JP6060790B2 - Method for producing substrate for cell culture - Google Patents

Description

  The present invention relates to a method for producing a cell culture substrate for culturing cells in a pattern.

  Cell culture techniques are used for the purpose of elucidating biochemical phenomena and properties of cells and producing useful substances. Furthermore, there are attempts to study the physiological activity and toxicity of artificially synthesized drugs using cultured cells, and research on regenerative medicine in which cultured cells and tissues are transplanted into patients through the process of culturing cells in vitro. It is actively done.

  On the other hand, a technique has been reported in which cultured cells are partially adhered and arranged on a substrate. 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, Patent Literature 1 attempts to arrange cultured cells on a surface obtained by patterning a cell-adhesive or cell-adhesive photosensitive hydrophilic polymer by photolithography. Furthermore, Patent Document 2 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, when performing 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 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. In addition, in a photolithography method using a photoresist, which is commonly used in the manufacture of semiconductor products, it is necessary to use a developer or the like for developing the photoresist, which may have an adverse effect on cell culture. Furthermore, in patterning by a photolithography method using a photomask as described in Patent Document 3, patterning defects are likely to occur when dust adheres to the photomask, and a cleaning step is necessary before exposure. Even if dust that cannot be removed by the cleaning process adheres to the photomask, the photomask is expensive and cannot be disposable, and an expensive apparatus such as an exposure apparatus is required.

Japanese Patent Laid-Open No. 3-7576 Japanese Patent Laid-Open No. 5-176653 JP 2007-312736 A

  An object of the present invention is to produce a cell culture substrate for culturing cells in a pattern at a low cost without using an expensive apparatus by a simple procedure.

  The present inventors have formed a functional polymer layer having a hydrolyzable bond with an acid or a base and controlling cell adhesiveness on a substrate, patterning a covering material, and functional polymer. It was found that the pattern of the functional polymer layer can be easily formed on the base material by carrying out the hydrolysis reaction by bringing the acid polymer or base into contact with only the exposed portion of the functional polymer layer.

That is, the present invention includes the following inventions.
(1) A method for producing a cell culture substrate,
Forming a functional polymer layer on a substrate having a bond that can be hydrolyzed by an acid or a base and controlling cell adhesion;
A process of forming a pattern of a covering material on the functional polymer layer by closely adhering the functional polymer layer and the patterned covering material;
A step of hydrolyzing a part of the functional polymer layer by bringing acid or base into contact with the functional polymer layer exposed by the pattern of the coating material, and a step of peeling the coating material from the functional polymer layer Including said method.
(2) The method according to (1), wherein the functional polymer has an ester bond as a bond that can be hydrolyzed by an acid or a base.
(3) The method according to (1) or (2), wherein the surface of the covering material to be in close contact with the functional polymer layer is a slightly adhesive surface having removability.
(4) The method according to (3), wherein the slightly sticky surface of the covering material is made of silicone rubber or silicone resin.
(5) The method according to any one of (1) to (4), wherein the functional polymer is an acrylic polymer or a methacrylic polymer.
(6) The method according to (5), wherein the functional polymer is selected from polyalkylene glycol (meth) acrylate and poly-N-isopropyl (meth) acrylamide.

  According to the present invention, it is possible to produce a cell culture substrate for culturing cells in a pattern at a low cost without using an expensive apparatus with a simple procedure.

It is a schematic diagram which shows one Embodiment of the manufacturing method of the base material for cell cultures of this invention.

As shown in FIG. 1, for example, the method for producing a cell culture substrate of the present invention comprises:
a) a step of forming a functional polymer layer 1 on a substrate 2 having a bond hydrolyzable by an acid or a base and controlling cell adhesion;
b) a step of closely contacting the functional polymer layer 1 and the pattern-formed coating material 4 to form a coating material pattern on the functional polymer layer;
c) a step of performing a hydrolysis reaction on a part of the functional polymer layer by bringing the functional polymer layer 5 exposed by the pattern of the coating material 4 into contact with an acid or a base 6, and d) the coating material 4 A step of peeling from the functional polymer layer.

  In the present invention, by using a functional polymer having a bond that can be hydrolyzed by an acid or a base, a pattern of the functional polymer layer can be easily formed on a substrate by hydrolysis using an acid or a base. Can do. Examples of the bond that can be hydrolyzed by an acid or a base include, but are not limited to, an ester bond, an amide bond, a thioester bond, an acetal bond, a hemiacetal bond, a ketal bond, and a hemiketal bond. Preferably, a functional polymer having an ester bond is used. The ester bond hydrolysis reaction is preferable because it proceeds under relatively mild conditions using a low-concentration acid or base without adding heat treatment, etc., so that damage to the substrate in the hydrolysis step can be suppressed. Moreover, since there are many types of molecules having an ester bond, it is preferable because the surface properties can be controlled in accordance with the experimental system.

  The functional polymer that controls the adhesion of cells is a functional polymer that provides an adhesive and / or non-adhesive surface to cells, such as a polymer that provides an adhesive surface to cells. And a polymer that provides a surface that is non-adhesive to cells and a polymer that provides a surface whose adhesion to cells changes.

As an index for determining cell adhesion, the cell adhesion spreading rate when cells are actually cultured can be used. The cell adhesion surface is preferably a surface having a cell adhesion extension rate of 60% or more, and more preferably a surface having a cell adhesion extension rate of 80% or more. If the cell adhesion extension rate is high, cells can be cultured efficiently. The cell adhesion extension rate in the present invention is such that cells to be cultured within a seeding density of 4000 cells / cm ² or more and less than 30000 cells / cm ² are seeded on the surface to be measured, and in an incubator at 37 ° C. and a CO ₂ concentration of 5%. It is defined as the ratio of cells that have adhered and spread when cultured for 14.5 hours ({(number of cells adhered) / (number of cells seeded)} × 100 (%)).

  Cell seeding is suspended in DMEM medium containing 10% FBS, seeded on the measurement object, and then slowly shaken the measurement object on which the cells are seeded so that the cells are distributed as uniformly as possible. It is done by. Furthermore, the measurement of the cell adhesion extension rate is performed after exchanging the medium immediately before the measurement to remove the non-adhered cells. When measuring cell adhesion spread rate, measure the location excluding the location where the cell density tends to be specific (for example, the center of the predetermined area where the density is likely to be high, the periphery of the predetermined area where the density is likely to be low) A place.

  On the other hand, cell non-adhesiveness refers to the property that cells are difficult to adhere. Cell non-adhesiveness is determined by whether or not cell adhesion or extension is unlikely to occur due to the chemical or physical properties of the surface. The non-cell-adhesive surface is preferably a surface having a cell adhesion extension rate as defined above of less than 60%, more preferably less than 40%, and even more preferably 5% or less. The surface is preferably 2% or less, and most preferably.

  Polymers that provide a non-adhesive surface to cells include hydrophilic polymers. In the present invention, the hydrophilic polymer refers to a hydrophilic polymer that suppresses cell adsorption, particularly nonspecific adsorption. The hydrophilic polymer refers to a polymer containing a hydrophilic functional group in the main chain or side chain of the polymer.

  Specific examples of the hydrophilic polymer having a bond that can be hydrolyzed by an acid or a base include polyalkylene glycol (particularly polyethylene glycol), polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene imine, polyallylamine, polyvinyl amine, polyvinyl acetate, poly Examples include acrylic acid, copolymers of these with other monomers, and (meth) acrylates such as graft polymers, preferably di (meth) acrylate. Among them, polyalkylene glycol (meth) acrylates having various molecular weights are commercially available and are excellent in biocompatibility, so that they can be suitably used.

  Other hydrophilic polymers such as polyalkylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyethyleneimine, polyallylamine, polyvinylamine, polyvinyl acetate, polyacrylic acid, copolymers of these with other monomers, and graft polymers One or more of these may be further mixed and used.

  The density and hydrophilicity of the hydrophilic polymer in the hydrophilic polymer layer formed on the substrate can be easily evaluated using the contact angle of water on the surface as an index. For example, if the water contact angle of the surface after immobilization of the hydrophilic polymer is typically 48 ° or less, preferably 40 ° or less, more preferably 30 ° or less, the hydrophilic polymer is present in sufficient density, It is considered that it has hydrophilicity and suppresses cell adsorption and adhesion, particularly nonspecific adsorption. In the present invention, the water contact angle refers to a water contact angle measured at 23 ° C.

  The coating liquid for forming the hydrophilic polymer layer as the functional polymer layer can be prepared by dissolving the hydrophilic polymer, preferably in an appropriate solvent. Only one type of hydrophilic polymer may be used, or a plurality of types may be used in combination. Specific examples of preferred solvents include methanol, ethanol, n-propanol, i-propanol, 2-butanol, n-butanol, and water, and one or more of them may be used. Other solvents such as 1-pentanol, 2-ethyl-1-butanol, 2-butoxyethanol, and ethylene (or diethylene) glycol or monoethyl ether thereof may be used. In the present invention, i-propanol is preferred. This is because i-propanol volatilizes easily at room temperature when applied to a thin film, so that the merit of drying described later can be obtained without a special drying step. In addition, even when polystyrene that is widely used for cell culture is selected as the base material, it is preferable because the surface is not affected.

  The amount of the hydrophilic polymer dissolved in the solvent is determined according to the molecular weight of the polymer, but the concentration is preferably 1% by weight or more, more preferably 5% by weight or more.

  As a functional polymer, a polymer that provides a surface with varying adhesion to cells includes a temperature-responsive polymer. The temperature-responsive polymer exhibits hydrophobicity at a cell culture temperature (usually about 37 ° C.), and exhibits hydrophilicity at the temperature at the time of recovery of the cultured cell sheet. The temperature at which the temperature-responsive polymer changes from hydrophobic to hydrophilic (critical solution temperature in water (T)) is not particularly limited, but from the viewpoint of ease of recovery of the cell sheet after culture, The temperature is preferably lower than the cell culture temperature. By including such a temperature-responsive polymer component, since cell scaffolds (cell adhesion surfaces) are sufficiently secured during cell culture, cell culture can be performed efficiently. On the other hand, at the time of collecting the cell sheet after culturing, the hydrophobic part is changed to hydrophilic, and the cultured cell sheet is separated from the cell culture substrate, thereby making it easier to collect the cell sheet. be able to. In particular, a temperature-responsive polymer that exhibits hydrophilicity at a temperature lower than a predetermined critical dissolution temperature and exhibits hydrophobicity at a temperature equal to or higher than the same temperature is preferable. The critical solution temperature in such a temperature-responsive polymer is particularly called the lower critical solution temperature. Specifically, a temperature-responsive polymer having a lower critical solution temperature T of 0 to 80 ° C., preferably 0 to 50 ° C. is preferable.

  Specific examples of the temperature-responsive polymer having a bond that can be hydrolyzed by an acid or a base include acrylic polymers and methacrylic polymers. Specific suitable polymers include, for example, poly-N-isopropylacrylamide (T = 32 ° C.), poly-Nn-propyl acrylamide (T = 21 ° C.), poly-Nn-propyl methacrylamide (T = 32 ° C.), poly-N-ethoxyethyl acrylamide (T = about 35 ° C.), poly-N-tetrahydrofurfuryl acrylamide (T = about 28 ° C.), poly-N-tetrahydrofurfuryl methacrylamide (T = about 35 ° C.) ), And poly-N, N-diethylacrylamide (T = 32 ° C.). Examples of other polymers include poly-N-ethylacrylamide, poly-N-cyclopropylacrylamide, and poly-N-cyclopropylmethacrylamide.

  As a coating liquid for forming a temperature-responsive polymer layer as a functional polymer layer, a (meth) acrylamide compound, an N- (or N, N-di) alkyl-substituted (meth) acrylamide derivative, a cyclic group (meta ) Monomers such as acrylamide derivatives, preferably dissolved in an appropriate solvent, can be used. The amount of the monomer contained in the coating liquid is preferably 5% by weight or more, more preferably 20% by weight or more, preferably 70% by weight or less, more preferably 50% by weight or less. As a coating solution for forming the temperature-responsive polymer layer, a solution in which a temperature-responsive polymer is dissolved in an appropriate solvent together with an appropriate crosslinking agent may be used.

  As the crosslinking agent, a crosslinking agent having two or more radiation-reactive functional groups is preferably used. After supplying a coating liquid containing a crosslinking agent having a radiation-reactive functional group together with a temperature-responsive polymer onto a substrate, irradiation with radiation (for example, an electron beam) causes the temperature-responsive polymer to pass through the crosslinking agent. Thus, it can be covalently bonded onto the substrate, and the temperature-responsive polymer can be firmly fixed. A radiation-reactive functional group, for example, an electron beam-reactive functional group, is not particularly limited as long as it can be covalently bonded to a substrate by irradiation with radiation (for example, an electron beam). Radiation-reactive functional groups are moieties that can form radicals under high energy irradiation, generate free radicals when exposed to a radiation source, and achieve crosslinking and grafting.

  The radiation-reactive functional group is appropriately selected depending on the temperature-responsive polymer to be used. For example, acryloyl group, methacryloyl group, azide group, primary, secondary or tertiary aliphatic group, alicyclic group, benzyl group, etc. And aromatic groups. The crosslinking agent may have only one kind of these radiation-reactive functional groups, or may have a plurality of kinds. It is preferable to use a crosslinking agent having an acryloyl group or a methacryloyl group as the radiation-reactive functional group. A crosslinking agent having an acryloyl group or a methacryloyl group is easily available, and is relatively easy to handle as compared with an azide group or the like.

  Specific examples of the crosslinking agent include polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, polypropylene glycol diacrylate, polypropylene glycol dimethacrylate, and ethoxylated glycerin (meth) acrylate such as ethoxylated glycerin diacrylate, ethoxylated glycerin triacrylate. Ethoxylated glycerin dimethacrylate, and ethoxylated glycerin trimethacrylate.

  The solvent used for preparing the coating liquid for forming the temperature-responsive polymer layer is not particularly limited, but those having a boiling point of 120 ° C. or less, particularly 60 to 110 ° C. under normal pressure are preferable. Specific examples of preferred solvents include methanol, ethanol, n-propanol, i-propanol, 2-butanol, n-butanol, and water, and one or more of them may be used. Of these, i-propanol is preferred for the same reason as described above. Other solvents such as 1-pentanol, 2-ethyl-1-butanol, 2-butoxyethanol, and ethylene (or diethylene) glycol or monoethyl ether thereof may be used.

  As the base material for forming the functional polymer layer, known or commercially available materials can be applied. When forming a polymer layer that provides a surface that is adhesive to cells as a functional polymer layer, by using a substrate having a non-cell-adhesive surface, the cell-adhesive surface and the cell non-adhesive A cell culture substrate having a patterned surface can be obtained. On the other hand, when forming a polymer layer that provides a non-adhesive surface to cells, a cell-adhesive surface and a non-cell-adhesive surface can be obtained by using a substrate having a cell-adhesive surface. A patterned cell culture substrate can be obtained.

  Specific examples of the substrate include inorganic materials such as metal, glass, ceramic and silicon, elastomers, and organic materials represented by plastics. Examples of plastic include polyester, polystyrene, low density polyethylene, medium density polyethylene, high density polyethylene, polypropylene, polyurethane, urethane acrylate, polymethyl methacrylate and other acrylic resins, polyamide (nylon), polycarbonate, polyacrylonitrile, polychlorinated. Examples include vinylidene, polyvinylidene fluoride, polyolefin, and polylactic acid. The substrate may be composed of a blend polymer or polymer alloy containing two or more of these materials. In addition, the base material can be hydrophilized using a treatment technique such as ozone treatment, plasma treatment, or sputtering.

  The shape of the substrate 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.

The coating amount of the coating liquid applied on the substrate for forming the temperature-responsive polymer layer is a coating amount necessary for the temperature-responsive polymer to exhibit a function (for example, temperature responsiveness), and is 50 mg / m. ^There should be ^two or more. The upper limit of the coating amount is not particularly limited, but is preferably less than 40 g / m ^{2 and} more preferably 10 g / m ² or less. The coating amount of the coating solution applied on the substrate for forming the hydrophilic polymer layer is the amount necessary for the hydrophilic polymer to exhibit its function (for example, suppression of cell non-adhesiveness or nonspecific adsorption). The amount may be 50 mg / m ² or more. The upper limit of the coating amount is not particularly limited, but is preferably less than 40 g / m ^{2 and} more preferably 10 g / m ² or less.

If the coating amount is too large, the coating thickness will increase due to the increased thickness, the thickness will increase, the penetration of radiation and the irradiation dose will not be stable, and there will be unevenness in the coating amount due to convection in the film derived from the irradiation energy. May occur. In order to shorten the washing time for washing free polymers and molecules, the coating amount is desirably 10 g / m ² or less.

  As a method for applying the coating liquid for forming the functional polymer to the base material, any known method may be used. For example, a coating method using a spin coater, a bar coater or the like, a spray coating method, a blade coating method. Gravure coating method, rod coating method, knife coding method, reverse roll coating method, offset gravure coating method and the like can be used. These are merely examples, and those skilled in the art can use those that are applicable.

  After supplying and applying a coating liquid on a base material, it is preferable to advance the binding reaction between the base material surface and the functional polymer by irradiation with radiation. The binding reaction here refers to a reaction in which a covalent bond between the base material and the functional polymer is formed by irradiation. Examples of the radiation used include α rays, β rays, γ rays, electron beams, ultraviolet rays, and the like. In the present invention, γ rays and electron beams are energy efficient, and electron beams are particularly preferable from the viewpoint of productivity. With respect to ultraviolet rays, it can be used by combining an appropriate polymerization initiator and an anchor agent with a substrate. The range of radiation dose is preferably 50 kGy to 400 kGy for electron beams, and 5 kGy to 50 kGy for γ rays. Usually, the dose range can be appropriately determined based on the temperature rise due to radiation and the Tg of the resin.

  In the functional polymer layer formed through the above-described steps, free polymer molecules, unreacted substances, and the like are present. Therefore, it is preferable to further include a washing step for washing in order to remove these free polymers and unreacted substances. Although it does not specifically limit as a washing | cleaning method, Typically, immersion washing | cleaning, swing washing | cleaning, shower washing | cleaning, spray washing | cleaning, ultrasonic washing | cleaning, etc. are mentioned. The cleaning liquid typically includes various water-based, alcohol-based, hydrocarbon-based, chlorine-based, acid / alkali cleaning liquids. The combination of the cleaning method and the cleaning liquid may be selected as appropriate.

  Subsequently, a patterned coating material is brought into close contact with the obtained functional polymer layer to form a pattern of the coating material on the functional polymer layer. Preferably, at least one surface is a slightly adhesive surface having removability, and the slightly adhesive surface (3 in FIG. 1) of the patterned coating material is closely attached to form a pattern of the coating material on the functional polymer layer. Form. Such a coating material can be temporarily adhered to the functional polymer layer and stably coated, so that the functional polymer layer can be protected from acid or base hydrolysis and easily after the hydrolysis reaction. Can be peeled off.

  Here, re-peelability refers to a property that can be easily peeled without causing damage to the substrate and leaving no adhesive on the surface of the substrate after being once adhered or adhered. Since the slightly tacky surface of the coating material in the present invention has slight tackiness, it comes into close contact with the functional polymer layer and is temporarily fixed during acid or base hydrolysis treatment. However, after the hydrolysis treatment is completed, the functional polymer layer and the coating material can be easily peeled off. Slight tackiness refers to tackiness that allows the covering material to be temporarily fixed on the functional polymer layer but can be easily peeled off.

  Examples of the material having the above characteristics include an elastomer material. The elastomer material includes a rubber material, a thermosetting resin-based elastomer material, and a thermoplastic resin-based elastomer material, and a thermosetting resin-based elastomer material is preferable from the viewpoint of heat resistance. The rubber material includes natural rubber and synthetic rubber (polybutadiene rubber, nitrile rubber, chloroprene rubber). Specific examples of the elastomer material include urethane rubber, nitrile rubber, silicone rubber, silicone resin (for example, polydimethylsiloxane), fluorine rubber, acrylic rubber, isoprene rubber, ethylene propylene rubber, chlorosulfonated polyethylene rubber, epichlorohydrin rubber, chloroprene. Examples thereof include rubber, styrene / butadiene rubber, butadiene rubber, and polyisobutylene rubber. Silicone rubber and silicone resin are particularly preferable because they are excellent in heat resistance, are not modified by acid or base, and are stable as a solid. Silicone rubber and silicone resin are also suitable for use as a cell culture substrate in that elasticity can be adjusted by the amount of the curing agent and they have moderate self-adhesiveness without using an adhesive.

  The shape of the covering material is not particularly limited as long as it is formed into a pattern that can partially cover the functional polymer layer. Preferably, it is a sheet form or a film form. The whole may be a coating material made of a material having re-peelability and slight adhesiveness as described above, or only a surface to be brought into contact with the functional polymer layer is made of a material having re-peelability and slight adhesiveness. It may be a material.

  When a coating material made entirely of an elastomer material is used, the thickness is preferably 10 μm or more, more preferably 50 μm or more, preferably 500 μm or less, more preferably 300 μm or less. A covering material having a thickness in the above range is preferable because it is easy to handle and can be easily cut into a desired pattern.

  The pattern of the covering material is not particularly limited as long as it is a two-dimensional pattern, and can be selected depending on the type of cells, 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 covering material in the shape of a graphic shape such as a circular shape and a quadrangular shape, or a shape in which the shape is cut out (a shape having an opening) And the like.

  Adhering the slightly sticky surface of the coating material to the functional polymer layer to form a coating material pattern on the functional polymer layer, and to the exposed functional polymer layer that is not covered with the coating material, By carrying out the hydrolysis reaction by bringing the acid or base into contact, the hydrolyzable bond is decomposed by the acid or base in the functional polymer, and the functional polymer can be removed from the substrate. Of the functional polymer layer, the region covered with the coating material is protected and thus does not undergo hydrolysis reaction. Therefore, when the coating material is peeled off after the hydrolysis treatment, the functional polymer layer remains in the region covered with the coating material, and the exposed region is a state in which the functional polymer layer is removed and the substrate surface is exposed. Become. As a result, a cell culture substrate in which the pattern of the functional polymer layer is formed in the same pattern as the coating material on the substrate is obtained.

  The acid used for the hydrolysis reaction is not particularly limited, and examples thereof include inorganic acids such as hydrochloric acid, nitric acid, and sulfuric acid, and organic acids such as formic acid, acetic acid, and propionic acid. Although it does not restrict | limit especially as a base used for a hydrolysis reaction, For example, sodium hydroxide, potassium hydroxide, lithium hydroxide, barium hydroxide, magnesium hydroxide, calcium hydroxide, etc. are mentioned. A 0.001 to 0.1 mol / L sodium hydroxide aqueous solution is preferably used. Such an aqueous sodium hydroxide solution is preferred because the washing step after hydrolysis is easy.

  The conditions for the hydrolysis reaction are not particularly limited, and are appropriately determined according to the type of functional polymer to be treated, the thickness of the functional polymer layer, the type of acid or base used, and the like. For example, when using 0.01 mol / L sodium hydroxide aqueous solution as a base, the conditions which process for 5 to 30 minutes at the temperature of 20-50 degreeC are mentioned.

  The substrate after the hydrolysis treatment is preferably washed to remove the remaining acid and base, decomposed polymer and the like after the covering material is peeled off. As a cleaning method, the same method as described above can be used.

  The cell culture substrate produced by the method of the present invention can be processed into a shape suitable for cell culture (for example, a dish shape). Moreover, you may affix on the cell culture surface of a dish-shaped container with an adhesive agent.

  Using the cell culture substrate produced by the method of the present invention, various cells, for example, epithelial cells and endothelial cells constituting each tissue and organ in the living body, skeletal muscle cells exhibiting contractility, smooth muscle cells, Cardiomyocytes, neurons constituting the nervous system, glial cells, fibroblasts, hepatocytes related to metabolism in the living body, non-hepatocytes and adipocytes, stem cells present in various tissues as cells having differentiation ability, and Can culture bone marrow cells, ES cells and the like.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the scope of the examples.

Example 1
A coating solution was prepared by dissolving in isopropyl alcohol such that polyethylene glycol diacrylate (PEGDA) (manufactured by Aldrich) was 5% by weight and polyethylene glycol 400 (PEG400) (manufactured by Kanto Chemical Co., Ltd.) was 40% by weight. The said coating liquid was apply | coated on the film (1.4 g / m < ² >) using the wire bar on the surface which gave the corona treatment to the commercially available polystyrene film (available from Sandick), and was made hydrophilic. This film was irradiated with an electron beam (irradiation dose of 200 kGy) to prepare a film sample in which a hydrophilic polymer was immobilized.

  The prepared film sample was immobilized on the bottom of the culture dish, and the surface was treated with 0.01 mol / L sodium hydroxide aqueous solution for 5 minutes. At this time, a part of the surface was covered with a sheet of polydimethylsiloxane (PDMS) to prevent contact with the aqueous sodium hydroxide solution.

Mouse fibroblasts (CCL163) were seeded when the culture dish was filled with 10% FBS-containing DMEM medium, and cultured overnight at 37 ° C. and 5% CO ₂ in a CO ₂ incubator. When the cells were taken out of the incubator and observed with an optical microscope, the area covered with the polydimethylsiloxane (PDMS) sheet maintained cell non-adhesiveness. Had adhered.

(Example 2)
A coating solution was prepared by dissolving in isopropyl alcohol such that poly-N-isopropylacrylamide (molecular weight 350,000) was 3% by weight and PEGDA was 0.10% by weight. The said coating liquid was apply | coated on the film (1.4 g / m < ² >) using the wire bar on the surface which gave the corona treatment to the commercially available polystyrene film (available from Sandick), and was made hydrophilic. This film was irradiated with an electron beam (irradiation dose of 120 kGy) to prepare a film sample in which a temperature-responsive polymer was immobilized.

  The prepared film sample was immobilized on the bottom of the culture dish, and the surface was treated with 0.01 mol / L sodium hydroxide aqueous solution for 5 minutes. At this time, a part of the surface was covered with gel poly (Panac), which is a slightly adhesive film, to prevent contact with an aqueous sodium hydroxide solution.

Bovine vascular endothelial cells were seeded when the culture dish was filled with 10% FBS-containing DMEM medium, and cultured in a CO ₂ incubator at 37 ° C. and 5% CO ₂ for 1 day. Then, it was stored in an incubator under 20 ° C. and 5% CO ₂ conditions. When the state of the cells was observed with an optical microscope after 30 minutes, the cells covered with the gel poly retained the temperature response performance, but the cells were detached, but the cells treated with the aqueous sodium hydroxide solution did not peel. It was.

1: Functional polymer layer 2: Substrate 3: Slightly adhesive surface 4: Coating material 5: Exposed functional polymer layer 6: Acid or base

Claims (6)

A method for producing a cell culture substrate,
Forming a functional polymer layer on a substrate having a bond that can be hydrolyzed by an acid or a base and controlling cell adhesion;
A process of forming a pattern of a covering material on the functional polymer layer by closely adhering the functional polymer layer and the patterned covering material;
A step of hydrolyzing a part of the functional polymer layer by bringing acid or base into contact with the functional polymer layer exposed by the pattern of the coating material, and a step of peeling the coating material from the functional polymer layer Including said method.   The method according to claim 1, wherein the functional polymer has an ester bond as a bond hydrolyzable by an acid or a base.   The method according to claim 1 or 2, wherein the surface of the covering material to be in close contact with the functional polymer layer is a slightly adhesive surface having removability.   4. The method according to claim 3, wherein the slightly sticky surface of the covering material is made of silicone rubber or silicone resin.   The method according to any one of claims 1 to 4, wherein the functional polymer is an acrylic polymer or a methacrylic polymer.   6. The method according to claim 5, wherein the functional polymer is selected from polyalkylene glycol (meth) acrylate and poly-N-isopropyl (meth) acrylamide.

Images