymers 0.000 description 1
- 239000011118 polyvinyl acetate Substances 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 125000002572 propoxy group Chemical group [*]OC([H])([H])C(C([H])([H])[H])([H])[H] 0.000 description 1
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- 210000000952 spleen Anatomy 0.000 description 1
- VEALVRVVWBQVSL-UHFFFAOYSA-N strontium titanate Chemical compound [Sr+2].[O-][Ti]([O-])=O VEALVRVVWBQVSL-UHFFFAOYSA-N 0.000 description 1
- 229920003048 styrene butadiene rubber Polymers 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
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- UQMOLLPKNHFRAC-UHFFFAOYSA-N tetrabutyl silicate Chemical compound CCCCO[Si](OCCCC)(OCCCC)OCCCC UQMOLLPKNHFRAC-UHFFFAOYSA-N 0.000 description 1
- LFQCEHFDDXELDD-UHFFFAOYSA-N tetramethyl orthosilicate Chemical compound CO[Si](OC)(OC)OC LFQCEHFDDXELDD-UHFFFAOYSA-N 0.000 description 1
- ZUEKXCXHTXJYAR-UHFFFAOYSA-N tetrapropan-2-yl silicate Chemical compound CC(C)O[Si](OC(C)C)(OC(C)C)OC(C)C ZUEKXCXHTXJYAR-UHFFFAOYSA-N 0.000 description 1
- ZQZCOBSUOFHDEE-UHFFFAOYSA-N tetrapropyl silicate Chemical compound CCCO[Si](OCCC)(OCCC)OCCC ZQZCOBSUOFHDEE-UHFFFAOYSA-N 0.000 description 1
- XOLBLPGZBRYERU-UHFFFAOYSA-N tin dioxide Chemical compound O=[Sn]=O XOLBLPGZBRYERU-UHFFFAOYSA-N 0.000 description 1
- 229910001887 tin oxide Inorganic materials 0.000 description 1
- 238000012090 tissue culture technique Methods 0.000 description 1
- 229910000348 titanium sulfate Inorganic materials 0.000 description 1
- XJDNKRIXUMDJCW-UHFFFAOYSA-J titanium tetrachloride Chemical compound Cl[Ti](Cl)(Cl)Cl XJDNKRIXUMDJCW-UHFFFAOYSA-J 0.000 description 1
- VXUYXOFXAQZZMF-UHFFFAOYSA-N titanium(IV) isopropoxide Chemical compound CC(C)O[Ti](OC(C)C)(OC(C)C)OC(C)C VXUYXOFXAQZZMF-UHFFFAOYSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
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- 229910001930 tungsten oxide Inorganic materials 0.000 description 1
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- 229920006305 unsaturated polyester Polymers 0.000 description 1
- 238000001771 vacuum deposition Methods 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/06—Plates; Walls; Drawers; Multilayer plates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0068—General culture methods using substrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2535/00—Supports or coatings for cell culture characterised by topography
- C12N2535/10—Patterned coating
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S977/00—Nanotechnology
- Y10S977/70—Nanostructure
- Y10S977/701—Integrated with dissimilar structures on a common substrate
- Y10S977/702—Integrated with dissimilar structures on a common substrate having biological material component
- Y10S977/703—Cellular
Definitions
- the present invention relates to a method for producing a cell culture substrate having cells adhered in a high-definition pattern, and an apparatus used for producing the cell culture substrate.
- Some cells particularly many animal cells, have an adhesion dependency of growing by adhering to something, and cannot survive for a long period of time in a floating state outside a living body. Cultivation of cells having such adhesion dependence requires a carrier for the cells to adhere to the cells. Generally, a plastic-made cell on which a cell adhesion protein such as collagen fibronectin is uniformly applied is generally used. A culture dish is used. These cell adhesion proteins are known to act on cultured cells, facilitating cell adhesion and affecting cell morphology.
- a technique for arranging cultured cells is to use a substrate having a patterned surface with different ease of adhesion to the cells, cultivate the cells on this surface, and allow the cells to adhere.
- a method is used in which cells are arranged by adhering the cells only to the processed surface.
- Patent Document 1 a charge holding medium having an electrostatic charge pattern formed thereon is applied to cell culture for the purpose of, for example, growing nerve cells in a circuit form. Further, Patent Document 2 attempts to arrange cultured cells on a surface obtained by patterning a non-cell-adhesive or cell-adhesive photosensitive hydrophilic polymer by a photolithography method.
- Patent Document 3 describes a cell culture substrate on which a substance such as collagen which affects cell adhesion rate and morphology is patterned, and a method for producing the substrate by photolithography. Has been disclosed. By culturing the cells on such a base material, more cells can be adhered to the surface on which collagen or the like is put on, thereby realizing the cell patterning.
- the pattern jungle of the cell culture site is required to have high definition depending on the application.
- a photolithography method or the like using a photosensitive material as described above, a high-definition pattern can be obtained, but the cell adhesive material needs to have photosensitivity.
- a micro'contact printing method is used as a method for forming a pattern of a high-definition cell adhesive material.
- Patent Document 1 JP-A-2-245181
- Patent Document 2 JP-A-3-7576
- Patent Document 3 JP-A-5-176753
- Patent Document 4 U.S. Pat.No. 5,512,131
- Patent Document 5 U.S. Pat.No. 5,900,160
- Patent Document 6 JP-A-9-240125
- Patent Document 7 JP-A-10-12545
- the present invention provides a light-shielding portion and a cell-adhesive material that has adhesiveness to cells and is decomposed or denatured by a photocatalyst accompanying energy irradiation so as to cover the light-shielding portion.
- a cell adhesion layer containing the substrate and forming a pattern for a puttering substrate
- the pattern-forming substrate is irradiated with the substrate-side force energy to form a pattern comprising a cell adhesion inhibitor in which the cell adhesive material has been decomposed or denatured and a cell adhesion portion other than the cell adhesion inhibitor.
- Energy irradiation process
- the cell adhesion layer is immersed in a cell culture solution in the cell adhesion step.
- cells can be adhered only to the cell adhesion portion, and cells can be cultured in a cell culture solution in a high-definition pattern.
- the puttering substrate having the light-shielding portion and the cell adhesive layer is formed in the puttering substrate forming step, the entire surface from the base material side in the energy irradiation step is formed.
- the cell adhesion material By irradiating the energy, the cell adhesion material can be decomposed or denatured only in the area where the light-shielding part is not formed, so that the cell adhesion material can be efficiently and highly precisely removed from the cell adhesion part and the cell adhesion inhibition part. Form a pattern You can do it.
- the cell adhesion layer may be a photocatalyst-containing cell adhesion layer containing a photocatalyst and the cell adhesion material.
- the photocatalyst contained in the photocatalyst-containing cell adhesive layer itself is excited, and the cell adhesive material can be decomposed or denatured. Therefore, a cell culture substrate can be manufactured with high manufacturing efficiency without having to separately form a layer containing a photocatalyst.
- the patterning substrate forming step includes forming at least a photocatalyst-containing layer containing a photocatalyst and the light-shielding portion on the base material, Alternatively, a step of forming the above-mentioned cell adhesion layer to form a substrate for patterning may be performed.
- the cell adhesion layer is formed on the photocatalyst containing layer, the possibility that the cells directly contact the photocatalyst when the cells are adhered to the cell adhesion portion in the cell adhesion step is low.
- a cell culture substrate in which cells are less likely to be affected by a photocatalyst over time can be manufactured.
- the cell adhesion inhibitor may be irradiated with energy during the cell adhesion step.
- the adhesion of the cell adhesion inhibitor to the cell can be further reduced by the action of the photocatalyst accompanying the energy irradiation, and the cell can be further prevented from adhering to the cell adhesion inhibitor. be able to.
- the cell adhesion inhibitor is irradiated with the substrate-side force energy to maintain the pattern of the cells adhered to the cell adhesion.
- a cell pattern maintenance step may be performed.
- the present invention provides a substrate support for supporting a substrate, a pH adjusting means for holding a cell culture solution containing the cells and the culture solution, and maintaining the pH of the cell culture solution, and the cell.
- a cell comprising: a cell culture solution holding unit having a temperature adjusting means for maintaining a temperature of a culture solution; and an energy irradiation unit for irradiating the substrate with energy.
- a culture substrate manufacturing apparatus Provided is a culture substrate manufacturing apparatus.
- the cell culture substrate manufacturing apparatus includes the cell culture solution holding unit, the substrate support unit, and the energy irradiation unit, when culturing cells in the cell culture solution, Proteins and cells attached to areas other than the area where cells are cultured can be removed by energy irradiation, etc., and a cell culture substrate on which cells are cultured in a high-definition pattern can be manufactured. And a cell culture substrate manufacturing apparatus. The invention's effect
- a cell culture substrate in which cells are cultured in a high-definition pattern in a cell culture solution can be produced, and cells can be cultured while maintaining a cell pattern for a long period of time. It can be done as a monkey.
- FIG. 1 is a process chart showing an example of a method for producing a cell culture substrate of the present invention.
- FIG. 2 is a process chart showing another example of the method for producing a cell culture substrate of the present invention.
- FIG. 3 is a schematic cross-sectional view showing one example of a patterning substrate formed in a patterning substrate forming step of the method for producing a cell culture substrate of the present invention.
- FIG. 4 is an explanatory view showing one example of the cell culture substrate manufacturing apparatus of the present invention.
- the present invention relates to a method for producing a cell culture substrate on which cells are adhered in a high-definition pattern, and an apparatus used for producing the cell culture substrate.
- a method for producing a cell culture substrate on which cells are adhered in a high-definition pattern and an apparatus used for producing the cell culture substrate.
- the method for producing the cell culture substrate of the present invention will be described.
- the light-shielding portion and the light-shielding portion are adhered to cells and are decomposed or denatured by a photocatalyst accompanying energy irradiation so as to cover the light-shielding portion.
- the pattern-forming substrate is irradiated with the substrate-side force energy to form a pattern comprising a cell adhesion inhibitor in which the cell adhesive material has been decomposed or denatured and a cell adhesion portion other than the cell adhesion inhibitor.
- Energy irradiation process
- the cell adhesive layer and the cell adhesion inhibitory part are formed in the cell adhesive layer in the energy irradiation step, the cell adhesive layer is formed in the cell adhesive step.
- the cells By immersing the cells in the cell culture solution, the cells can be easily adhered only to the cell adhesion portion, and the cells can be cultured in the desired pattern in the cell culture solution.
- proteins and cells attached to the cell adhesion inhibitor are removed. The cells can be cultured in a high-definition pattern.
- a cell culture layer is formed. Since a light-shielding portion is formed between the substrate and the substrate, in the energy irradiation step, the entire surface of the substrate is irradiated with energy to decompose the cell adhesive material only in an area where the light-shielding portion is not formed. Thus, the cell adhesion part and the cell adhesion inhibition part can be easily formed in a high-definition pattern.
- a first embodiment of the method for producing a cell culture substrate of the present invention will be described.
- a light-shielding portion and a photocatalyst accompanying energy irradiation are adhered to cells and are decomposed or denatured so as to cover the light-shielding portion.
- the pattern-forming substrate is irradiated with the substrate-side force energy to form a pattern comprising a cell adhesion inhibitor in which the cell adhesive material has been decomposed or denatured and a cell adhesion portion other than the cell adhesion inhibitor.
- Energy irradiation process
- the cell adhesion layer is a photocatalyst-containing cell adhesion material containing a photocatalyst and the cell adhesion material.
- a light-shielding portion 2 is formed on a substrate 1, and a photocatalyst containing a photocatalyst and a cell adhesive material is covered so as to cover the light-shielding portion 2.
- a patterning substrate forming step of forming the containing cell adhesive layer 3 to form a patterning substrate is performed (FIG. L (a)).
- energy 4 is irradiated from the substrate 1 side of the puttering substrate (FIG. 1 (b)), and the cell adhesive material is decomposed or denatured by the action of the photocatalyst accompanying the energy irradiation, and adheres to the cells.
- the energy that forms a pattern consisting of the cell adhesion inhibitory part 5 with reduced properties and the cell adhesion part 6 where the energy 4 is unirradiated and has good adhesion to cells (Fig. 1 (c)).
- An irradiation step is performed.
- the cell culture containing the cells and the culture solution In a nutrient solution 7 a cell adhesion step (FIG. 1 (d)) for adhering the cells 8 to the cell adhesion parts 6 is performed, and a cell culture substrate having the cells 8 adhered to only the cell adhesion parts 6 with high definition is manufactured. It is done.
- the photocatalyst-containing cell adhesion layer contains a photocatalyst and a cell adhesion material
- the photocatalyst-containing cell adhesion layer itself is irradiated with energy during an energy irradiation step or the like.
- the cell adhesive material can be decomposed or denatured, and the production efficiency of the cell culture substrate can be improved without the necessity of separately forming a layer containing the photocatalyst.
- the patterning substrate forming step in the present embodiment includes the step of forming a light-shielding portion and a light-shielding portion on the base material so as to cover the light-shielding portion and adhere to cells and decompose by the action of a photocatalyst accompanying energy irradiation.
- this is a step of forming a photocatalyst-containing cell adhesion layer containing a cell adhesion material to be denatured and a photocatalyst to form a substrate for putterjung.
- the method of forming each member is not particularly limited. Forming a part, and then applying a coating liquid for forming a photocatalyst-containing cell adhesive layer containing a photocatalyst and a cell adhesive material.
- the photocatalyst-containing cell adhesion layer formed in this step is formed on a base material described later so as to cover a light-shielding portion described later, and contains a photocatalyst and a cell adhesive material.
- the formation method and the like are not particularly limited as long as such a layer can be formed.
- a photocatalyst and a photocatalyst-containing cell adhesion layer containing the cell adhesion material spin coating, spray coating, dip coating It can be formed by applying by a wet method such as a method, a roll coating method, a bead coating method, a die coating method and the like.
- the thickness of the photocatalyst-containing cell adhesion layer is selected appropriately depending on the type of the cell culture substrate, etc. Normally, about 0.01 m-1.0 ⁇ m, and especially about 0.1 ⁇ m 0.3 ⁇ m or so.
- the type of the cell adhesive material used in this step is not particularly limited as long as it has adhesiveness to cells and is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation.
- having adhesiveness to cells means that they adhere well to cells. If the adhesiveness to cells differs depending on the type of cells, etc., it means that they adhere well to target cells. .
- the cell adhesive material used in this step has such an adhesive property to cells, and is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation, and has an adhesive property to cells. Those that disappear or those that change to those having cell adhesion inhibitory properties that inhibit adhesion to cells are used.
- the above-mentioned materials having adhesive properties to cells include a material having adhesive properties to cells due to physical properties and a material having adhesive properties to cells due to biochemical properties. There are two types; ⁇ .
- Physically deteriorating factors that determine the adhesiveness between cells and a material having adhesiveness to cells based on physicochemical properties include surface free energy and electrostatic interaction. For example, when the adhesiveness to cells is determined by the surface free energy of the material, if the material has a surface free energy within a predetermined range, the adhesiveness between the cells and the material becomes good, and if the material falls outside the range, the material has good surface free energy. Adhesion between the cells and the material will be reduced. As the change in cell adhesiveness due to such surface free energy, for example, the experimental results shown in the lower part of Yoshinobu Raft, supervised by CMC Publishing Noo Materials, p. 109, are known.
- Materials having adhesiveness to cells due to such factors include, for example, hydrophilized polystyrene, poly (N-isopropylacrylamide), and the like. like this
- a material is used, the surface free energy is changed by the action of a photocatalyst accompanying the energy irradiation, for example, the functional group on the surface of the above material is replaced or decomposed, and the material has adhesiveness to cells. No, or have a cell adhesion inhibitory property.
- the adhesiveness between cells and a material is determined by an electrostatic interaction or the like
- the adhesiveness to cells is determined by, for example, the amount of positive charge of the material.
- the material having an adhesive property to cells by such an electrostatic interaction include basic polymers such as polylysine, aminopropyltriethoxysilane, N- (2-aminoethyl) -3-aminopropyltrimethoxysilane. And condensates containing them.
- the above-mentioned material is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation, for example, the amount of positive charges present on the surface can be changed, and the adhesion to cells can be improved. Having no, or having cell adhesion inhibitory properties.
- Examples of the material having adhesive properties to cells due to its biological properties include those having good adhesive properties with specific cells or those having good adhesive properties with many cells.
- Examples include fibronectin, laminin, tenascin, vitronectin, a peptide containing an RGD (arginine-glycine-aspartate) sequence, a peptide containing a YIGSR (tyrosine isoleucine glycine-serine arginine) sequence, collagen, atelocollagen, gelatin and the like.
- Such a cell adhesive material varies depending on the type of the above-mentioned material, etc., and is usually 0.01% to 95% by weight, particularly 1% to 10% by weight in the photocatalyst-containing cell adhesive layer. % Is preferably contained. Thereby, the region containing the cell adhesive material can be a region having good adhesion to cells.
- the photocatalyst contained in the photocatalyst-containing cell adhesion layer formed in this step will be described.
- the photocatalyst used in this step is the same as the above-mentioned cell adhesive material There is no particular limitation as long as it can be decomposed or denatured by the action of the photocatalyst accompanying the gamma irradiation.
- the action mechanism of a photocatalyst typified by titanium oxide as described below is not necessarily clear, but a direct reaction with a compound near a carrier force generated by light irradiation, or It is thought that active oxygen species generated in the presence of oxygen, water, and water change the chemical structure of organic matter.
- the carrier exerts an effect on the above-mentioned cell adhesive material in an energy irradiation step described later.
- titanium dioxide TiO 2
- zinc oxide ZnO
- tin oxide SnO 2
- strontium titanate known as optical semiconductors
- the titanium dioxide is particularly preferably used because it has a high band gap energy, is chemically stable, is toxic, and is easily available. Titanium dioxide includes anatase type and rutile type, and any of them can be used in this embodiment. Anatase type titanium dioxide is preferred. The excitation wavelength of anatase-type titanium dioxide is below 380 nm.
- anatase type titanium dioxide examples include, for example, anatase type titania sol of peptized hydrochloride type (STS-02 (average particle diameter: 7 nm) manufactured by Ishihara Sangyo Co., Ltd.) and ST-K01 manufactured by Ishihara Sangyo Co., Ltd. ), Nitrate peptized anatase-type titazole (TA-15 (average particle size: 12 nm) manufactured by Nissan Chemical Industries, Ltd.), and the like.
- STS-02 average particle diameter: 7 nm
- ST-K01 manufactured by Ishihara Sangyo Co., Ltd.
- TA-15 Nitrate peptized anatase-type titazole manufactured by Nissan Chemical Industries, Ltd.
- a photocatalyst having a preferred average particle size of 50 nm or less, preferably 20 nm or less, since the smaller the particle size of the photocatalyst, the more effectively the photocatalytic reaction occurs.
- a visible light responsive titanium oxide may be used as the titanium oxide. Visible light responsive titanium oxide is also excited by the energy of visible light, and examples of such visible light responsive methods include a method of nitriding titanium oxide.
- the titanium oxide (TiO 2) is subjected to nitriding treatment to form a band gear of titanium oxide (TiO 2). A new energy level is formed inside the gap, and the band gap is narrowed. As a result, the excitation wavelength of ordinary titanium oxide (TiO 2) is 380 nm.
- TiO 2 titanium oxide
- Nitriding of titanium oxide is referred to as crystallizing titanium oxide (TiO 2).
- TiO 2 titanium oxide
- Processing such as arranging nitrogen atoms at the grain boundaries of the body.
- TiO 2 nitriding titanium oxide
- the amount of the photocatalyst contained in the photocatalyst-containing cell adhesion layer formed according to this embodiment is in the range of 5 to 95% by weight, preferably 10 to 60% by weight, and more preferably 20 to 40% by weight. Can be set with. This is because in the energy irradiation step described later, the cell adhesive material of the photocatalyst-containing cell adhesive layer in the area irradiated with the energy can be decomposed or denatured.
- the photocatalyst used in the present embodiment preferably has low adhesiveness to cells.
- the area where the photocatalyst is exposed due to the decomposition of the cell adhesive material or the like can be used as an area having low adhesiveness to cells.
- the photocatalyst-containing cell adhesion layer to be formed may contain, for example, a binder or the like that improves the strength, resistance, or the like, if necessary, for example, without using the cell adhesion material or the photocatalyst alone. Good.
- a material having a cell adhesion-inhibiting property of inhibiting adhesion to cells at least after an energy irradiation step described later at least, as the binder.
- the energy This is because the adhesiveness with the cells of the cell adhesion inhibitor, which is the area irradiated with energy in the irradiation step, can be reduced.
- a material having the above-mentioned cell adhesion inhibitory property even before the energy irradiation, and having the cell adhesion inhibitory property by the action of a photocatalyst accompanying the energy irradiation may be.
- the binder a material which has cell adhesion inhibitory property particularly by the action of a photocatalyst accompanying energy irradiation. Accordingly, in the region before the energy irradiation, only the region irradiated with the energy that does not inhibit the adhesion of the cell adhesive material to the cell has the lower adhesion to the cell. It's a character that comes out.
- an organic substituent whose main skeleton has a high binding energy so as not to be decomposed by the photoexcitation of the photocatalyst and which is decomposed by the action of the photocatalyst is used.
- an organopolysiloxane which exhibits a large strength by hydrolyzing and polycondensing a black hole or an alkoxysilane by a sol-gel reaction or the like; (2) water repellency / oil repellency And organopolysiloxanes obtained by cross-linking reactive silicones having excellent properties.
- Y is an alkyl group, fluoroalkyl group, butyl group, amino group, phenol group or epoxy group, or an organic group containing them, and X represents an alkoxyl group, an acetyl group or a halogen.
- ⁇ is an integer from 0 to 3.
- the organopolysiloxane is one or more hydrolytic condensates or cohydrolytic condensates of the silicon compound represented by
- the carbon number of the organic group represented by ⁇ is preferably in the range of 120.
- Alkoxy group represented by X is a methoxy group, an ethoxy group, a propoxy group, or a butoxy group. Preferably, there is.
- Examples of the reactive silicone of the above (2) include compounds having a skeleton represented by the following general formula. [0061] [Formula 1]
- n is an integer of 2 or more
- R 1 and IT are each a substituted or unsubstituted alkyl, aryl, aryl, or cyanoalkyl group having 120 carbon atoms, and the molar ratio of the whole Less than 40% are burs, fouls and halogenated fouls.
- the surface energy is lowest when R 2 is a methyl group, the methyl group is preferably at least 60% in a preferred molar ratio.
- the chain terminal or the side chain has at least one or more reactive group such as a hydroxyl group in the molecular chain.
- the surface of the region irradiated with the energy can be made highly hydrophilic by the action of the photocatalyst accompanying the energy irradiation. Thereby, the adhesion to the cells is inhibited, and the cells do not adhere to the energy-irradiated region.
- the contact angle with water before irradiation with energy is preferably in the range of 15 ° to 120 °, particularly preferably in the range of 20 ° to 100 °. . Accordingly, the present invention is a glass which does not inhibit the adhesiveness of the above-mentioned cell adhesive material to cells.
- the contact angle force with water is preferably S10 ° or less.
- the contact angle with water as used herein is measured using a contact angle measuring instrument (CA-Z type manufactured by Kyowa Interface Science Co., Ltd.) using a contact angle with water or a liquid having an equivalent contact angle. Measurement (micro-syringe force 30 seconds after dropping the droplet), and obtained from the results or as a graph. Further, a stable organosilicon conjugate, which does not undergo a crosslinking reaction such as dimethylpolysiloxane, may be mixed with the above-mentioned organopolysiloxane in a binder.
- a contact angle measuring instrument CA-Z type manufactured by Kyowa Interface Science Co., Ltd.
- the adhesiveness to cells is reduced or the change is assisted by causing a change in the wettability of the area irradiated with energy. It may contain a decomposed substance or the like.
- Examples of such a decomposed substance include a surfactant that is decomposed or the like by the action of a photocatalyst accompanying energy irradiation, becomes hydrophilic, and reduces the adhesiveness to cells. it can.
- a surfactant that is decomposed or the like by the action of a photocatalyst accompanying energy irradiation, becomes hydrophilic, and reduces the adhesiveness to cells. It can.
- Specific examples include hydrocarbons such as NIKKOL BL, BC, BO, and BB series from Nikko Chemicals, ZONYL FSN and FSO from DuPont, Surflon S-141, 145 from Asahi Glass, and Dainippon Japan.
- Megafac F-141, 144 manufactured by Ink Chemical Industry Co., Ltd., Futergent F-200, F251, manufactured by Neos Co., Ltd., Dudyne DS-401, 402, manufactured by Daikin Industries, Ltd., Florad manufactured by Threeem Co., Ltd.
- silicone-based nonionic surfactants such as FC-170 and 176
- cationic surfactants ion-based surfactants
- amphoteric surfactants can also be used.
- polyvinyl alcohol unsaturated polyester, acrylic resin, polyethylene, diaryl phthalate, ethylene propylene diene monomer, epoxy resin, phenol resin, polyurethane, melamine resin , Polycarbonate, polychlorinated vinyl, polyamide, polyimide, styrene butadiene rubber, chloroprene rubber, polypropylene, polybutylene, polystyrene, polyvinyl acetate, nylon, polyester, polybutadiene, polybenzimidazole, polyacryl-tolyl, epi Examples thereof include oligomers and polymers such as chlorhydrin, polysulfide, and polyisoprene.
- such a binder is contained in the photocatalyst-containing cell adhesion layer in an amount of 5 wt% to 95 wt%, particularly 40 wt% to 90 wt%, particularly 60 wt% to 80 wt%. It is preferable to be contained within the range.
- the shape of the light-shielding portion formed in this step is not particularly limited as long as it is provided between the photocatalyst-containing cell adhesive layer and a base material described later.
- the energy irradiation step it can be formed in a region to be a cell adhesion portion, that is, a region where cells are finally cultured.
- the energy irradiation is performed in the energy irradiation step described below.
- the photocatalyst-containing cell adhesive layer on the region where the light-shielding portion is formed is not irradiated with energy, and the cell adhesive material in this region can be prevented from being decomposed.
- the method for forming such a light-shielding portion is not particularly limited as long as it can shield the energy irradiated in the energy irradiation step described later. It may be formed by forming a metal thin film such as chromium having a thickness of about 1000 to 2000 A by a method or the like, and patterning the thin film. As the patterning method, a usual patterning method such as sputtering can be used.
- a method in which a layer in which light-shielding particles such as carbon fine particles, metal oxides, inorganic pigments, and organic pigments are contained in a resin binder may be formed in a pattern.
- the resin binder used may be one or a mixture of two or more resins such as polyimide resin, acrylic resin, epoxy resin, polyacrylamide, polybutyl alcohol, gelatin, casein, and cellulose.
- An oily resin, or an OZW emulsion resin composition for example, an emulsion whose reactivity is emulsified can be used.
- the thickness of the resin light-shielding portion can be set within a range of 0.5 to 10 m.
- a method of patterning the resin light-shielding portion a commonly used method such as a photolithography method and a printing method can be used.
- the light-shielding portion may be formed on the surface of the substrate on which the photocatalyst-containing cell adhesive layer is formed or may be formed on the opposite surface. Good.
- a primer layer may be formed between the photocatalyst-containing cell adhesive layer and the light-shielding portion.
- the function and function of this primer layer are not always clear, the formation of the primer layer prevents the degradation or denaturation of the cell adhesive material in the photocatalyst-containing cell adhesive layer by the action of the photocatalyst.
- impurities from the openings existing between the light shielding parts, in particular, patterning the light shielding parts It is considered to have a function of preventing the diffusion of residues generated at the time and diffusion of impurities such as metals and metal ions.
- the cell adhesion material can be decomposed or denatured with high sensitivity in the energy irradiation step described later, and as a result, the cell adhesion portion and the cell adhesion inhibition portion can be separated with high definition. It can be formed in a pattern.
- the primer layer prevents impurities present not only in the light-shielding portions but also in the openings formed between the light-shielding portions from affecting the action of the photocatalyst. Is preferably formed over the entire light-shielding portion including the opening.
- the primer layer in the present embodiment is not particularly limited as long as the primer layer is formed so that the light-shielding portion does not come into contact with the photocatalyst-containing cell adhesion layer.
- the material constituting the primer layer is not particularly limited, but an inorganic material that is not easily decomposed by the action of a photocatalyst is preferable.
- Specific examples include amorphous silica.
- the precursor of the amorphous silica is represented by the general formula SiX, wherein X is a halogen, a methoxy group, an ethoxy group, or an acetyl group.
- Silanols which are silicon compounds that are groups, and hydrolysates thereof, or polysiloxanes having an average molecular weight of 3000 or less are preferable.
- the thickness of the primer layer is preferably in the range of 0.001 ⁇ m to 1 ⁇ m.
- the force is preferably in the range of 0.001 ⁇ m to 0.1 ⁇ m.
- the substrate used in this step is capable of forming the above-mentioned photocatalyst-containing cell adhesive layer and the above-mentioned light shielding portion, and is capable of transmitting the energy irradiated in the energy irradiation step described later.
- the material is not particularly limited, for example, inorganic materials such as glass and quartz, and organic materials represented by plastics can be used.
- the flexibility and the like of the substrate are appropriately selected depending on the type and use of the finally obtained cell culture substrate.
- the energy irradiation step of the present embodiment will be described.
- the puttering substrate is irradiated with the above-mentioned substrate-side power, and the cell adhesion material is decomposed or denatured.
- This is a step of forming a pattern including a cell adhesion portion.
- the substrate-side power of the puttering substrate is also irradiated with energy, and the cell adhesion material is decomposed or denatured, so that the cell adhesion inhibiting portion whose adhesion to cells is reduced, and the energy not yet applied.
- the method of irradiating the energy is not particularly limited as long as the cell adhesive material remains due to the irradiation and a cell adhesion portion having good adhesion to cells can be formed.
- the cell adhesion inhibitor contains a photocatalyst, a decomposed product or a modified product of the cell adhesion material, and the like.
- the substrate-side power is also irradiated with energy to the entire surface, and a region where the light-shielding portion is formed is a cell adhesion inhibiting portion and the light-shielding portion is formed.
- the area where the cells fall is the cell adhesion area.
- energy is irradiated using, for example, a photomask to decompose or denature the cell adhesive material only in the target region.
- a cell adhesion inhibitor can be formed.
- energy irradiation may be performed in a cell culture solution containing cells and a culture solution used in a cell adhesion step described below.
- the cell adhesion step and the energy irradiation step which will be described later, can be performed in the same apparatus or the like, and the production efficiency of the cell culture substrate can be improved.
- the above-mentioned buttering substrate may be entirely immersed in a cell culture solution and subjected to energy irradiation.However, in the present embodiment, in particular, only the photocatalyst-containing cell adhesive layer of the above-mentioned puttering substrate, Preferably, it is contacted with a cell culture.
- the contact between the photocatalyst-containing layer and the cell culture medium means that the entire photocatalyst-containing layer and the cell adhesion layer can be simply brought into contact with the cell culture medium by simply contacting the surface of the photocatalyst-containing cell adhesion layer with the cell culture medium. It shall include immersion.
- Irradiation energy irradiation exposure refers to irradiation of a line of energy capable of decomposing or denaturing a cell adhesive material by the action of a photocatalyst accompanying the energy irradiation. And is not limited to light irradiation.
- the wavelength of light used for such energy irradiation is set to a range of 400 nm or less, preferably 380 nm or less. This is because, as described above, a preferred photocatalyst used as a photocatalyst is titanium dioxide, and as the energy for activating the photocatalytic action by the titanium dioxide, light of the above-described wavelength is preferred. It is.
- Examples of the light source that can be used for such energy irradiation include a mercury lamp, a metal halide lamp, a xenon lamp, an excimer lamp, and various other light sources.
- a mercury lamp a mercury lamp
- a metal halide lamp a xenon lamp
- an excimer lamp an excimer lamp
- various other light sources such as a mercury lamp, a metal halide lamp, a xenon lamp, an excimer lamp, and various other light sources.
- the substrate has a light-shielding portion in the same pattern as the cell adhesion portion, it can be carried out by irradiating the entire surface with energy from the substrate side.
- the energy irradiation amount at the time of energy irradiation is an irradiation amount necessary for the cell-adhered material to be decomposed or denatured by the action of a photocatalyst.
- the cell adhesion step of the present embodiment is a step of adhering the above-mentioned cells to the above-mentioned cell adhesion part in a cell culture solution containing cells and a culture solution.
- the cell adhesion layer is immersed in the cell culture solution containing the cells and the culture solution thereof, for example, so that the cell adhesion portion is formed.
- Cells can be adhered only to the part. Real truth In the embodiment, the cells can be adhered in the cell culture solution, and the cells can be cultured in a desired pattern.
- the cells used in such a cell culture solution include, for example, nervous tissue, liver, kidney, spleen, blood vessels, brain, cartilage, and other non-adherent cells such as blood cells, which are present in living organisms. Any tissue and cells derived therefrom can be used.
- non-adherent cells a technique for modifying a cell membrane in order to perform adhesive fixation has been devised in recent years, and such a technique can be used in this embodiment as necessary. Is possible.
- each tissue as described above is formed by cells having various functions, it is necessary to select and use desired cells.
- the liver it is formed from epithelial cells, endothelial cells, Kupffer cells, fibroblasts, fat-ingesting cells, and the like, in addition to hepatic parenchymal cells.
- the adhesiveness to the cell adhesive material varies depending on the type of cells, it is necessary to select the cell adhesive material used in the photocatalyst-containing cell adhesive layer and the composition ratio thereof according to the cell type.
- the content of such cells may be appropriately selected depending on the type of cells, etc., and is usually 10 4 cells / ml—10 8 cells / ml, especially 10 5 cells / ml in a normal cell culture medium. It is preferable that the content be within the range of 10 7 cells / ml. Thereby, the cell can efficiently adhere cells to the cell adhesion portion.
- the culture solution used in this step is appropriately selected depending on the type of the above-mentioned cells.
- "Tissue culture technology, third edition, basic edition” p3—p5 Japanese Society for Tissue Culture) Edited
- commercially available products such as an Eagle's medium and a fisher's medium can also be used.
- the cell adhesion inhibitor may be continuously or intermittently irradiated with energy. This makes it possible to remove cells and the like adhered to the cell adhesion inhibitor by the action of the photocatalyst contained in the photocatalyst-containing cell adhesion layer, and to achieve a high-definition pattern only in the cell adhesion part. This is because the cells can be adhered in a shape.
- the energy irradiated is the photocatalyst
- the energy applied in the energy irradiation step is not particularly limited as long as it is an energy capable of removing cells and the like adhering to the cell adhesion inhibitor by the action of the above. .
- the entire surface of the substrate-side cap is irradiated with energy, so that the surface of the cell adhesion inhibition portion is exposed.
- the cells can be removed.
- a cell pattern maintaining step of maintaining the pattern of the cells adhered to the cell adhesion portion by irradiating the inhibitor with energy may be performed.
- the method of irradiating the substrate-side force energy is not particularly limited as long as the method is capable of irradiating the substrate-side force energy and removing cells and the like adhered on the cell-adhered portion.
- the cell culture may be performed in a state where the cell culture solution and the cell adhesion layer are in contact with each other, or may be performed after the cell culture substrate to which the cells are adhered is pulled out of the cell culture solution. .
- the method of irradiating the energy in the cell pattern maintaining step and the like can be the same as in the above-described energy irradiating step, and thus the detailed description is omitted here.
- a second embodiment of the method for producing a cell culture substrate of the present invention will be described.
- a light-shielding portion and a photocatalyst which is adherent to cells and is exposed to energy so as to cover the light-shielding portion are provided on a substrate.
- a cell adhesive layer containing a cell adhesive material that is decomposed or denatured by use and forming a puttering substrate for use as a puttering substrate
- the pattern-forming substrate is irradiated with the substrate-side force energy to form a pattern comprising a cell adhesion inhibitor in which the cell adhesive material has been decomposed or denatured and a cell adhesion portion other than the cell adhesion inhibitor.
- Energy irradiation process
- a light-shielding portion 2 is formed on a substrate 1, and a photocatalyst-containing layer 9 containing a photocatalyst is formed so as to cover the light-shielding portion 2.
- a patterning substrate forming step is performed in which the cell adhesion layer 10 is formed on the photocatalyst-containing layer 9 and used as a patterning substrate (FIG. 2 (a)).
- energy 14 is applied from the substrate 1 side of the puttering substrate (FIG. 2 (b)), and is contained in the cell bonding layer 10 by the action of the photocatalyst contained in the photocatalyst containing layer 9.
- It consists of a cell adhesion inhibitor 5 whose cell adhesion material has been degraded or denatured to reduce its adhesion to cells, and a cell adhesion part 6 where energy 4 has not been irradiated and which has good adhesion to cells.
- An energy irradiation step for forming a pattern (FIG. 2 (c)) is performed.
- the cell adhesion section 6 is subjected to the cell adhesion step of adhering the cells 8 (FIG. 2 (d)), so that only the cell adhesion section 6 is formed. It is possible to manufacture a cell culture substrate to which the cells 8 are adhered with high definition.
- a cell adhesion layer 10 may be formed so as to cover the light shielding part 2.
- the cell adhesive layer containing the cell adhesive material and the photocatalyst containing layer containing the photocatalyst are separately formed, in the energy irradiation step or the like, from the substrate side When energy is applied, the photocatalyst in the photocatalyst-containing layer is excited, and the cell adhesive material in the adjacent cell adhesive layer is decomposed or denatured.
- the cells adhered in the cell adhesion step are adhered on the cell adhesion layer, there is little possibility that the cells directly contact the photocatalyst. This has the advantage that cells are less likely to be affected by the photocatalyst over time.
- each step in the method for manufacturing a cell culture substrate of the present embodiment will be described.
- the patterning substrate forming step in the present embodiment includes forming a photocatalyst-containing layer having at least a photocatalyst and the above-mentioned light-shielding portion on a base material, having an adhesive property to cells, and an action of the photocatalyst accompanying energy irradiation.
- This is a step of forming a cell adhesion layer containing a cell adhesion material that is decomposed or denatured by the above process to obtain a patterning substrate.
- the configuration and the like of the patterning substrate are not particularly limited.
- light is shielded on the substrate.
- a photocatalyst-containing layer is formed so as to cover the light-shielding portion
- a cell adhesion layer is formed on the photocatalyst-containing layer
- the photocatalyst-containing layer is formed on a substrate for use as a patterning substrate.
- a light-shielding portion may be formed on the photocatalyst-containing layer, and a cell adhesive layer may be formed so as to cover the light-shielding portion.
- V does not affect the photocatalyst function in the photocatalyst-containing layer on the region where the light-shielding portion is formed when energy is irradiated from the substrate side in the energy irradiation step described below. This is because this region can be used as a cell adhesion portion.
- the light-shielding portion may be formed on a surface of the substrate opposite to the surface on which the photocatalyst-containing layer and the cell adhesion layer are formed.
- the cell adhesion layer formed in this step is a layer having a cell adhesive material having at least adhesive property to cells.
- a specific cell adhesive material a material similar to the cell adhesive material used for the photocatalyst-containing cell adhesive layer described in the first embodiment can be used, and thus a detailed description is omitted here.
- the cell adhesive layer formed in this step also contains the material having the cell adhesion inhibitory property described in the photocatalyst-containing cell adhesive layer of the first embodiment. This is a force that makes it possible to lower the adhesiveness to the cells of the cell adhesion inhibitor, which is the area irradiated with energy in the energy irradiation step described later.
- the formation of such a cell adhesion layer can be carried out by applying a coating solution for forming a cell adhesion layer containing the above-mentioned cell adhesion material by a general application method or the like. Since the method can be the same as the method for forming the photocatalyst-containing cell adhesive layer described in the embodiment, the description is omitted here.
- the cell adhesion material-containing liquid containing the cell catalyst material contains the photocatalyst-containing liquid.
- the layer may be soaked to form a cell adhesion layer on the photocatalyst-containing layer by an adsorption method.
- the energy irradiation step described below can be performed in a liquid, and the cell adhesion step is performed in a cell culture solution. Therefore, for example, when a cell adhesive material is weak against drying or the like! ⁇ A material or oxygen is weak!
- the energy irradiation step is performed in the cell adhesive material-containing liquid or the cell culture solution. After that, by performing the cell adhesion step, it is possible to prevent the cell adhesion material from drying or denaturing during the production of the cell culture substrate, and to adhere the target cells in a pattern. Because it becomes.
- the cell adhesive layer in such a liquid containing a cell adhesive material.
- the cell adhesive material include materials whose functions are reduced by drying, such as proteins. For example, collagen, fibronectin, ⁇ -globulin and the like can be mentioned.
- the solvent used for the cell adhesive material-containing liquid include water, alcohol, ethylene daryl, acid, buffer, and medium.
- the thickness of the cell adhesion layer formed in this step is appropriately selected depending on the type of the cell culture substrate, etc. Normally, about 0.1 OOl / zm-l.O / zm, Above all 0.005 ⁇ m — 0.1 ⁇ m or so.
- the photocatalyst-containing layer formed in this step is not particularly limited as long as it is a layer containing at least a photocatalyst, and may be a layer having only photocatalyst power, and may contain other components such as a binder. It may be a layer or the like.
- the photocatalyst used in the present embodiment can be the same as that used for the photocatalyst-containing cell adhesive layer in the first embodiment, and in this embodiment, in particular, titanium oxide is used. Is preferred! / ,.
- Examples of a method for forming a photocatalyst-containing layer in which only a photocatalyst is effective include a method using a vacuum film-forming method such as a sputtering method, a CVD method, and a vacuum evaporation method.
- a vacuum film-forming method such as a sputtering method, a CVD method, and a vacuum evaporation method.
- amorphous titania is formed on a base material, and then the crystalline titania is formed by firing.
- a method of changing the phase to titania may be used.
- the amorphous titanium used herein includes, for example, hydrolysis, dehydration condensation of inorganic salts of titanium such as titanium tetrachloride and titanium sulfate, tetraethoxytitanium, tetraisopropoxytitanium, tetra-n-propoxytitanium, and tetrabutoxytitanium.
- Organic titanium conjugates such as titanium and tetramethoxytitanium can be obtained by hydrolysis and dehydration condensation in the presence of an acid. Then, it can be modified to anatase type titania by baking at 400 ° C to 500 ° C, and modified to rutile type titania by baking at 600 ° C to 700 ° C.
- a binder a binder having a high binding energy such that the main skeleton of the binder is not decomposed by the photoexcitation of the photocatalyst is preferable.
- the above-mentioned cell adhesion layer is used as such a binder.
- organopolysiloxanes described in the above section is used.
- the photocatalyst-containing layer is formed by dispersing a photocatalyst and an organopolysiloxane as a binder in a solvent together with other additives as necessary. It can be formed by preparing a coating solution and applying the coating solution onto a substrate.
- a coating solution As the solvent to be used, alcohol-based organic solvents such as ethanol and isopropanol are preferable.
- the coating can be performed by a known coating method such as spin coating, spray coating, dip coating, roll coating, bead coating, and die coating.
- a UV-curable component is included as a binder, the photocatalyst-containing layer can be formed by performing a curing treatment by irradiating ultraviolet rays.
- an amorphous silica precursor can be used as a binder.
- This amorphous silica precursor is represented by the general formula SiX, where X is a halogen, methoxy, ethoxy, or acetyl group.
- silicon compounds such as hydroxyl groups, silanols which are hydrolysates thereof, and polysiloxanes having an average molecular weight of 3000 or less!
- Specific examples include tetraethoxysilane, tetraisopropoxysilane, tetra-n-propoxysilane, tetrabutoxysilane, tetramethoxysilane and the like.
- the precursor of the amorphous silica and the particles of the photocatalyst are uniformly dispersed in a non-aqueous solvent, and the transparent substrate is hydrolyzed with moisture in the air to form silanol.
- the photocatalyst-containing layer can be formed by dehydration-condensation polymerization at room temperature. If the dehydration-condensation polymerization of silanol is performed at 100 ° C. or higher, the degree of polymerization of silanol increases, and the strength of the film surface can be improved.
- These binders can be used alone or in combination of two or more.
- the content of the photocatalyst in the photocatalyst containing layer can be set in the range of 5 to 60% by weight, preferably 20 to 40% by weight.
- the thickness of the photocatalyst-containing layer is preferably in the range of 0.05-10 / zm.
- the photocatalyst-containing layer may contain, in addition to the above-mentioned photocatalyst and binder, a surfactant and the like used for the above-mentioned cell adhesion layer.
- the surface of the photocatalyst-containing layer preferably has low adhesiveness to cells, for example, because the surface has high hydrophilicity. Accordingly, in the energy irradiation step described later, when the photocatalyst-containing layer is exposed by disassembling the cell adhesion layer, the area can be made into an area having low adhesion to cells.
- the above-mentioned puttering substrate is irradiated with energy from the substrate side, and the cell adhesion material is decomposed or denatured, and the cell adhesion inhibitor other than the cell adhesion inhibitor.
- This is a step of forming a pattern composed of the parts.
- the substrate-side power of the puttering substrate is also irradiated with energy, and the cell adhesion material is decomposed or denatured, and the cell adhesion-inhibiting portion whose adhesion to cells is reduced, and the energy is not increased.
- the method of irradiating the energy is not particularly limited as long as the cell adhesive material remains due to the irradiation and a cell adhesion portion having good adhesion to cells can be formed. At this time, for example, when the cell adhesion material is decomposed by the action of a photocatalyst accompanying energy irradiation, a small amount of the cell adhesion material is contained in the cell adhesion inhibitor!
- the cell adhesion layer is completely decomposed and removed to expose the photocatalyst-containing layer.
- the cell adhesive material is modified by the action of a photocatalyst accompanying energy irradiation, the denatured product or the like is contained in the cell adhesion inhibitor.
- the entire surface of the substrate side is also irradiated with energy so that a region where the light-shielding portion is formed is a cell adhesion inhibiting portion and the light-shielding portion is formed.
- the area where the cells fall is the cell adhesion area.
- energy is irradiated using, for example, a photomask to decompose or denature the cell adhesive material only in the target region.
- a cell adhesion inhibitor can be formed.
- this step is performed in the cell adhesive material-containing liquid containing the cell adhesive material.
- the energy irradiation may be performed in a cell culture solution containing cells and a culture solution used in a cell adhesion step described below.
- the whole of the puttering substrate may be immersed in the cell culture solution and subjected to energy irradiation.
- the base material of the puttering substrate may be immersed in the cell culture solution. It is preferred not to make contact. Thereby, the transmission efficiency of the irradiated energy can be improved, and the cell culture substrate can be manufactured efficiently.
- the cell adhesion step of the present embodiment is a step of adhering cells to a cell adhesion portion in the cell adhesion layer in a cell culture solution containing cells and a culture solution.
- the cell adhesion step in the present embodiment can be the same as the cell adhesion step in the above-described first embodiment, and a detailed description thereof will be omitted.
- energy may be applied to the cell adhesion inhibition portion.
- steps may be appropriately performed as needed.
- energy is irradiated to the above-mentioned cell adhesion inhibitor from the above-mentioned substrate side.
- a cell pattern maintaining step of maintaining the pattern of the cells adhered to the cell adhesion portion may be performed.
- proteins or cells adhere to the cell adhesion inhibitor after the cell adhesion step these cells can be removed by the action of the photocatalyst accompanying the energy irradiation. is there.
- the cell pattern maintaining step can be performed in the same manner as in the first embodiment, and thus the detailed description thereof will be omitted.
- the plate manufacturing apparatus includes a substrate support for supporting the substrate, a cell culture solution containing the cells and the culture solution, a pH adjusting means for maintaining the pH of the cell culture solution, and a temperature of the cell culture solution.
- a cell culture solution holding unit having a temperature adjusting means for maintaining the temperature, and an energy irradiation unit for irradiating the substrate with energy.
- the cell culture substrate manufacturing apparatus of the present invention includes a substrate support 22 for supporting a substrate 21, a cell culture solution holding portion 23 for holding a cell culture solution 7, an energy storage device for the substrate.
- the cell culture solution holding unit 23 includes a pH adjusting means (not shown) for maintaining pH in the cell culture solution 7, and a cell culture solution 7. It has a temperature adjusting means (not shown) for maintaining the medium temperature.
- the cell culture substrate manufacturing apparatus includes the cell culture solution holding unit, the substrate support unit, and the energy irradiation unit, for example, a cell culture using a layer containing a photocatalyst is performed.
- a cell culture using a layer containing a photocatalyst is performed.
- protein and cells attached to the area other than the area where the cells are cultured on the substrate can be removed by energy irradiation, etc., and cell culture in which cells are cultured in a high-definition pattern
- a cell culture substrate manufacturing apparatus capable of manufacturing a substrate can be provided.
- the substrate used in the present invention includes a base material, a light-shielding portion formed on the base material, and a light-shielding portion formed on the base material so as to cover the light-shielding portion.
- the substrate be a patterning substrate having a cell adhesive layer containing a cell adhesive material that has a cell adhesive material that is decomposed or denatured by the action of a photocatalyst accompanying energy irradiation. This allows the substrate supporting portion to support the patterning substrate, and also irradiates the substrate side power with energy by the energy irradiating portion, thereby forming a light shielding portion.
- a pattern consisting of a cell adhesion inhibitor in which the cell adhesion material in the cell adhesion layer is decomposed or denatured, and a cell adhesion portion other than the cell adhesion inhibitor.
- the cells can be attached only on the cell adhesion section, and the cells can be cultured in a desired pattern.
- the power that can be done.
- the above-mentioned energy irradiation may be performed with the cell adhesive layer immersed in the cell culture medium or not. You may go in a state.
- the cell culture solution holding unit and the energy irradiation unit are included in one device, the cell culture solution holding unit and the energy irradiation unit are used when the cells are adhered in the cell culture solution, After adhesion, energy can be irradiated to the cell adhesion inhibitor, so even if proteins or cells adhere to the cell adhesion inhibitor, cells and the like are removed by the action of the photocatalyst accompanying the energy irradiation. Thus, cells can be adhered with high definition only on the cell adhesion portion.
- the substrate supporting portion in the present invention is not particularly limited as long as it can support the substrate, and preferably adjusts the height and position of the substrate as freely as possible.
- the substrate supporting portion may be provided with a temperature adjusting means or the like for maintaining the temperature of the substrate. This makes it possible, for example, to maintain the activity of the cells adhered on the cell adhesion portion, and is a force capable of producing a high-quality cell culture substrate.
- the cell culture solution holding unit in the present invention holds a cell and a cell culture solution containing the culture solution, and maintains a pH of the cell culture solution, and a temperature control unit that maintains the temperature of the cell culture solution.
- a means it is not particularly limited, and may have a stirring means and the like as necessary.
- the pH adjusting means and the temperature adjusting means are provided in the cell culture solution holding section for holding the cell culture solution, the pH and temperature in the cell culture solution are maintained. It can keep cells constant and prevent cells from dying or becoming less active.
- a pH adjusting means the one used as a pH adjusting means in a general cell culture device can be used, and the description is omitted here.
- the temperature adjusting means the cell culture solution in the cell culture solution holding section is also controlled. Any device that can maintain the temperature can be used as a temperature adjusting device in a general cell culture device, and thus the description thereof is omitted.
- the energy irradiating unit in the present invention irradiates the substrate supported by the substrate supporting unit with energy using, for example, a layer containing a photocatalyst, and removes cells in the energy-irradiated region to form a cell pattern.
- the cell adhesion material contained in the cell adhesion layer of the patterning substrate is decomposed or denatured by the action of a photocatalyst. It is preferred that is possible.
- the light source that can be used for such energy irradiation is not particularly limited as long as it can activate the photocatalyst.
- a light source such as a laser such as excimer or YAG may be used. By using these light sources, the photocatalyst can be excited, and the cell adhesion portion can be formed.
- the cell culture substrate manufacturing apparatus of the present invention is not particularly limited as long as it has the above-described substrate support, cell culture solution holding unit, and energy irradiation unit. You may have. Further, the cell culture substrate manufacturing apparatus of the present invention is preferably used for the above-described “A. Method for manufacturing cell culture substrate” and the like.
- the present invention is not limited to the above embodiment.
- the above embodiment is an exemplification, and has substantially the same configuration as the technical idea described in the claims of the present invention. Included in the technical scope of the invention.
- the incubator was modified with a commercially available pH and temperature adjustment function, a mercury lamp was installed on the ceiling, and a power supply with a timer was installed outside the incubator.
- jigs with adjustable height that can support the cell culture substrate at the four corners were installed on the ceiling.
- a cylinder with a 5% CO 95% Air regulator was inserted through a flow meter.
- a 3 cm square quartz glass substrate with a stripe-shaped light-shielding layer with a light-shielding portion of 80 m and an opening of 300 m was provided on the surface of the substrate by a general procedure for manufacturing a chrome mask.
- the photocatalyst coating solution is applied to the surface of the glass substrate by spin coating, and the substrate is dried at a temperature of 150 ° C for 10 minutes to cause hydrolysis and polycondensation reactions.
- a photocatalyst-containing layer having a thickness of 0 and firmly fixed in siloxane was formed on the substrate.
- fibronectin F-4759 Sigma
- 200 ml of pure water 0.2 mg
- this aqueous solution is dropped on the photocatalyst layer of the substrate provided with the photocatalyst containing layer at a ratio of 300 1 per lcm 2 of the substrate area. This was allowed to stand at 4 ° C for 24 hours. Further, the substrate was washed twice with PBS to obtain a puttering substrate having a photocatalyst-containing layer and a cell adhesive material-containing layer on the substrate. The substrate was immediately immersed in PBS and immediately moved to the next step.
- the liver extracted from the rat was transferred to a petri dish, subdivided into 5 mm-sized pieces using a scalpel, 20 ml of DMEM medium was added, lightly suspended with a pipette, and filtered with a cell strainer. The resulting coarsely dispersed cell suspension was centrifuged at 500-600 rpm for 90 seconds, and the supernatant was removed by suction. DMEM medium was newly added to the remaining cells and centrifuged again. By repeating this operation three times, substantially uniform hepatic parenchymal cells were obtained. To the obtained hepatocytes, 20 ml of DMEM medium was added and suspended to prepare a hepatocyte suspension.
- Wavmouth MB752Z1 medium (containing L-glutamine and no NaHCO) (Gibco)
- the liver parenchymal cell suspension prepared above was suspended in the Waymouth MB752Z1 medium solution prepared similarly, and then seeded on the above-mentioned cell culture puttering substrate placed in a petri dish. Turn off the germicidal light in the above incubator at 37 ° C, 5% CO for 24 hours.
- liver parenchymal cells were allowed to adhere to the entire surface of the substrate.
- Non-adherent cells and dead cells were removed by washing the substrate twice with PBS.
- the culture medium was newly added to the Petri dish, and the cell-adhered substrate was supported by the jig with the cell-adhered substrate facing down, and the cell-adhered surface was immersed in the medium.
- the culture of the cells was continued for up to 48 hours while exchanging the medium, and the cells were observed with an optical microscope. As a result, it was confirmed that the cells adhered along the cell adhesion part on the cell culture patterning substrate.
- the incubator was modified with a commercially available pH and temperature adjustment function, a mercury lamp was installed on the floor, and a power supply with a timer was installed outside the incubator. Instead of the shelves originally provided in the incubator, she made and mounted shelves to support petri dishes with metal wires of about 3 mm in diameter. 5% CO 95% Air Bonus with Regulator
- the vessel was connected to the incubator via a flow meter.
- Example 2 Using the same substrate as in Example 1, the same procedure as in Example 1 was performed.
- the center of the bottom of the polystyrene petri dish was cut out at a diameter of about 2.5 cm, and the substrate with the photocatalyst layer was attached thereto.
- Example 2 The same operation as in Example 1 was performed using the above-mentioned petri dish to form a substrate for puttering.
- the Petri dish with a pattern was set on the wire shelf with care so that the center of the Petri dish was not pressed.
- Example 1 The same experiment as in Example 1 was performed, and the same result as in Example 1 was obtained in this example.
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Priority Applications (1)
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US10/594,174 US7687251B2 (en) | 2004-03-26 | 2005-03-24 | Method for producing cell culture substrate and apparatus for producing cell culture substrate |
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JP2004091653A JP4456393B2 (ja) | 2004-03-26 | 2004-03-26 | 細胞培養基板の製造方法および細胞培養基板製造装置 |
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JP4571229B2 (ja) * | 2009-01-29 | 2010-10-27 | エンパイア テクノロジー ディベロップメント エルエルシー | 細胞培養システム、細胞培養方法、細胞培養容器、及び細胞培養容器の製造方法 |
JP2012125218A (ja) * | 2010-12-17 | 2012-07-05 | National Institute Of Advanced Industrial Science & Technology | 細胞分別用マイクロチップおよび細胞分別方法ならびに細胞分別装置 |
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US20060019390A1 (en) * | 2004-01-28 | 2006-01-26 | Dai Nippon Printing Co., Ltd. | Patterning substrate and cell culture substrate |
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JP5819056B2 (ja) * | 2010-11-18 | 2015-11-18 | 大日本印刷株式会社 | 細胞培養用基材 |
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JP7343119B2 (ja) | 2019-04-26 | 2023-09-12 | 株式会社片岡製作所 | 細胞培養基材、細胞培養容器、細胞の培養方法、細胞の製造方法、細胞培養基材の製造方法、および細胞培養容器の製造方法 |
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JP4571229B2 (ja) * | 2009-01-29 | 2010-10-27 | エンパイア テクノロジー ディベロップメント エルエルシー | 細胞培養システム、細胞培養方法、細胞培養容器、及び細胞培養容器の製造方法 |
JPWO2010086976A1 (ja) * | 2009-01-29 | 2012-07-26 | エンパイア テクノロジー ディベロップメント エルエルシー | 細胞培養システム、細胞培養方法、細胞培養容器、及び細胞培養容器の製造方法 |
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JP2012125218A (ja) * | 2010-12-17 | 2012-07-05 | National Institute Of Advanced Industrial Science & Technology | 細胞分別用マイクロチップおよび細胞分別方法ならびに細胞分別装置 |
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US20070141697A1 (en) | 2007-06-21 |
JP2005270055A (ja) | 2005-10-06 |
JP4456393B2 (ja) | 2010-04-28 |
US7687251B2 (en) | 2010-03-30 |
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