Culture substrate, method for producing culture substrate, method for producing cell, and reusable culture substrate

Abstract

Provided is a culture substrate which comprises, on a metal substrate that has undergone oxygen plasma processing and silanization processing, a temperature-responsive polymer having a glass transition temperature of greater than 121°C.

Description

Culture substrate, method for producing culture substrate, method for producing cells, and culture substrate for repeated use

[0001] The present invention relates to a culture substrate, a method for producing a culture substrate, a method for producing cells, and a culture substrate for repeated use.

[0002] Typically, cell culture is performed on glass surfaces or on the surfaces of synthetic polymer materials that have been subjected to various treatments. For example, various polystyrene vessels that have been surface-treated (e.g., gamma-ray irradiation, silicone coating, etc.) are widely used as cell culture vessels. Conventionally, cells cultured and grown using such cell culture vessels are detached and recovered from the vessel surface by treatment with proteolytic enzymes such as trypsin or chemicals. However, when recovering cells grown using such treatments with proteolytic enzymes or chemicals, the processing steps become complicated, increasing the possibility of contamination with impurities. Furthermore, the treatments can damage the cells themselves and proteins surrounding the cells, such as the extracellular matrix (ECM), causing denaturation. Therefore, there is a demand for using cells cultured with membrane proteins and ECM proteins still attached, so as to prevent the loss of cell function.

[0003] Currently, as a cell culture technique in biochemistry, medicine, immunology, etc., a technology has been put into practical use that controls cell adhesion and detachment by temperature change, thereby enabling non-invasive cell detachment without the use of proteolytic enzymes or chemicals (enzyme- and chemical-free), and is used in a variety of bioapplications, including cell sheet detachment. Patent Document 1 discloses a cell culture support material having a first layer coated with a cell adhesive substance containing a natural cell adhesive substance and / or a synthetic cell adhesive substance, and a second layer coated with a homopolymer or copolymer whose critical solution temperature in water is in the range of 0 to 80°C. Examples of materials for the substrate to be coated with the cell adhesive substance include glass, modified glass, polymeric compounds such as polystyrene and polymethyl methacrylate, ceramics, and metals.

[0004] Japanese Patent Application Publication No. 5-38278

[0005] Conventionally, the base material of temperature-responsive culture supports has generally been a polymer compound, and cell culture supports using such polymer compounds are typically disposable and discarded after a single use. Therefore, when producing a three-dimensional tissue from cells, it is necessary to prepare cells, cell sheets, etc. using a large number of disposable cell culture supports, which places a heavy burden on the environment and is costly, making it difficult to put into practical use.

[0006] Furthermore, since the above-mentioned polymer compounds have low thermal conductivity, when cells are detached using a temperature-responsive culture support made of a polymer compound, it is difficult to efficiently change the temperature of the culture medium to detach the cells.

[0007] The present invention aims to provide a culture substrate that can be repeatedly used by autoclaving, which was not possible with conventional disposable temperature-responsive cell culture supports, has a low environmental impact, does not require enzyme or chemical treatment, allows cells to be non-invasively detached without damaging the cells themselves or proteins around the cells such as the extracellular matrix (ECM), allows a large number of cells to be recovered at low cost, and allows efficient temperature control of the culture medium; a method for producing the culture substrate; a method for producing cells using the culture substrate; and a culture substrate for repeated use.

[0008] <1> The culture substrate of the present invention is a culture substrate having a temperature-responsive polymer having a glass transition temperature of more than 121°C on a metal substrate that has been treated with oxygen plasma and silanized.

[0009] Such a culture substrate can be reused after autoclaving because the temperature-responsive polymer maintains its temperature responsiveness even after autoclaving. Furthermore, such a culture substrate can be reused after autoclaving because the immobilized temperature-responsive polymer is not easily peeled off, allowing for the production of large amounts of cells at low cost.

[0010] As shown in Figure 1, a culture substrate in which a temperature-responsive polymer is modified on a metal substrate is a temperature-responsive culture substrate that allows cells to be cultured at a temperature higher than the hydration temperature of the temperature-responsive polymer (e.g., 37°C when poly-N-isopropylacrylamide (PIPAAm) is used), and then the cells or cell sheet can be detached and recovered without damaging their structure or function by simply lowering the temperature to a temperature lower than the dehydration temperature of the temperature-responsive polymer (e.g., 20°C when PIPAAm is used).

[0011] Thermoresponsive polymers are polymers whose solubility in water changes dramatically with temperature: they are soluble in water at low temperatures, but when heated to a certain temperature (lower critical solution temperature: LCST), they become insoluble, aggregate, precipitate, and become cloudy (Figure 2). This is because at temperatures below the LCST, the polymer chains are hydrated and elongated due to the strong interaction between the amide bonds and water, and adopt a random coil conformation. Conversely, at temperatures above the LCST, dehydration occurs, and the polymer chains aggregate into a globular state due to hydrophobic interactions (Figure 2).

[0012] As an example of the behavioral changes of the temperature-responsive polymer immobilized on the culture substrate of the present invention and the cells on the temperature-responsive polymer, Figure 2 shows the state change due to temperature change of PIPAAm (which has both hydrophilic and hydrophobic moieties in the side chain and an LCST of 32°C) and the behavioral changes of cells cultured on that PIPAAm. At temperatures higher than the LCST, the substrate surface becomes hydrophobic, allowing cells to adhere, but at temperatures lower than the LCST, the substrate surface becomes hydrophilic, making it non-adhesive and causing cells adhered to the substrate surface to detach, making it possible to recover cells simply by changing the temperature (Figure 2).

[0013] Furthermore, by using a metal substrate as the substrate to be modified with the temperature-responsive polymer, the culture substrate of the present invention does not deform even under autoclave conditions (saturated steam conditions of 121°C or higher and 2 atmospheres), where a typical temperature-responsive cell culture substrate cannot maintain its shape, and therefore can be used as a reusable culture substrate.

[0014] Therefore, the culture substrate of the present invention can be reused by autoclaving, has a low environmental impact, does not require enzyme or chemical treatment, and allows cells to be detached non-invasively without damaging the cells themselves or proteins surrounding the cells, such as the extracellular matrix (ECM), allowing for the recovery of a large number of cells at low cost, and provides a temperature-responsive culture support that can efficiently control the temperature of the culture medium and substrate.Furthermore, since the culture substrate of the present invention has a metal substrate, cells can be efficiently detached by rapidly changing the temperature of the metal substrate using a Peltier element or the like.

[0015] <2> In the culture substrate of the present invention, the temperature-responsive polymer is preferably poly-N-isopropylacrylamide (PIPAAm).

[0016] Such a culture substrate facilitates cell culture and recovery, since the LCST of PIPAAm is close to the body temperature of the animal.

[0017] <3> In the culture substrate of the present invention, the metal substrate is preferably made of titanium.

[0018] Such culture substrates use titanium, which is lightweight, strong, and corrosion-resistant, for the metal base, making them easy to handle, ultrasonically clean, autoclave, and reuse, allowing for the production of large quantities of cells at low cost.

[0019] <4> In addition, in the culture substrate of the present invention, it is preferable that the metal substrate and the temperature-responsive polymer are connected via a silicon atom.

[0020] In such a culture substrate, the metal substrate and the temperature-responsive polymer are easily bonded via a covalent bond, and the temperature-responsive polymer immobilized on the metal substrate is unlikely to peel off.

[0021] <5> Furthermore, the culture substrate of the present invention can comprise a base having a recess and a lid covering the base, and the temperature-responsive polymer can be disposed on the bottom surface of the recess in the base.

[0022] Such a culture substrate is easy to use for general cell culture.

[0023] <6> The method for producing a culture substrate of the present invention also includes a silane agent treatment step of treating the surface of the oxygen plasma-treated metal substrate with a silane agent, and a temperature-responsive polymer bonding step of bonding a temperature-responsive polymer having a glass transition temperature of more than 121°C to the metal substrate.

[0024] Such a method for producing a culture substrate can be used repeatedly by autoclaving, has a low environmental impact, does not require enzyme or chemical treatment, and allows cells to be detached non-invasively without damaging the cells themselves or proteins surrounding the cells, such as the extracellular matrix (ECM), allowing for the recovery of a large number of cells at low cost, and can produce a temperature-responsive culture support that can efficiently control the temperature of the culture medium. Furthermore, because the temperature-responsive polymer is easily immobilized on the metal substrate via a covalent bond, the immobilized temperature-responsive polymer is less likely to peel off, making it possible to provide a culture substrate that appropriately exhibits temperature responsiveness even after autoclaving.

[0025] <7> In the method for producing a culture substrate of the present invention, the temperature-responsive polymer is preferably poly-N-isopropylacrylamide (PIPAAm).

[0026] According to such a method for producing a culture substrate, the LCST of PIPAAm is close to the body temperature of an animal, so that a culture substrate that allows easy cell culture and recovery can be produced.

[0027] <8> In the method for producing a culture substrate of the present invention, the metal substrate is preferably made of titanium.

[0028] This method of producing a culture substrate uses titanium, which is lightweight, strong, and corrosion-resistant, for the metal base, making it easy to perform culture operations, ultrasonically clean, and autoclave treatment for reuse, and it is possible to produce a culture substrate that can be used to produce large quantities of cells at low cost.

[0029] <9> The cell production method of the present invention also includes a cell production step of seeding and culturing cells on the surface of a culture substrate having a temperature-responsive polymer with a glass transition temperature of more than 121°C on a metal substrate that has been oxygen plasma-treated and silanized.

[0030] Such a cell production method allows for efficient temperature control of the culture medium and substrate, thereby enabling efficient production of large numbers of cells. Furthermore, by autoclaving and repeatedly using the temperature-responsive culture support, cells can be detached non-invasively, with minimal environmental impact, without the need for enzyme or chemical treatment, and without damaging the cells themselves or proteins surrounding the cells, such as the extracellular matrix (ECM), allowing for the production of large numbers of cells at low cost. Furthermore, the immobilized temperature-responsive polymer in the culture substrate used is resistant to detachment, and the culture substrate maintains appropriate temperature responsiveness even after autoclaving, allowing for the production of large numbers of cells at low cost. Furthermore, by rapidly changing the temperature of the metal substrate using a Peltier element or the like, cells can be rapidly detached, allowing for efficient cell production.

[0031] <10> In addition, in the cell production method of the present invention, the cells can be produced in a sheet form in the cell production step.

[0032] Such a method for producing cells makes it possible to produce a cell sheet.

[0033] <11> Furthermore, the cell production method of the present invention preferably includes, after the cell production step, a cell detachment step of detaching the cells from the temperature-responsive polymer by changing the temperature.

[0034] Such a cell production method makes it possible to recover cells without the need for enzymatic or chemical treatment, and without damaging the cells themselves or proteins surrounding the cells, such as the extracellular matrix (ECM).

[0035] <12> In the cell production method of the present invention, the temperature-responsive polymer is preferably poly-N-isopropylacrylamide (PIPAAm).

[0036] In such a method for producing cells, the LCST of PIPAAm is close to the body temperature of an animal, making it easy to culture and recover the cells.

[0037] <13> In the cell production method of the present invention, the metal substrate is preferably made of titanium.

[0038] In this cell production method, titanium, which is lightweight, strong, and corrosion-resistant, is used for the metal substrate of the culture medium, making it easy to operate the culture, ultrasonically clean, and autoclave the material for reuse, making it easy to produce large quantities of cells at low cost.

[0039] <14> In the method for producing a cell of the present invention, the temperature-responsive polymer is preferably connected to the metal substrate via a silicon atom.

[0040] In such a cell production method, the metal substrate and the temperature-responsive polymer are easily bonded via a covalent bond, and the temperature-responsive polymer fixed on the metal substrate is not easily peeled off, so the culture substrate can be easily autoclaved and reused, making it easy to produce large quantities of cells at low cost.

[0041] <15> Furthermore, in the cell production method of the present invention, the culture substrate may comprise a base having a recess and a lid covering the base, and the temperature-responsive polymer may be present on the bottom surface of the recess in the base.

[0042] Such a method for producing cells makes general cell culture easy.

[0043] <16> The repeated use culture substrate of the present invention contains a temperature-responsive polymer having a glass transition temperature of more than 121°C.

[0044] Such a reusable culture substrate allows the temperature-responsive polymer to exhibit appropriate temperature responsiveness even after autoclaving, allows for repeated use by autoclaving, has a low environmental impact, does not require enzyme or chemical treatment, and allows cells to be detached non-invasively without damaging the cells themselves or proteins surrounding the cells such as the extracellular matrix (ECM), allows for the recovery of a large number of cells at low cost, and can provide a temperature-responsive culture support that can efficiently control the temperature of the culture medium.

[0045] <17> In the repeated use culture substrate of the present invention, the temperature-responsive polymer is preferably poly-N-isopropylacrylamide (PIPAAm).

[0046] In such a repeatedly usable culture substrate, the LCST of PIPAAm is close to the body temperature of an animal, making it easy to culture and recover cells.

[0047] According to the present invention, it is possible to provide a temperature-responsive culture support that can be reused by autoclaving, has a low environmental impact, does not require enzyme or chemical treatment, non-invasively detaches cells without damaging the cells themselves or proteins surrounding the cells such as the extracellular matrix (ECM), allows the recovery of a large number of cells at low cost, and can efficiently control the temperature of the culture medium.

[0048] 1 is a schematic diagram showing an example of non-invasive cell detachment using a temperature-responsive polymer; FIG. 2 is a diagram showing an example of a state change of a temperature-responsive polymer whose morphology changes with temperature change; FIG. 3 is a diagram explaining an example of a flow for immobilizing a temperature-responsive polymer on a metal substrate; FIG. 4 is a diagram showing an example of a peak obtained by surface analysis of a metal substrate surface to which a temperature-responsive polymer is immobilized by XPS; FIG. 5 is a diagram showing an example of an evaluation of the surface wettability of a metal substrate surface to which a temperature-responsive polymer is not bonded; FIG. 6 is a diagram showing an example of an evaluation of the surface wettability of a metal substrate surface to which a temperature-responsive polymer is bonded; FIG. 7 is a diagram showing an example of the shape of a metal substrate before and after autoclaving; FIG. 8 is a diagram showing an example of the shape of a temperature-responsive cell culture substrate before and after autoclaving; FIG. 9 is a diagram showing an example of a change in adhesion behavior of cells cultured on an autoclaved surface of a temperature-responsive polymer-bonded metal substrate due to a temperature change; FIG. 10 is a diagram showing an example of cells cultured in sheet form on an autoclaved surface of a temperature-responsive polymer-bonded metal substrate, and then recovered by a temperature change; FIG. 11 is a diagram showing an example of cells cultured in sheet form on an autoclaved surface of a temperature-responsive polymer-bonded metal substrate, and then recovered by a temperature change. 1 is a diagram showing an example of recovery of cells cultured in sheet form on the surface of a temperature-responsive polymer-bonded metal substrate that has been autoclaved once, by a temperature change; FIG. 2 is a diagram showing an example of recovery of cells cultured in sheet form on the surface of a temperature-responsive polymer-bonded metal substrate that has been autoclaved twice, by a temperature change; FIG. 3 is a diagram showing an example of change in adhesion behavior of cells cultured on the surface of a temperature-responsive polymer-bonded metal substrate that has been autoclaved three times, by a temperature change; FIG. 4 is a diagram showing an example of recovery of cells cultured in sheet form on the surface of a temperature-responsive polymer-bonded metal substrate that has been autoclaved four times, by a temperature change; FIG. 5 is a diagram showing an example of recovery of cells cultured in sheet form on the surface of a temperature-responsive polymer-bonded metal substrate that has been autoclaved five times, by a temperature change.

[0049] Conventionally, the substrate of a temperature-responsive culture support generally uses a polymer compound, and cell culture supports using such polymer compounds are typically disposable and discarded after a single use. Therefore, for example, when creating a three-dimensional tissue from cells, a large number of disposable cell culture supports must be used to create the cells and cell sheets that make up the tissue, resulting in high costs and making practical application difficult. This also places a heavy burden on the environment. Furthermore, because polymer compounds have low thermal conductivity, when using a temperature-responsive culture support that uses a polymer compound, it is difficult to efficiently change the temperature of the culture medium or substrate when detaching cells.

[0050] In order to solve these problems, the present inventors have developed a culture substrate that can be reused by autoclaving after use and that allows the temperature of the culture medium and substrate to be rapidly changed in response to changes in external temperature, a method for producing the same, a method for producing cells using the culture substrate, and a culture substrate for repeated use.

[0051] (Culture Substrate) The culture substrate of the present invention has a temperature-responsive polymer with a glass transition temperature of above 121°C on a metal substrate that has been treated with oxygen plasma and silanized. The culture substrate of the present invention can be repeatedly used by autoclaving, has a low environmental impact, does not require enzyme or chemical treatment, and allows cells to be non-invasively detached without damaging the cells themselves or proteins surrounding the cells, such as the extracellular matrix (ECM), allowing a large number of cells to be recovered at low cost, and can provide a temperature-responsive culture support that can efficiently control the temperature of the culture medium.

[0052] The "temperature-responsive polymer" of the present invention is a polymer with a glass transition temperature of more than 121°C. It is also a temperature-responsive polymer that does not denature under autoclave sterilization conditions. The temperature-responsive polymer does not lose its temperature-responsive function even when autoclaved while immobilized on the culture substrate. If the culture substrate of the present invention does not contain the temperature-responsive polymer, cells cannot be recovered solely by changing the temperature of the culture medium, making it difficult to efficiently recover cells that retain pericellular proteins such as the extracellular matrix (ECM), and further making it difficult to secure a sufficient cell scaffold (cell adhesion surface) during cell culture.

[0053] The substrate used in the culture substrate of the present invention is a "metal substrate" that has been treated with oxygen plasma and silanized. There are no particular limitations on the metal substrate that can be used in the present invention, as long as it can bind a temperature-responsive polymer, and any suitable metal substrate can be used depending on the purpose. If the substrate of the culture substrate is not a material that does not deform at high temperatures, such as metal, but rather a polymer compound or the like that is used in ordinary culture substrates, the culture substrate will deform significantly under autoclave sterilization conditions.

[0054] The shape of the culture substrate of the present invention can be, for example, a dish shape or a film shape. When a film-shaped substrate is used, a temperature-responsive polymer layer is formed on the surface of the film-shaped substrate, and then the substrate can be processed into a shape suitable for cell culture (e.g., a dish shape). During processing, if necessary, a member made of another material can be used in combination with the substrate. When a dish-shaped substrate is used, it is sufficient that at least the inner bottom surface of the dish, which will be the cell adhesion surface, is coated with a temperature-responsive polymer.

[0055] The culture substrate of the present invention may comprise a base having a recess and a lid covering the base, with the temperature-responsive polymer being disposed on the bottom surface of the recess. Alternatively, a plate-shaped base to which the temperature-responsive polymer is bonded may be provided with walls made of a material that can be used for the culture substrate of the present invention, such as metal, ceramic, glass, or silicone such as polydimethylsiloxane (PDMS).

[0056] <Autoclave Sterilization Conditions> "Autoclave treatment" refers to sterilization treatment using a high-pressure steam sterilizer, which cleans and / or sterilizes objects in heated water or in an atmosphere of steam generated from the heated water, at high internal pressure. In this specification, "under autoclave sterilization conditions" refers to "saturated steam conditions at 121°C or higher and 2 atmospheres." The autoclave treatment is generally performed for 15 to 20 minutes under the autoclave sterilization conditions, but there is no limit to the sterilization time as long as sufficient sterilization is achieved, and the sterilization time can be increased or decreased depending on the content and purpose of the objects to be sterilized.

[0057] <Temperature-responsive polymer> The temperature-responsive polymer contained in the culture substrate of the present invention is a temperature-responsive polymer having a glass transition temperature of more than 121° C. The glass transition temperature of the temperature-responsive polymer contained in the culture substrate of the present invention is preferably 125° C. or higher, more preferably 130° C. or higher, and even more preferably 135° C. or higher.

[0058] The term "temperature-responsive" in "temperature-responsive polymer" means that its solubility in water changes dramatically with temperature; it dissolves in water at low temperatures, but becomes insoluble and aggregates or precipitates when heated to a certain temperature (lower critical solution temperature: LCST). The "glass transition temperature" is the temperature at which a substance that is liquid at high temperatures suddenly increases in viscosity within a certain temperature range as the temperature drops, loses almost all fluidity, and turns into an amorphous solid. If the glass transition temperature of the temperature-responsive polymer used in the present invention is 121°C or lower, the polymer may be denatured by autoclaving, and the temperature-responsive polymer on the metal substrate may no longer exhibit appropriate temperature responsiveness after autoclaving the culture substrate.

[0059] <<Measurement of Glass Transition Temperature (Tg) of Polymer>> The glass transition temperature of a polymer can be measured by differential scanning calorimetry (DSC). The glass transition temperature of a polymer may be measured, for example, under the following measurement conditions. When measuring a highly hygroscopic sample, the sample may be heat-treated in the DSC apparatus or the like before measurement to remove excess moisture, and the heat treatment temperature may be from room temperature to 120°C.

[0060] - Measurement conditions - Apparatus: Q1000 manufactured by TA Instruments Data processing: "Universal Analysis 2000" manufactured by TA Instruments Atmosphere: Nitrogen (50 mL / min) Temperature and calorific value calibration: High-purity indium (Tm = 156.61°C, ΔHm = 28.71 J / g) Temperature range: Room temperature to 300°C Heating rate: 10°C / min Sample amount: Approximately 5 mg Sample container: Aluminum standard container

[0061] In addition, when a large endotherm resulting from enthalpy relaxation (volume relaxation) overlaps with the glass transition signal in the DSC curve of the measurement sample, the glass transition temperature of the measurement sample can be determined by assuming a baseline on the curve showing the glass transition signal in the DSC curve and reading the central temperature of the stepped signal.

[0062] The temperature-responsive polymer that can be used in the culture substrate of the present invention is preferably hydrophobic at the cell culture temperature (usually about 37°C) and hydrophilic at the temperature at which the cultured cells are harvested, in order to convert the hydrophobic moieties to hydrophilic moieties at the time of cell harvesting after culture, thereby detaching the cultured cells from the cell culture substrate and making cell harvesting even easier; poly-N-isopropylacrylamide is particularly preferred. The temperature at which the temperature-responsive polymer changes from hydrophobic to hydrophilic (critical solution temperature (T) in water) is not particularly limited, but from the perspective of ease of cell harvesting after culture, a temperature lower than the cell culture temperature is preferred. In particular, a temperature-responsive polymer that is hydrophilic at temperatures below a predetermined lower critical solution temperature and hydrophobic at temperatures equal to or higher than that temperature is preferred; the critical solution temperature of such a temperature-responsive polymer is particularly referred to as the lower critical solution temperature (LCST).

[0063] The temperature-responsive polymer in the present invention preferably has a lower critical solution temperature (LCST) of 0 to 80° C., more preferably 0 to 50° C. If the LCST is 0° C. or higher, the cell proliferation rate generally decreases significantly or cells are less likely to die, whereas if the LCST is 80° C. or lower, cells are less likely to die.

[0064] The temperature-responsive polymer used in the present invention may be either a homopolymer or a copolymer. Specifically, it can be obtained by homopolymerization or copolymerization of the following monomers:

[0065] Examples of monomers that can be used in the present invention include (meth)acrylamide compounds such as N-isopropylacrylamide, N-(or N,N-di)alkyl-substituted (meth)acrylamide derivatives, (meth)acrylamide derivatives having a cyclic group, and vinyl ether derivatives, and one or more of these may be used. When one type of monomer is used alone, the polymer formed on the substrate will be a homopolymer, and when multiple types of monomers are used together, the polymer formed on the substrate will be a copolymer, and both forms are encompassed by the present invention. In the case of a copolymer, any two or more of these can be used.

[0066] Furthermore, when it is necessary to adjust T depending on the type of proliferating cells, when it is necessary to enhance the interaction between the coating substance and the cell culture support, or when it is necessary to adjust the balance of hydrophilicity and hydrophobicity of the cell support, other monomers may be added and copolymerized. Furthermore, grafts, copolymers, or block copolymers of the above polymers used in the present invention with other polymers, or mixtures of the polymers of the present invention with other polymers or copolymers may be used. Furthermore, crosslinking is also possible to the extent that the original properties of the polymer are not impaired. Since the cells are cultured and detached at this time, the detachment is carried out in the range of 5°C to 50°C. Therefore, examples of the temperature-responsive polymer include poly-N-n-propylacrylamide (LCST of homopolymer is 21°C), poly-N-n-propylmethacrylamide (LCST of homopolymer is 27°C), poly-N-isopropylacrylamide (LCST of homopolymer is 32°C), poly-N-isopropylmethacrylamide (LCST of homopolymer is 43°C), poly-N-cyclopropylacrylamide (LCST of homopolymer is 45°C), poly-N-ethoxyethylacrylamide (LCST of about 35°C), poly-N-ethoxyethylmethacrylamide (LCST of about 45°C), poly-N-tetrahydrofurfurylacrylamide (LCST of about 28°C), poly-N-tetrahydrofurfurylmethacrylamide (LCST of about 35°C), poly-N,N-ethylmethylacrylamide (LCST of homopolymer is 56°C), and poly-N,N-diethylacrylamide (LCST of homopolymer is 32°C).

[0067] Examples of polymers for copolymerization used in the present invention include, but are not limited to, hydrous polymers such as polyacrylamide, poly-N,N-diethylacrylamide, poly-N,N-dimethylacrylamide, polyethylene oxide, polyacrylic acid and salts thereof, polyhydroxyethyl methacrylate, polyhydroxyethyl acrylate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose, and carboxymethyl cellulose.

[0068] <<Use of Temperature-Responsive Polymer in Reusable Culture Substrates>> The temperature-responsive polymer having a glass transition temperature of greater than 121°C used in the present invention can be used in reusable substrates. This is because a reusable culture substrate can be provided by bonding the temperature-responsive polymer to a substrate that can be used repeatedly. The temperature-responsive polymer in the reusable culture substrate of the present invention is a temperature-responsive polymer having a glass transition temperature of greater than 121°C. The glass transition temperature of the temperature-responsive polymer in the reusable culture substrate of the present invention is preferably 125°C or higher, more preferably 130°C or higher, and even more preferably 135°C or higher. Furthermore, the temperature-responsive polymer in the reusable culture substrate of the present invention is preferably poly-N-isopropylacrylamide (PIPAAm). Because the LCST of PIPAAm is near the body temperature of an animal, using PIPAAm as the temperature-responsive polymer in the reusable substrate of the present invention facilitates cell culture and makes it easy to recover a large number of cells at low cost. Furthermore, there are no limitations on the material of the substrate used in the reusable culture substrate of the present invention, as long as it can be autoclaved and used repeatedly, and any material suitable for the purpose of the present invention can be used. However, from the perspective of repeated use as a culture substrate, it is preferable that the substrate does not deform when autoclaved. For example, substrates made of metal, ceramics, glass, or silicone such as polydimethylsiloxane (PDMS) can be used, but metal substrates are more preferable.

[0069] <Metal Substrate> The substrate used in the culture substrate of the present invention is a metal substrate. The metal substrate usable in the present invention is not particularly limited as long as it can be bonded to a temperature-responsive polymer, and an appropriate metal substrate can be used depending on the purpose. Furthermore, it is not particularly limited as long as it contains a material to which the temperature-responsive polymer can be introduced via a covalent bond by electron beam irradiation, radiation irradiation, or the like. Only the surface may contain a material that can be covalently bonded to the temperature-responsive polymer by electron beam irradiation, radiation irradiation, or the like, or the entire substrate may contain such a material. The metal substrate used in the culture substrate of the present invention can be treated with oxygen plasma to produce hydrophilic reactive groups on its surface. Examples of reactive groups that can be produced by oxygen plasma treatment of the metal substrate include hydroxyl groups. The metal substrate and the temperature-responsive polymer are preferably bonded via a covalent bond, and this bonding treatment preferably uses a silane agent (silanization treatment). The metal substrate and the temperature-responsive polymer in the present invention are preferably connected via a silicon atom, and it is preferable that a silicon atom is included in the bond between the metal substrate and the temperature-responsive polymer. The metal substrate used in the culture substrate of the present invention is preferably one that has been subjected to oxygen plasma treatment and then silanization treatment using a silane agent, which allows the temperature-responsive polymer to be easily bonded to the metal substrate via a covalent bond.

[0070] The material constituting the metal substrate in the present invention is not particularly limited as long as it is capable of cell culture and can be bonded to the temperature-responsive polymer via a covalent bond. However, if it is capable of forming an oxide film on its surface, a hydroxyl group that serves as a reactive group can be generated by plasma treatment or the like in the process of immobilizing the temperature-responsive polymer. Therefore, it is preferable that the metal be a metal capable of forming an oxide film on its surface, and the metal substrate may be made of an alloy containing two or more of these materials.

[0071] The metal substrate in the present invention is preferably made of titanium. Examples of metals used for the metal substrate in the present invention include pure titanium and titanium alloys, more preferably titanium alloys, and even more preferably alloys of titanium, aluminum, and vanadium. Examples of titanium alloys include Ti-6Al-4V (Ti6Al4V), Ti-6Al-4VELI (Ti6Al4VELI), Ti-6Al-2Nb-1Ta (Ti6Al2Nb1Ta), Ti-15Zr-4Nb-4Ta (Ti15Zr4Nb4Ta), Ti-6Al-7Nb (Ti6Al7Nb), Ti-3Al-2.5V (Ti3Al2.5V), Ti-13Nb-13Zr (Ti13Nb13Zr), Ti-15Mo-5Zr-3Al (Ti15Mo5Zr3Al), Ti-12Mo-6Zr-2Fe (Ti12Mo6Zr2Fe), and Ti-15Mo (Ti15Mo). Titanium is lightweight, strong, and corrosion-resistant, making it suitable for culturing, ultrasonic cleaning, and autoclaving, and is also suitable for reuse. Titanium is also suitable as a metallic biomaterial and is non-toxic to cells.

[0072] In addition to the above, examples of metals that can be used for the metal substrate in the present invention include stainless steel, cobalt-chromium alloys, pure iron, steel, cast iron, nickel alloys, gold, platinum, pure aluminum, aluminum alloys, pure copper, copper alloys (bronze, cupronickel, phosphor bronze, brass), pure cobalt, cobalt alloys, magnesium alloys, silver, tungsten, zinc, zinc alloys, lead, tin, pure tantalum, tantalum alloys, pure niobium, niobium alloys, molybdenum alloys, zirconium (alloys), gold-silver-palladium alloys, etc. Stainless steel and cobalt-chromium alloys can be used as metallic biomaterials and are not toxic to cells, so are preferred as metals for use in the present invention.

[0073] Furthermore, as long as the object of the present invention is not impaired, any layer or intermediate layer of the metal substrate of the present invention may be provided on the surface or may be subjected to any treatment. For example, the surface of the metal substrate may be made hydrophilic by using a treatment technique such as ozone treatment, plasma treatment, or sputtering.

[0074] The culture substrate of the present invention may further comprise a cell-adhesive substance, including a natural cell-adhesive substance and / or a synthetic cell-adhesive substance. The cell-adhesive substance may be located between the temperature-responsive polymer layer and the metal substrate, or on the metal substrate to which the temperature-responsive polymer is bound, as long as the cell-adhesive substance is located within a range that allows sufficient bonding between the metal substrate and the temperature-responsive polymer. However, from the viewpoint of facilitating bonding when bonding the temperature-responsive polymer to the metal substrate, the cell-adhesive substance is preferably located on the metal substrate to which the temperature-responsive polymer is bound.

[0075] The cell adhesive substance that can be used in the culture substrate of the present invention is not limited as long as it has affinity / adhesion to cells, and any substance can be used depending on the purpose. Examples of natural cell adhesive substances include oligosaccharides, gelatin, collagen, fibronectin, laminin, fibrin, and cell adhesion peptides that are components of these substances. Synthetic cell-adhesive substances can be obtained by homopolymerizing a monomer containing a cell-adhesive group, copolymerizing two monomers containing cell-adhesive groups, or copolymerizing a monomer containing a cell-adhesive group with a monomer not containing a cell-adhesive group. Examples of such cell-adhesive groups include carboxylic acid groups and their salts, anhydrides, sulfonic acid groups and their salts, sulfonate esters, sulfonamides, phosphate groups and their salts, amino groups, hydroxyl groups, long-chain alkyl groups, mercapto groups, ether groups, thioether groups, polyether groups, ketone groups, aldehyde groups, acyl groups, cyano groups, nitro groups, acylamino groups, halogen groups, glycidyl groups, allyl groups, and phosphobetaine groups and sulfobetaine groups that contain these cell-adhesive groups in the same monomer. These natural or synthetic cell-adhesive substances can be used alone or in combination in the present invention.

[0076] (Method for manufacturing a culture substrate) The method for manufacturing a culture substrate of the present invention comprises a silane agent treatment step of treating the surface of an oxygen plasma-treated metal substrate with a silane agent, and a temperature-responsive polymer bonding step of bonding a temperature-responsive polymer having a glass transition temperature of above 121° C. to the metal substrate. If the method for manufacturing a culture substrate of the present invention does not include the step of treating the surface of the metal substrate with a silane agent, the subsequent temperature-responsive polymer bonding step will not be easy to perform, making it difficult to fix the temperature-responsive polymer to the metal substrate and potentially causing it to peel off after fixation. This may reduce the number of times the culture substrate can be autoclaved and reused, and may reduce the amount of cultured cells that can be recovered non-invasively at low cost.

[0077] In the temperature-responsive polymer bonding step, when a monomer unit of a temperature-responsive polymer is used to bond the temperature-responsive polymer onto a metal substrate, the temperature-responsive polymer may be applied in any state of gas, liquid, or solid, but it is preferable to apply a solution of the monomer unit of the temperature-responsive polymer to the metal substrate.

[0078] The concentration of the solution of the monomer units of the temperature-responsive polymer applied to the metal substrate in the temperature-responsive polymer bonding step is preferably 5 to 65 wt %, more preferably 10 to 55 wt %, and even more preferably 30 to 50 wt %. The concentration of the solution of the monomer units of the temperature-responsive polymer is reflected in the thickness and density of the temperature-responsive polymer layer formed on the metal substrate, and affects the conditions suitable for cell detachment and cell adhesion. If the concentration of the solution of the monomer units of the temperature-responsive polymer is 5 wt % or more, cells are easily detached when the medium temperature is changed for cell detachment, while if it is 65 wt % or less, the adhesion of cultured cells to the bottom surface of the culture substrate is good. From the viewpoint of efficiency in cell culture, a concentration of the solution of the monomer units of the temperature-responsive polymer of 30 to 50 wt % is particularly suitable.

[0079] The temperature-responsive polymer used in the present invention is a polymer with a glass transition temperature of greater than 121°C. If the glass transition temperature of the temperature-responsive polymer used in the present invention is 121°C or lower, the polymer may be denatured by autoclaving, and the temperature-responsive polymer on the metal substrate may no longer exhibit appropriate temperature responsiveness after autoclaving the culture substrate. The glass transition temperature of the temperature-responsive polymer used in the present invention is preferably 125°C or higher, more preferably 130°C or higher, and even more preferably 135°C or higher. The temperature-responsive polymer used in the method for producing a culture substrate of the present invention is preferably poly-N-isopropylacrylamide (PIPAAm). Because the LCST of PIPAAm is near the body temperature of an animal, a culture substrate suitable for culturing and recovering cells can be produced.

[0080] The metal base of the culture substrate used in the method for producing a culture substrate of the present invention is preferably made of titanium. Titanium is a lightweight, strong, and corrosion-resistant metal, which makes it easy to handle culture operations, ultrasonically clean, and autoclave-treat for reuse, making it possible to produce a culture substrate that can produce large amounts of cells at low cost.

[0081] An example of a method for producing a culture substrate of the present invention will be described in detail below with reference to FIG. 3. The present invention is produced, for example, mainly by the following steps [1] to [5]. [1] A polished substrate surface is subjected to a hydrophilization treatment to generate hydrophilic reactive groups on the substrate surface (in FIG. 3, the Ti6Al4V substrate surface is subjected to oxygen plasma treatment to generate hydroxyl groups). [2] A surface treatment is performed using a silane agent (in FIG. 3, 3-(methacryloyloxy)propyltrimethoxysilane) under vacuum at appropriate temperature and time conditions (silane agent treatment step). [3] The silane-treated surface is washed and dried under vacuum conditions. [4] After cooling the silane-treated substrate to room temperature (approximately 25°C), a solution of monomer units of a temperature-responsive polymer is dropped onto the sample. After the solution spreads over the surface, the substrate and the temperature-responsive polymer are bonded by a chemical reaction (in FIG. 3, electron beam irradiation is performed for a certain period of time under a nitrogen atmosphere) (temperature-responsive polymer bonding step). [5] After bonding the substrate and the temperature-responsive polymer, the substrate is placed in cold water, washed, and dried to obtain the desired substrate surface to which the temperature-responsive polymer is bonded.

[0082] The substrate, metal substrate, and hydrophilization treatment that can be used in [1] above are as described above. The surface of the substrate used in [1] above may be subjected to blasting or microfabrication in addition to polishing, as long as the object of the present invention is not hindered. By changing the surface properties through microfabrication, the orientation of the seeded cells can be controlled.

[0083] The silane agent used in [2] above is not particularly limited as long as it can achieve the object of the present invention. Examples include those having a vinyl group, an epoxy group, a styryl group, a methacryl group, an acrylic group, an amino group, an isocyanurate group, a ureido group, a mercapto group, an isocyanate group, or an acid anhydride. Among these, those having an acrylic group, a methacryl group, an amino group, a hydroxyl group, a mercapto group, or a styryl group are preferred because of their high reactivity with the polymer.

[0084] Specific examples of the silane agent that can be used in the present invention include vinyltrimethoxysilane, vinyltriethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-(methacryloyloxy)propyl Trimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, N-2-(aminoethyl)-3-aminopropylmethyldimethoxysilane, N-2-(aminoethyl)-3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-triethoxysilyl-N-(1,3-dimethyl-butylidene)propylamine, N-phenyl-3-aminopropyltrimethoxysilane Examples of compounds include trimethoxysilane, N-(vinylbenzyl)-2-aminoethyl-3-aminopropyltrimethoxysilane hydrochloride, tris-(trimethoxysilylpropyl)isocyanurate, 3-ureidopropyltrialkoxysilane, 3-mercaptopropylmethyldimethoxysilane, 3-mercaptopropyltrimethoxysilane, 3-isocyanatopropyltriethoxysilane, and 3-trimethoxysilylpropylsuccinic anhydride. From the viewpoint of immobilizing the polymer, however, vinyltrimethoxysilane, vinyltriethoxysilane, and It is preferable to use 3-methacryloxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-(methacryloyloxy)propyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-mercaptopropylmethyldimethoxysilane, and 3-mercaptopropyltrimethoxysilane.

[0085] When a monomer unit of a temperature-responsive polymer is bonded to the surface of a substrate, the monomer may be in any state of gas, liquid, or solid. When a homopolymer or copolymer is used for coating, the polymer may be in any state of solution or solid. When these are bonded by a chemical reaction, electron beam irradiation (EB), gamma ray irradiation, ultraviolet irradiation, plasma treatment, corona treatment, or, if the substrate has a suitable reactive functional group, commonly used organic reactions such as radical reaction, anionic reaction, or cationic reaction can be used, but electron beam irradiation (EB) is preferred. Note that Figure 3 illustrates the bond between the substrate surface and the silane agent, and the Si bond at this bond is represented by -O-Si-O(CH ２ ) ３ Although it is indicated as -, Si normally has four bonds, and therefore Si has bonds that are not shown in FIG. 3, but for convenience of notation, they are omitted in FIG.

[0086] In the above [4], it is presumed that after a solution of monomer units of a temperature-responsive polymer is dropped onto the surface of a substrate, the substrate is irradiated with, for example, an electron beam, whereby radicals are generated from the monomer units of the temperature-responsive polymer and the organic components of the silane agent, and polymerization and crosslinking of the monomer and / or polymer and fixation of a polymer gel via the organic components of the silane agent occur almost simultaneously, resulting in the formation of a temperature-responsive polymer layer on the surface of the substrate.

[0087] The bond between the substrate surface and the temperature-responsive polymer can be confirmed by surface analysis of the metal substrate after immobilization of the temperature-responsive polymer using X-ray photoelectron spectroscopy (XPS). In XPS analysis of the substrate surface after immobilization of the temperature-responsive polymer, waveform separation of the C1s spectrum reveals a signal derived from the temperature-responsive polymer (in Figure 4, the peak due to the carbon atom of the carbonyl group of PIPAAm (peak IV due to the carbon atom of IV in the chemical structural formula in Figure 4)). This confirms that the temperature-responsive polymer has been chemically bonded to the metal substrate surface (Figure 4). Furthermore, the nitrogen element derived from the temperature-responsive polymer, which was not detected on the metal substrate surface before immobilization of the temperature-responsive polymer, is detected on the metal substrate on which the temperature-responsive polymer has been immobilized, confirming that a gel layer of the temperature-responsive polymer has been immobilized.

[0088] Furthermore, by dropping water onto the surface of a metal substrate before and after immobilizing a temperature-responsive polymer, changing the temperature, and measuring and comparing the contact angle before and after the temperature change to confirm whether or not there was a change in the contact angle of water, it was possible to confirm that the metal substrate surface onto which a temperature-responsive polymer was immobilized is a surface that changes between hydrophobic and hydrophilic properties (Figure 5). A larger contact angle indicates a more hydrophobic property, and a smaller contact angle indicates a more hydrophilic property.

[0089] Furthermore, similarly to the above, when the surface of a temperature-responsive polymer-bonded metal substrate after autoclaving under autoclave sterilization conditions is analyzed by XPS, carbon atoms and nitrogen atoms derived from the temperature-responsive polymer are confirmed, confirming that even after autoclaving, the culture substrate of the present invention has the temperature-responsive polymer chemically bonded and immobilized on the metal substrate.

[0090] Furthermore, by culturing cells using the autoclaved culture substrate of the present invention and confirming that cells can be detached by changing the temperature of the culture medium, it was confirmed that the temperature-responsive effect of the temperature-responsive polymer in the culture substrate of the present invention continues to be maintained even when autoclaved under autoclave sterilization conditions. Furthermore, since the temperature-responsive effect is maintained even after autoclaving, it was confirmed that the temperature-responsive polymer of the present invention does not denature even under autoclave sterilization conditions, and that it maintains its temperature-responsive effect even after being bonded to a metal substrate without denature.

[0091] The thickness of the gel containing the temperature-responsive polymer on the substrate surface to which the temperature-responsive polymer is bound after drying in the above [5] is preferably 2 to 40 nm, more preferably 3 to 35 nm, and even more preferably 4 to 20 nm. If the thickness of the gel containing the temperature-responsive polymer is 2 nm or more, cells are easily detached when the temperature of the medium is changed for cell detachment, and this tendency is even stronger if the thickness is 3 nm or 4 nm or more. If the thickness of the gel containing the temperature-responsive polymer is 40 nm or less, the adhesion of cultured cells to the bottom surface of the culture substrate is good, and if the thickness is 35 nm or 20 nm or less, this tendency is even stronger. From the viewpoint of efficiency in cell culture, the thickness of the polymer gel containing the temperature-responsive polymer is particularly preferably in the range of 20±5 nm.

[0092] The method for producing a culture substrate of the present invention can further include a step of applying a cell-adhesive substance containing a natural cell-adhesive substance and / or a synthetic cell-adhesive substance. A solution of the cell-adhesive substance diluted to an appropriate ratio is added to the surface of the metal substrate after immobilization of the temperature-responsive polymer, and the solution is left for 1 to 10 minutes, followed by washing with water or a buffer solution 1 to 3 times, thereby obtaining a culture substrate further containing a cell-adhesive substance in the present invention. The cell-adhesive substances containing natural cell-adhesive substances and / or synthetic cell-adhesive substances that can be used in this step are as described above.

[0093] The cell adhesive substance may be added to the culture substrate of the present invention during the manufacturing process of the culture substrate of the present invention before autoclaving, or the cell adhesive substance may be added to the culture substrate of the present invention after autoclaving.

[0094] (Cell Production Method) The cell production method of the present invention includes a cell production step in which cells are seeded and cultured on the surface of a culture substrate having a temperature-responsive polymer with a glass transition temperature above 121°C on a metal substrate that has been oxygen plasma-treated and silanized. In cell production, when recovering a large number of cells non-invasively without enzymatic or chemical treatment to avoid damaging the cells themselves or proteins surrounding the cells, such as the extracellular matrix (ECM), without using a culture substrate having a temperature-responsive polymer with a glass transition temperature above 121°C on a metal substrate, it is difficult to efficiently control the temperature of the culture substrate, the solution used for cell culture (e.g., culture medium, PBS, physiological saline, serum), the temperature-responsive polymer, and the recovered cells, and it is also difficult to autoclave and reuse them. This necessitates the use of large quantities of disposable culture substrates, which increases the environmental burden and makes it difficult to efficiently produce a large number of cells at low cost. Furthermore, the cell production method of the present invention preferably further includes a cell detachment step, following the cell production step, in which the cells are detached from the temperature-responsive polymer by changing the temperature. When using the cell production method of the present invention, the culture substrate used has a metal base, which allows for efficient temperature changes in the medium and substrate, making it possible to rapidly detach cells and produce more cells more efficiently. Furthermore, the use of a Peltier element or the like allows for faster temperature changes in the metal base, resulting in more efficient cell production.

[0095] The temperature-responsive polymer used in the present invention is a polymer with a glass transition temperature of greater than 121°C. If the glass transition temperature of the temperature-responsive polymer used in the present invention is 121°C or lower, the polymer may be denatured by autoclaving, and the temperature responsiveness may not be properly exhibited. The glass transition temperature of the temperature-responsive polymer used in the present invention is preferably 125°C or higher, more preferably 130°C or higher, and even more preferably 135°C or higher. The temperature-responsive polymer used in the cell production method of the present invention is preferably poly-N-isopropylacrylamide (PIPAAm). The LCST of PIPAAm is close to the body temperature of an animal, making it suitable for cell culture and recovery.

[0096] The metal substrate of the culture substrate used in the cell production method of the present invention is preferably made of titanium. Titanium is a lightweight, strong, and corrosion-resistant metal, making it easy to handle in culture, ultrasonically clean, and autoclave-treat for reuse, making it easy to produce large amounts of cells at low cost.

[0097] Furthermore, the surface of the metal substrate of the culture substrate used in the cell production method of the present invention is treated with oxygen plasma and a silane agent. By treating the surface of the metal substrate with oxygen plasma, it becomes easier to introduce a temperature-responsive polymer onto the surface of the metal substrate via a covalent bond. Treating the surface of the metal substrate with a silane agent makes it easy to immobilize the temperature-responsive polymer onto the metal substrate of the culture substrate used in cell production via a covalent bond, and the immobilized temperature-responsive polymer is less likely to peel off, making it easy to autoclave the culture substrate and reuse it, facilitating the production of large quantities of cells at low cost.

[0098] The temperature-responsive polymer is preferably connected to the metal substrate via a silicon atom. In a culture substrate used for cell production, bonding the temperature-responsive polymer to the metal substrate via a silicon atom facilitates covalent bonding between the metal substrate and the temperature-responsive polymer, making it difficult for the temperature-responsive polymer immobilized on the metal substrate to peel off. This facilitates autoclaving and reuse of the culture substrate, facilitating the production of large quantities of cells at low cost.

[0099] In the cell production method of the present invention, the culture substrate may include a base having a recess and a lid covering the base, and the temperature-responsive polymer may be provided on the bottom surface of the recess in the base. Use of such a culture substrate for cell production facilitates general cell culture.

[0100] As an example of the cell production method of the present invention, poly-N-isopropylacrylamide (PIPAAm) is used as a temperature-responsive polymer, and will be explained using Figures 1 and 2. PIPAAm is known to have a lower critical dissolution temperature of 32°C. In its free state, PIPAAm undergoes dehydration in water at temperatures above 32°C, causing the polymer chains to aggregate and precipitate, resulting in a cloudy appearance. Conversely, at temperatures below 32°C, the polymer chains become hydrated and dissolved in water (Figure 2, top). The culture substrate used in the present invention is a culture substrate in which a temperature-responsive polymer with a glass transition temperature above 121°C is bound and immobilized to the surface of a culture substrate having a metal substrate treated with oxygen plasma and silanized (Figure 1). Therefore, at temperatures above 32°C, the polymer on the substrate surface also dehydrates in the same way, but because the polymer chains are coated and immobilized on the substrate surface, the substrate surface becomes hydrophobic (Figure 2, bottom right). Conversely, at temperatures below 32°C, the polymer on the substrate surface becomes hydrated by the water contained in the medium, but because the polymer chains are coated and fixed on the substrate surface, the substrate surface becomes hydrophilic (Figure 2, bottom left). At this time, the hydrophobic surface is suitable for cells to attach and grow, while the hydrophilic surface becomes too hydrophilic for cells to attach, and the cultured cells or cell sheet can be detached simply by cooling (Figure 1).

[0101] The cell production method of the present invention will be described in more detail below. The temperature-responsive polymer and metal substrate that can be used for the culture substrate of the present invention are as described above.

[0102] <Cells> The type of cells used in the present invention is not particularly limited, and cells from the site where the obtained cell sheet laminate is to be transplanted, or cells derived from the organ or tissue to be evaluated, may be used. For example, when the purpose is to regenerate myocardial tissue or to evaluate myocardial function, examples of cells to be used include one or a mixture of two or more types of cells selected from the group consisting of cardiomyocytes, cardiomyocytes, myoblasts, mesenchymal stem cells, vascular endothelial cells, vascular endothelial progenitor cells, fibroblasts, bone marrow-derived cells, and adipose-derived cells, and the type of cells is not particularly limited.

[0103] When the purpose is to regenerate liver tissue, to create an artificial liver that mimics liver tissue, or to evaluate the function of liver tissue, the cells to be used may include, for example, one or a mixture of two or more types of cells selected from the following: hepatic parenchymal cells, sinusoidal endothelial cells, Kupffer cells, stellate cells, pit cells, bile duct epithelial cells, vascular endothelial cells, vascular endothelial progenitor cells, fibroblasts, bone marrow-derived cells, adipose-derived cells, and mesenchymal stem cells, and there is no limitation on the type of cells.

[0104] When the purpose is the regeneration of kidney tissue, the production of an artificial kidney simulating kidney tissue, or a method for evaluating kidney function, examples of cells to be used include one or a mixture of two or more types of cells selected from the group consisting of kidney cells, granular cells, collecting duct epithelial cells, parietal epithelial cells, podocytes, mesangial cells, smooth muscle cells, tubular cells, interstitial cells, glomerular cells, vascular endothelial cells, vascular endothelial progenitor cells, fibroblasts, bone marrow-derived cells, adipose-derived cells, and mesenchymal stem cells, and there is no limitation on the type of cells.

[0105] When the purpose is the regeneration of adrenal tissue, the production of an artificial adrenal gland that mimics the adrenal gland, or a method for evaluating adrenal function, examples of cells that can be used include adrenal medulla cells, adrenal cortical cells, zona glomerulosa cells, zona fasciculata cells, epireticular cells, vascular endothelial cells, vascular endothelial progenitor cells, fibroblasts, bone marrow-derived cells, adipose-derived cells, and mesenchymal stem cells, or a mixture of two or more types of cells, and there is no limitation on the type of cells.

[0106] When the purpose is to regenerate skin or to evaluate skin function, examples of cells to be used include epidermal keratinocytes, melanocytes, arrector pili muscle cells, hair follicle cells, vascular endothelial cells, vascular endothelial progenitor cells, fibroblasts, bone marrow-derived cells, adipose-derived cells, and mesenchymal stem cells, or a mixture of two or more types of cells, and there is no limitation on the type of cells.

[0107] When the purpose is to regenerate mucosal tissue or to evaluate the function of mucosal tissue, for example, the cells used may be cells collected from tissues that constitute the mucosa. Examples of the types of mucosa include buccal mucosa, gastric mucosa, intestinal mucosa, olfactory epithelium, oral mucosa, and uterine mucosa. Examples of the cells collected from mucosal tissue include one type or a mixture of two or more types of cells, and the types are not limited in any way.

[0108] The content ratio of the above cells is not particularly limited. In this case, if vascular endothelial cells, vascular endothelial precursor cells, etc. are mixed into the cell group, it is advantageous because a vascular network can be efficiently constructed within the cell group.

[0109] The cells used in the present invention include, for example, cells collected directly from biological tissue, cells collected directly and differentiated in a culture system, or cell lines, but the type is not limited thereto. The origin of these cells is not particularly limited, and examples include humans, rats, mice, guinea pigs, marmosets, rabbits, dogs, cats, sheep, pigs, goats, monkeys, chimpanzees, and immunodeficient animals thereof. However, when the multilayered cell sheet of the present invention is used for human treatment, it is preferable to use cells derived from humans, pigs, monkeys, or chimpanzees.

[0110] The cells used in the present invention are not particularly limited, and may be, for example, cells that have been fluorescently stained and / or dyed using at least one method, such as a reagent, protein, or gene. When cells into which a reporter gene has been introduced are used, the activity of the cells, cell sheet, or cell sheet laminate can be determined by detecting the fluorescence produced by the reporter protein produced upon expression of the reporter gene, or the fluorescence emitted when the reporter protein reacts with a specific substrate. The reporter gene or reporter protein used is not particularly limited, and examples include green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), DsReD, β-glucuronidase, LacZ, Kaede, luciferase, and alkaline phosphatase.

[0111] The method for introducing a gene into a cell may be any conventional method, and is not particularly limited, and examples thereof include the lipofection method, the viral vector method, the calcium phosphate method, the electroporation method, the DEAE-dextran method, the microinjection method, etc. Furthermore, cells derived from a transgenic animal in which a reporter gene has been introduced into the host genome using these gene introduction methods may also be used.

[0112] The promoter sequence that regulates the expression of the reporter gene is not particularly limited, and may be appropriately selected depending on the purpose of detecting the expression of the reporter gene.

[0113] Culture conditions other than temperature may be those of a conventional method and are not particularly limited. For example, the medium used may be a medium supplemented with a known serum such as fetal calf serum (FCS), or a serum-free medium without such serum.

[0114] In the cell production method of the present invention, the number of cells seeded on the surface of the culture substrate during culture varies depending on the animal species of the cells used and the type of tissue from which they are derived, but is generally 1×10 ４ ~10 x 10 ４ cell / cm ２ is preferred, and 2 × 10 ４ ~8 x 10 ４ cell / cm ２ is more preferable, and 3×10 ４ ~5 x 10 ４ cell / cm ２ Even more preferably, the seeding concentration is 1×10 ４ cell / cm ２ If the number of cells is 10×10 or more, cells generally grow easily and the degree of expression of the functions of the obtained cells is unlikely to deteriorate. ４ cell / cm ２ This is preferable because the cells have not yet reached confluence.

[0115] In the present invention, cultured cells can be detached and recovered from the culture substrate by lowering the temperature of the culture substrate to which the cultured cells are attached to below the lower critical dissolution temperature of the temperature-responsive polymer on the metal substrate of the culture substrate. This can be done in a medium or in another isotonic solution, and can be selected according to the purpose.

[0116] For the purpose of detaching and recovering cells more quickly and efficiently, methods such as gently tapping or shaking the substrate, or stirring the medium using a pipette, may be used alone or in combination.

[0117] Furthermore, if necessary, an instrument for transferring cultured cells may be used. Such an instrument is not limited in terms of material or shape as long as it can capture the detached cell sheet, but materials such as polyvinylidene difluoride (PVDF), silicone, polyvinyl alcohol, urethane, cellulose and its derivatives, chitin, chitosan, collagen, gelatin, fibrin glue, etc. are typically used, and these materials may be used in the form of a membrane, porous membrane, nonwoven fabric, or woven fabric in contact with the cells.

[0118] The cells produced in the present invention are not damaged by proteolytic enzymes such as dispase and trypsin or chemicals when the cultured cells are recovered. Therefore, the cells detached from the substrate contain adhesive proteins. This allows the cultured cells to adhere well and engraft efficiently when applied to tissue.

[0119] <Cell Sheet> In the cell production method of the present invention, the cells can be produced in a sheet form in the cell production step to produce a cell sheet.

[0120] For example, the protease dispase is known to be able to detach cells while maintaining 10-40% of the desmosome structure between cells, but it also destroys most of the basement membrane-like proteins and other proteins between the cells and the substrate, resulting in cell sheets generally obtained using protease enzymes or chemicals with weak strength. In contrast, the cell sheet produced according to the present invention causes less destruction of the basement membrane-like proteins, desmosome structure, and basement membrane-like proteins between the cells and the substrate, and when the cultured cell sheet is detached, the desmosome structure between the cells can be maintained to a certain extent. Therefore, the cell sheet detached from the substrate contains adhesive proteins, which allow cultured cells to adhere well when applied to tissue, resulting in efficient engraftment.

[0121] Furthermore, if necessary, a jig for moving the cell sheet may be used. Such a jig is not limited in terms of material or shape as long as it can capture the detached cell sheet, but materials such as polyvinylidene difluoride (PVDF), silicone, polyvinyl alcohol, urethane, cellulose and its derivatives, chitin, chitosan, collagen, gelatin, fibrin glue, etc. are typically used, and these materials may be used in the form of a membrane, porous membrane, nonwoven fabric, or woven fabric in contact with the cell sheet.

[0122] <Cell Sheet Laminate> The method for producing the cell sheet laminate of the present invention is not particularly limited, and can be obtained, for example, by detaching cultured cells in a sheet form and, if necessary, laminating the cultured cell sheets together using a cultured cell transfer device. In this case, the temperature of the medium is not particularly limited as long as it is higher than the lower critical dissolution temperature of the temperature-responsive polymer used in the culture substrate.

[0123] Thus, the present invention can also provide a thick multilayered cell sheet. While the layering method is not particularly limited, it is preferable to layer three or fewer cell sheets in multiple batches rather than layering cell sheets all at once. The number of layers may be determined appropriately depending on the purpose for which the multilayered cell sheet is to be used, but is preferably five or more layers, more preferably ten or more layers, and even more preferably fifteen or more layers. Increasing the thickness of the multilayered cell sheet allows for more pronounced benefits from the effects of the present invention and is also advantageous in that a larger number of cells can be transplanted into the recipient.

[0124] Examples of the present invention will be described below, but the present invention is not limited to these examples. Unless otherwise specified, various preparations and evaluations were carried out under conditions of room temperature of 25°C and humidity of 50%.

[0125] <Measurement of Glass Transition Temperature of Polymer> The glass transition temperature of poly-N-isopropylacrylamide (PIPAAm) was measured by differential scanning calorimetry (DSC). Since the measurement sample was highly hygroscopic, the measurement sample was heat-treated in a DSC apparatus at room temperature to 120°C prior to measurement to remove excess moisture, and then the glass transition temperature was measured. Furthermore, when a large endotherm resulting from enthalpy relaxation (volume relaxation) overlaps with the glass transition signal in the DSC curve of the measurement sample, a baseline was assumed on the curve showing the glass transition signal in the DSC curve, and the central temperature of the stepwise signal was read and used as the glass transition temperature of each sample. The measurement method and conditions for the glass transition temperature of each polymer are as follows:

[0126] - Measurement conditions - Apparatus: Q1000 manufactured by TA Instruments Data processing: "Universal Analysis 2000" manufactured by TA Instruments Atmosphere: Nitrogen (50 mL / min) Temperature and calorific value calibration: High-purity indium (Tm = 156.61°C, ΔHm = 28.71 J / g) Temperature range: Room temperature to 300°C Heating rate: 10°C / min Sample amount: Approximately 5 mg Sample container: Aluminum standard container

[0127] The glass transition temperature of poly-N-isopropylacrylamide (PIPAAm) measured under the above conditions was 137.4°C.

[0128] <Changes in polymer after autoclave treatment> Approximately 5 mg of poly-N-isopropylacrylamide (PIPAAm) was placed in a glass container, the container's opening was covered with aluminum foil, and the container was then autoclaved (121°C, 2 atmospheres, 15 minutes). After the temperature had sufficiently decreased, each glass container was removed and each polymer was observed. No change in appearance of poly-N-isopropylacrylamide (PIPAAm) was observed before and after autoclaving.

[0129] <Preparation of Culture Substrate> A titanium metal substrate used as the culture substrate was prepared by cutting a 24 mm diameter rolled round bar (manufactured by Daido Steel Co., Ltd.) into a 4 mm thick piece. The cut surface was polished with emery paper and then buffed with a colloidal silica suspension to a mirror finish to produce a disk-shaped Ti6Al4V (64 titanium: φ24 mm). The surface of the Ti6Al4V disk was subjected to oxygen plasma treatment at 400 W for 3 minutes in an oxygen / argon mixed gas environment at 100 mTorr to hydrophilize the surface. Subsequently, 3-(methacryloyloxy)propyltrimethoxysilane (manufactured by Tokyo Chemical Industry Co., Ltd.) (3 ml) was added to a separable flask as a silane agent. A disk-shaped Ti6Al4V having a hydrophilic surface was placed in the separable flask to which the silane agent had been added, so as to avoid contact with the silane agent solution as much as possible. The separable flask was then placed in a vacuum state and left to stand at 70°C for 3 hours, thereby treating the hydrophilic surface of the disk-shaped Ti6Al4V with the silane agent (Figure 3).

[0130] The silanized surface of the obtained Ti6Al4V disk was washed with toluene, methanol, and ultrapure water, in that order, and then dried under vacuum at 160°C. After drying, the silanized Ti6Al4V disk was left to cool at room temperature. Purified N-isopropylacrylamide (KJ Chemicals Co., Ltd.) was used as the monomer of the temperature-responsive polymer, and 10 wt%, 30 wt%, 40 wt%, and 50 wt% N-isopropylacrylamide solutions were dissolved in 2-propanol to prepare the polymer. The 10 wt% N-isopropylacrylamide solution was prepared so that the 2-propanol concentration was 90 wt%, the 20 wt% N-isopropylacrylamide solution was prepared so that the 2-propanol concentration was 80 wt%, the 30 wt% N-isopropylacrylamide solution was prepared so that the 2-propanol concentration was 70 wt%, the 40 wt% N-isopropylacrylamide solution was prepared so that the 2-propanol concentration was 60 wt%, and the 50 wt% N-isopropylacrylamide solution was prepared so that the 2-propanol concentration was 50 wt%. 20 μl of each N-isopropylacrylamide solution was dropped onto the surface of separate silanized Ti6Al4V disks.

[0131] After the N-isopropylacrylamide solution was spread on the surface of the silanized Ti6Al4V disk, an electron beam (0.25 MGy) was irradiated onto the Ti6Al4V surface to which the N-isopropylacrylamide solution of each concentration had been added under a nitrogen atmosphere using an electron beam irradiation device (Curetron, manufactured by NHV Corporation).

[0132] After electron beam irradiation, the Ti6Al4V disks were placed in cold water and allowed to stand, then washed with ultrapure water and dried at 45°C to obtain a culture substrate on which poly(N-isopropylacrylamide) (PIPAAm) was immobilized on Ti6Al4V. The immobilized poly(N-isopropylacrylamide) (PIPAAm) was in a gel state.

[0133] Hereinafter, Ti6Al4V treated with 10 wt%, 30 wt%, 40 wt%, and 50 wt% N-isopropylacrylamide solutions will be referred to as "10PIPAAm-Ti6Al4V," "30PIPAAm-Ti6Al4V," "40PIPAAm-Ti6Al4V," and "50PIPAAm-Ti6Al4V." "10PIPAAm-Ti6Al4V" was used as the culture substrate prepared in Example 1, "30PIPAAm-Ti6Al4V" in Example 2, "40PIPAAm-Ti6Al4V" in Example 3, and "50PIPAAm-Ti6Al4V" in Example 4.

[0134] <Surface Analysis of Temperature-Responsive Polymer-Bound Metal Substrate> In order to confirm whether the temperature-responsive polymer PIPAAm was bound to the metal substrate Ti6Al4V, the 10PIPAAm-Ti6Al4V prepared in Example 1 was subjected to surface analysis by X-ray photoelectron spectroscopy using an X-ray photoelectron spectrometer (K-Alpha, manufactured by ThermoFisher Scientific). When the C1s spectrum was subjected to waveform separation in the surface analysis by X-ray photoelectron spectroscopy, a signal (peak IV in FIG. 4 ) derived from the carbonyl group of PIPAAm (carbon atom IV in the chemical structural formula of FIG. 4 ) was confirmed, indicating that PIPAAm was bound to the surface of Ti6Al4V via a covalent bond and chemically immobilized thereon.

[0135] <Evaluation of Surface Wettability of Temperature-Responsive Polymer-Bonded Metal Substrate (Contact Angle Measurement)> 5 μL of Mill Q water was dropped using a syringe onto the surface of the 40PIPAAm-Ti6Al4V prepared in Example 3 under predetermined temperature conditions (37°C and 20°C). The contact angle was measured 30 seconds after the drop. A high-end automatic contact angle meter (DSA100 manufactured by KRUSS) was used to measure the contact angle under a humidity condition of 50% (Example 5). For comparison, Ti6Al4V without PIPAAm bonded thereto ( FIG. 5A ) was also measured in the same manner (Comparative Example 1). As shown in FIG. 5B , the contact angle of the 40PIPAAm-Ti6Al4V surface with PIPAAm immobilized thereon changed with temperature. At 37°C, the contact angle was approximately 65° to 73°, indicating hydrophobicity, and at 20°C, the contact angle was 49° to 54°, indicating hydrophilicity (Example 5). On the other hand, for Ti6Al4V without PIPAAm (Fig. 5A), no significant change in contact angle with temperature was observed (Comparative Example 1).The above results indicate that the 40PIPAAm-Ti6Al4V surface is a surface whose hydrophobicity / hydrophilicity changes with temperature.

[0136] <Changes in a commercially available temperature-responsive cell culture substrate before and after autoclave (AC) treatment> A commercially available temperature-responsive cell culture substrate (manufactured by CellSeed Co., Ltd., registered trademark: UpCell) and 10PIPAAm-Ti6Al4V prepared in Example 1 were autoclaved (121°C, 2 atmospheres, 15 minutes). After autoclaving, the commercially available temperature-responsive cell culture substrate (registered trademark: UpCell) deformed and became cloudy ( FIG. 6B ), whereas 10PIPAAm-Ti6Al4V maintained its original shape without deformation ( FIG. 6A ).

[0137] <Changes in elemental composition of the surface of a temperature-responsive polymer-bonded metal substrate before and after autoclave treatment> The surfaces of Ti6Al4V before PIPAAm bonding treatment and the 10PIPAAm-Ti6Al4V surface prepared in Example 1 before and after autoclave treatment were analyzed by X-ray photoelectron spectroscopy (XPS) using an X-ray photoelectron spectrometer (K-Alpha, manufactured by ThermoFisher Scientific). In an unautoclave-treated state, no N element was detected on the Ti6Al4V surface before PIPAAm bonding treatment (Comparative Example 2), but N atoms derived from PIPAAm were detected in 10PIPAAm-Ti6Al4V bonded with PIPAAm, confirming that PIPAAm was bonded and immobilized on the Ti6Al4V surface. Furthermore, it was confirmed that N atoms remained and the PIPAAm gel layer was maintained even when 10PIPAAm-Ti6Al4V was autoclaved one or more times. The elemental composition analysis of the 10PIPAAm-Ti6Al4V surface by X-ray photoelectron spectroscopy (XPS) before and after autoclaving was performed in Example 6, and the results of Example 6 are shown in Table 1. In Table 1, "ND" means "Not Detected."

[0138]

[0139] Similarly as described above, the surfaces of Ti6Al4V before PIPAAm bonding treatment and the 50PIPAAm-Ti6Al4V surface prepared in Example 4 before and after autoclave treatment were analyzed by X-ray photoelectron spectroscopy (XPS) using an X-ray photoelectron spectrometer (K-Alpha manufactured by ThermoFisher Scientific). While N elements were not detected on the Ti6Al4V surface before PIPAAm bonding treatment (Comparative Example 3), N atoms derived from PIPAAm were detected on the 50PIPAAm-Ti6Al4V surface to which PIPAAm had been bonded, confirming that PIPAAm was bonded and immobilized on the Ti6Al4V surface. Furthermore, even when autoclaving of 50PIPAAm-Ti6Al4V was performed once or multiple times, N atoms remained, confirming that the PIPAAm gel layer was maintained. An elemental composition analysis of the 50PIPAAm-Ti6Al4V surface by X-ray photoelectron spectroscopy (XPS) before and after autoclaving was performed in Example 7, and the results of Example 7 are shown in Table 2. In Table 2, "ND" means "Not Detected."

[0140]

[0141] The results in Tables 1 and 2 show that PIPAAm remains fixed on the surface of Ti6Al4V to which PIPAAm has been bound, even after multiple autoclave treatments.

[0142] <Confirmation of cell detachment properties due to temperature change on culture substrate after autoclaving> The surface of the 50PIPAAm-Ti6Al4V prepared in Example 4 was autoclaved (once), and then mouse myoblasts (C2C12) that had been labeled in advance with calcein AM (C396-Cellstain (registered trademark)-Calcein-AM solution, manufactured by Dojindo Laboratories, Inc.) were prepared. A cell culture medium was prepared by adding bovine serum (30 w / v% albumin solution, bovine serum-derived, fatty acid-free, manufactured by Wako Pure Chemical Industries, Ltd., 017-22231) to a concentration of 10% and penicillin streptomycin (15140-122 PENICILLIN STREPTOMYCIN, manufactured by Life Technologies Corporation) as an antibiotic to a concentration of 1% to Dulbecco's Modified Eagle Medium (D-MEM / Ham's F-12 (containing L-glutamine and phenol red), manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 048-29785). Calcein AM-labeled C2C12 cells were added to the culture medium and suspended at a concentration of 1.0 × 10 ４ cell / cm ２ The cells were seeded on the 50PIPAAm-Ti6Al4V surface so that the surface area was 100 μm and cultured for 24 hours at 37°C in a cell culture incubator (MCO-5AC-PJ, Panasonic Corporation). After culture, cells on the 50PIPAAm-Ti6Al4V surface were observed at 5x magnification using a fluorescence microscope (Eclipse LV100ND, Nikon Corporation). The cells were observed to be attached in a spread-out state (Figure 7, left). The cells were then placed in a cell culture incubator (MCO-96, Sanyo Electric Co., Ltd. (now Panasonic Holdings Corporation)) at 20°C for 30 minutes to lower the temperature of the culture substrate to 20°C. The cells on the 50PIPAAm-Ti6Al4V surface were again observed using a fluorescence microscope. Many of the cells were found to have a rounded shape, adsorbed to the 50PIPAAm-Ti6Al4V surface, or floating in the medium (Figure 7, right).

[0143] These results demonstrate that the PIPAAm-bound Ti6Al4V surface retains its temperature-responsive effect on cell detachment even after autoclaving. Therefore, the temperature-responsive polymer bound to the metal substrate of the present invention maintains its temperature-responsive effect on cell detachment even under autoclave sterilization conditions without denaturation.

[0144] <Recovery of cell sheets cultured on autoclaved culture substrates> Each of the 30PIPAAm-Ti6Al4V, 40PIPAAm-Ti6Al4V, and 50PIPAAm-Ti6Al4V disks prepared in Examples 2 to 4 was autoclaved (once), and then a silicone rubber ring was attached to the disk. Mouse myoblasts (C2C12) pre-labeled with calcein AM (C396-Cellstain (registered trademark)-Calcein-AM solution, manufactured by Dojindo Laboratories, Inc.) were suspended in the same cell culture medium as above, and 1.5 × 10 ５ cells / cm ２ The cells were seeded on the surface of each disk so that the cells were in the shape of a globular substance. Then, the cells were cultured in a cell culture incubator (PHC Holdings Corporation CO ２ The cells were cultured for 48 hours at 37°C in an incubator (MCO-18AIC (UV)). After the culture, the temperature was changed to 20°C in the same manner as above, and it was confirmed that cells could be recovered in sheet form from the 40PIPAAm-Ti6Al4V and 50PIPAAm-Ti6Al4V surfaces (Figure 8). The cell sheet recovered from the 40PIPAAm-Ti6Al4V surface was free of damage and was confirmed to be a single cell sheet in which the cells were continuously bonded across the surface (Figure 9).

[0145] The above results suggest that the efficiency of cell detachment when the temperature is changed varies depending on the solution concentration of the monomer units of the temperature-responsive polymer used when bonding the temperature-responsive polymer to a metal substrate in the present invention.

[0146] <Recovery of cell sheets or cells cultured on culture substrates after multiple autoclave treatments> "Pure titanium" was used as the titanium metal substrate used in the culture substrate, and a disk-shaped Ti "50PIPAAm-Ti" was prepared in the same manner as in Example 4, and this was used as the culture substrate of Example 8.

[0147] After autoclaving the 50PIPAAm-Ti disk of Example 8, a silicone rubber ring was attached to it, and mouse-derived myoblast cells (C2C12) were suspended in the same cell culture medium as above, and 1.0 × 10 ５ cells / cm ２ The cells were seeded on the surface of a 50PIPAAm-Ti disk so that the density of the cells was 100%. The cells were then placed in a cell culture incubator (PHC Holdings Corporation, CO ２ The cells were cultured in an incubator (MCO-18AIC (UV)) at 37°C for 48 hours. After that, the cells were placed in a cell culture incubator (MCO-96, manufactured by Sanyo Electric Co., Ltd. (now Panasonic Holdings Corporation)) at 20°C. Within 10 to 20 minutes, the cells detached from the 50PIPAAm-Ti surface in a sheet-like form. It was confirmed that this cell sheet was a single cell sheet in which the cells were continuously bonded on the surface ( FIG. 10 ).

[0148] Next, after washing the culture substrate in the usual way and subjecting it to a second autoclave treatment, a silicone rubber ring was attached, and mouse-derived myoblast cells (C2C12) were suspended in the ring in the same cell culture medium as above, and 1.0 × 10 ５ cells / cm ２ The cells were seeded on the surface of a 50PIPAAm-Ti disk so that the density of the cells was 100%. The cells were then placed in a cell culture incubator (PHC Holdings Corporation, CO ２ The cells were cultured in an incubator (MCO-18AIC (UV)) at 37°C for 48 hours. After that, the cells were placed in a cell culture incubator (MCO-96, manufactured by Sanyo Electric Co., Ltd. (now Panasonic Holdings Corporation)) at 20°C. Within 10 to 20 minutes, the cells detached from the 50PIPAAm-Ti surface in a sheet-like form. It was confirmed that this cell sheet was a single cell sheet in which the cells were continuously bonded across the surface ( FIG. 11 ).

[0149] Next, after washing this culture substrate in the usual way and autoclaving it for the third time, a silicone rubber ring was attached to the culture substrate. Mouse myoblasts (C2C12) pre-labeled with calcein AM (C396-Cellstain (registered trademark)-Calcein-AM solution, manufactured by Dojindo Laboratories, Inc.) were suspended in the same cell culture medium as above, and 1.0 × 10 ４ cell / cm ２ The cells were seeded on the surface of 50PIPAAm-Ti disks so that the cells were aligned in a uniform pattern, and cultured for 24 hours at 37°C in a cell culture incubator (MCO-5AC-PJ, manufactured by Panasonic Corporation). When the cells on the 50PIPAAm-Ti surface were observed at 5x magnification using a fluorescence microscope (ECLIPSE LV100ND, manufactured by Nikon Corporation), the cells were observed to be attached in a spread-out state. The cultured cells were then placed in a cell culture incubator (MCO-96, manufactured by SANYO Electric Co., Ltd. (now Panasonic Holdings Corporation)) at 20°C. When the cells on the 50PIPAAm-Ti surface were again observed using a fluorescence microscope, it was confirmed that many of the cells had become round within 10 to 20 minutes and were adsorbed to the 50PIPAAm-Ti surface or floating in the medium (Figure 12).

[0150] Next, after washing the culture substrate in the usual way and autoclaving it for the fourth time, a silicone rubber ring was attached to the culture substrate. Mouse-derived myoblast cells (C2C12) were suspended in the same cell culture medium as above, and 1.0 × 10 ５ cells / cm ２ The cells were seeded on the surface of a 50PIPAAm-Ti disk so that the density of the cells was 100%. The cells were then placed in a cell culture incubator (PHC Holdings Corporation, CO ２ The cells were cultured in an incubator (MCO-18AIC (UV)) at 37°C for 48 hours. After that, the cells were placed in a cell culture incubator (MCO-96, manufactured by Sanyo Electric Co., Ltd. (now Panasonic Holdings Corporation)) at 20°C. Within 10 to 20 minutes, the cells detached from the 50PIPAAm-Ti surface in a sheet-like form. It was confirmed that this cell sheet was a single cell sheet in which the cells were continuously bonded across the surface ( FIG. 13 ).

[0151] Next, after washing the culture substrate in the usual way and autoclaving it for the fifth time, a silicone rubber ring was attached to the culture substrate. Mouse-derived myoblast cells (C2C12) were suspended in the same cell culture medium as above, and 1.0 × 10 ５ cells / cm ２ The cells were seeded on the surface of a 50PIPAAm-Ti disk so that the density of the cells was 100%. The cells were then placed in a cell culture incubator (PHC Holdings Corporation, CO ２ The cells were cultured in an incubator (MCO-18AIC (UV)) at 37°C for 48 hours. After that, the cells were placed in a cell culture incubator (MCO-96, manufactured by Sanyo Electric Co., Ltd. (now Panasonic Holdings Corporation)) at 20°C. Within 10 to 20 minutes, the cells detached from the 50PIPAAm-Ti surface in a sheet-like form. It was confirmed that this cell sheet was a single cell sheet in which the cells were continuously bonded across the surface ( FIG. 14 ).

[0152] The above results demonstrate that the culture substrate of the present invention is a reusable culture substrate in which the temperature-responsive polymer exhibits appropriate temperature responsiveness even after multiple autoclave treatments, and cells and cell sheets can be recovered by temperature changes.

[0153] Although the embodiments of the present invention have been described in detail above, the present invention is not limited to these embodiments, and various modifications and changes are possible within the scope of the gist of the present invention described in the claims.

[0154] Furthermore, aspects of the present invention are as follows, for example: <1> A culture substrate comprising a temperature-responsive polymer having a glass transition temperature of greater than 121°C on a metal substrate that has been oxygen plasma-treated and silanized. <2> The culture substrate according to <1>, wherein the temperature-responsive polymer is poly-N-isopropylacrylamide. <3> The culture substrate according to any one of <1> to <2>, wherein the metal substrate is made of titanium. <4> The culture substrate according to any one of <1> to <3>, wherein the metal substrate and the temperature-responsive polymer are connected via a silicon atom. <5> The culture substrate according to any one of <1> to <4>, comprising a substrate having a recess and a lid that covers the substrate, wherein the temperature-responsive polymer is located on the bottom surface of the recess in the substrate. <6> A method for producing a culture substrate, comprising: a silane agent treatment step of treating the surface of a metal substrate that has been oxygen plasma-treated with a silane agent; and a temperature-responsive polymer bonding step of bonding a temperature-responsive polymer having a glass transition temperature of greater than 121°C to the metal substrate. <7> The method for producing a culture substrate according to <6>, wherein the temperature-responsive polymer is poly-N-isopropylacrylamide. <8> The method for producing a culture substrate according to any one of <6> to <7>, wherein the metal substrate is made of titanium. <9> A method for producing cells, comprising a cell production step of seeding and culturing cells on the surface of a culture substrate having a temperature-responsive polymer having a glass transition temperature of greater than 121°C on a metal substrate that has been oxygen plasma treated and silanized to produce cells. <10> The method for producing cells according to <9>, wherein the cells are produced in a sheet form in the cell production step. <11> The method for producing cells according to any one of <9> to <10>, comprising, after the cell production step, a cell detachment step of detaching the cells from the temperature-responsive polymer by changing the temperature. <12> The method for producing cells according to any one of <9> to <11>, wherein the temperature-responsive polymer is poly-N-isopropylacrylamide. <13> The method for producing cells according to any one of <9> to <12>, wherein the metal substrate is made of titanium. <14> The method for producing a cell according to any one of <9> to <13>, wherein the temperature-responsive polymer is connected to the metal substrate via a silicon atom.<15> The method for producing cells according to any one of <9> to <14>, wherein the culture substrate comprises a base having a recess and a lid covering the base, and the temperature-responsive polymer is present on the bottom surface of the recess in the base. <16> A culture substrate for repeated use, comprising a temperature-responsive polymer having a glass transition temperature of higher than 121°C. <17> The culture substrate for repeated use according to <16>, wherein the temperature-responsive polymer is poly-N-isopropylacrylamide.

[0155] The culture substrate according to any one of <1> to <5>, the method for producing a culture substrate according to any one of <6> to <8>, the method for producing cells according to any one of <9> to <15>, and the culture substrate for repeated use according to any one of <16> to <17> can solve the conventional problems and achieve the object of the present invention.

[0156] This application claims priority based on Japanese Patent Application No. 2023-012270, filed with the Japan Patent Office on January 30, 2023, and incorporates the entire contents of said application by reference.

Claims

1. A culture substrate comprising a temperature-responsive polymer having a glass transition temperature of more than 121°C on a metal substrate that has been oxygen plasma-treated and silanized, the temperature-responsive polymer being bonded to the metal substrate via a covalent bond.

2. The culture substrate according to claim 1 , wherein the temperature-responsive polymer is poly-N-isopropylacrylamide.

3. The culture substrate according to claim 1 , wherein the metal substrate is made of titanium.

4. The culture substrate according to claim 1 , wherein the metal substrate and the temperature-responsive polymer are connected via a silicon atom.

5. The device comprises a base having a recess and a lid covering the base, The culture substrate according to claim 1 , wherein the temperature-responsive polymer is present on a bottom surface of the recess in the base.

6. a silane agent treatment step of treating the surface of the oxygen plasma-treated metal substrate with a silane agent; and a temperature-responsive polymer bonding step of bonding a temperature-responsive polymer having a glass transition temperature of more than 121°C onto the metal substrate via a covalent bond.

7. The method for producing a culture substrate according to claim 6, wherein the temperature responsive polymer is poly-N-isopropylacrylamide.

8. The method for producing a culture substrate according to claim 6, wherein the metal substrate is made of titanium.

9. A method for producing cells, comprising a cell production step of seeding and culturing cells on a surface of a culture substrate having a temperature-responsive polymer having a glass transition temperature of above 121°C, via a covalent bond, on a metal substrate that has been oxygen plasma treated and silanized, to produce the cells.

10. The method for producing cells according to claim 9 , wherein the cells are produced in a sheet form in the cell production step.

11. The method for producing cells according to claim 9 , further comprising, after the cell production step, a cell detachment step of detaching the cells from the temperature-responsive polymer by changing the temperature.

12. The method for producing cells according to claim 9 , wherein the temperature-responsive polymer is poly-N-isopropylacrylamide.

13. The method for producing cells according to claim 9 , wherein the metal substrate is made of titanium.

14. The method for producing a cell according to claim 9 , wherein the temperature-responsive polymer is connected to the metal substrate via a silicon atom.

15. The culture substrate comprises a base having a recess and a lid covering the base, The method for producing a cell according to claim 9 , wherein the temperature-responsive polymer is present on a bottom surface of the recess in the substrate.

16. A culture substrate for repeated use, characterized in that it has a temperature-responsive polymer having a glass transition temperature of more than 121°C on a metal substrate that has been treated with oxygen plasma and silanized, and the temperature-responsive polymer is bonded to the metal substrate via a covalent bond.

17. The culture substrate for repeated use according to claim 16, wherein the temperature-responsive polymer is poly-N-isopropylacrylamide.