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

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

Technical Field

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

Background Art

[0002] Generally, cell culture is often performed on the surface of glass or on the surface of synthetic polymer materials that have been subjected to various treatments. For example, various containers with a surface treatment using polystyrene as a material (such as γ-ray irradiation, silicone coating, etc.) are popular as cell culture containers. Conventionally, cells cultured and grown using such cell culture containers are detached and recovered from the container surface by treatment with proteolytic enzymes such as trypsin or chemicals. However, when recovering cells grown by treatment with the above-mentioned proteolytic enzymes or chemicals, the treatment process becomes complicated, the possibility of impurity contamination increases, and the grown cells may be damaged and denatured by the above treatment to the proteins around the cells such as the cells themselves and the extracellular matrix (ECM). Therefore, there is a demand to use cells cultured while the membrane proteins of the cell membrane and proteins such as ECM remain attached so that the original functions of the cells are not impaired.

[0003] Currently, as a cell culture technique in biochemistry, medicine, immunology, etc., a technique for non-invasively detaching cells without using the above-mentioned proteolytic enzymes or chemicals (enzyme / chemical-free) by controlling the adhesiveness and detachability of cells by temperature change has been put into practical use and is used in various bio-applications including the detachment of cell sheets. Patent Document 1 discloses a cell culture support material in which a cell-adhesive material containing a cell-adhesive natural substance and / or a cell-adhesive synthetic substance is coated as a first layer, and the surface is coated with a homopolymer or copolymer having a lower critical solution temperature for water in the range of 0 to 80°C as a second layer. As the material of the substrate coated with the cell-adhesive material, glass, modified glass, polystyrene, a polymer compound such as polymethyl methacrylate, ceramics, metal, etc. are disclosed.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Conventionally, as the substrate of a temperature-responsive culture support, those using a polymer compound are common. The specifications of a cell culture support using such a polymer compound are usually disposable and are discarded once used. Therefore, when producing a three-dimensional tissue from cells, etc., it is necessary to prepare cells, cell sheets, etc. using a very large number of disposable cell culture supports to produce a tissue, resulting in a high environmental load and high cost, making it difficult to put into practical use.

[0006] Furthermore, since the above polymer compound has a low thermal conductivity, when detaching cells using a temperature-responsive culture support made of the polymer compound as a material, it is difficult to efficiently change the temperature of the medium for detaching cells.

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

Means for Solving the Problems

[0008] <1>The culture substrate of the present invention is a culture substrate having a temperature-responsive polymer with a glass transition temperature exceeding 121°C on a metal substrate that has been subjected to oxygen plasma treatment and silanization treatment.

[0009] For such a culture substrate, since the temperature-responsive polymer appropriately exhibits temperature responsiveness even after autoclaving, it can be autoclaved and reused. Also, for such a culture substrate, the fixed temperature-responsive polymer is difficult to peel off, can be autoclaved and reused, and a large number of cells can be produced at low cost.

[0010] As shown in FIG. 1, a culture substrate with a temperature-responsive polymer modified on a metal substrate is such that after culturing cells at a temperature higher than the temperature at which the temperature-responsive polymer hydrates (for example, 37°C when using poly-N-isopropylacrylamide (PIPAAm)), the temperature-responsive polymer is lowered to a temperature lower than the temperature at which the temperature-responsive polymer dehydrates (for example, 20°C when using PIPAAm), and the cells or cell sheet can be detached and recovered without damaging their structure and function. It is a temperature-responsive culture substrate.

[0011] A temperature-responsive polymer is a polymer whose solubility in water changes dramatically with temperature changes. It dissolves in water at low temperatures, but when the temperature is raised to a certain temperature (lower critical solution temperature: LCST), it becomes insoluble, aggregates and precipitates, resulting in turbidity (Figure 2). This is because at temperatures lower than the LCST, the polymer chains are hydrated and stretched due to the strong interaction between the amide bond sites and water, taking on a random coil conformation. Conversely, at temperatures higher than the LCST, dehydration occurs, and the polymer chains aggregate into a globular state due to hydrophobic interactions (Figure 2).

[0012] As an example showing 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 due to temperature changes, Figure 2 shows the state changes of PIPAAm (which has both a hydrophilic part and a hydrophobic part in the side chain, and the LCST is 32 °C) due to temperature changes, and the behavioral changes of the cells cultured on the PIPAAm. When the temperature is higher than the LCST, the substrate surface becomes hydrophobic and cells can adhere. However, when the temperature is lower than the LCST, the substrate surface becomes hydrophilic and the surface becomes non-adhesive to cells, causing the cells adhered to the substrate surface to detach. Therefore, cells can be recovered only by changing the temperature (Figure 2).

[0013] In addition, by using a metal substrate as the substrate for modifying the temperature-responsive polymer, even under autoclave conditions (121 °C or higher, saturated steam conditions of 2 atmospheres) where the shape cannot be maintained with a normal temperature-responsive cell culture substrate, the culture substrate of the present invention will not deform. Therefore, it can be made into a reusable culture substrate.

[0014] Therefore, with the culture substrate of the present invention, it can be repeatedly used by autoclave treatment, has a low environmental impact, does not require enzyme or chemical treatment, non-invasively detaches cells without damaging the cells themselves and the proteins around the cells such as the extracellular matrix (ECM), and can recover a large number of cells at low cost. A temperature-responsive culture support that can efficiently control the temperature of the culture medium and the substrate can be provided. In addition, 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> It is preferable that the temperature-responsive polymer of the culture substrate of the present invention is poly-N-isopropylacrylamide (PIPAAm).

[0016] With such a culture substrate, since the LCST of PIPAAm is near the body temperature of animals, cell culture and recovery are easy.

[0017] <3> Further, it is preferable that the metal substrate of the culture substrate of the present invention is made of titanium.

[0018] With such a culture substrate, since titanium, which is lightweight, high-strength, and excellent in corrosion resistance, is used for the metal substrate, the culture operation is easy, ultrasonic cleaning can be performed, autoclave treatment can be carried out and it is easy to reuse, and a large number of cells can be produced at low cost.

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

[0020] With such a culture substrate, the metal substrate and the temperature-responsive polymer are likely to be bonded via a covalent bond, and the temperature-responsive polymer fixed on the metal substrate is difficult to peel off.

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

[0022] With such a culture substrate, it is easy to use for general cell culture.

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

[0024] With such a method for manufacturing a culture substrate, it can be repeatedly used by autoclaving, has a low environmental load, does not require enzyme or chemical treatment, can non-invasively detach cells without damaging the cells themselves and proteins around the cells such as the extracellular matrix (ECM), can recover a large number of cells at low cost, and can manufacture a temperature-responsive culture support that can efficiently control the temperature of the culture medium. In addition, since the temperature-responsive polymer is likely to be fixed onto the metal substrate via a covalent bond, the fixed temperature-responsive polymer is less likely to peel off, and a culture substrate that appropriately exhibits temperature responsiveness even after autoclaving can be provided.

[0025] <7>Moreover, in the method for manufacturing the culture substrate of the present invention, it is preferable that the temperature-responsive polymer is poly-N-isopropylacrylamide (PIPAAm).

[0026] With such a method for manufacturing a culture substrate, since the LCST of PIPAAm is near the body temperature of animals, a culture substrate that is easy to culture and recover cells can be manufactured.

[0027] <8>Moreover, in the method for manufacturing the culture substrate of the present invention, it is preferable that the metal substrate is made of titanium.

[0028] With such a method for manufacturing a culture substrate, since titanium, which is lightweight, high-strength, and excellent in corrosion resistance, is used for the metal substrate, a culture substrate that is easy to perform culture operations, can be ultrasonically cleaned, can be autoclaved and reused easily, and can manufacture a large number of cells at low cost can be manufactured.

[0029] <9>Moreover, the method for producing cells of the present invention 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 exceeding 121°C on a metal substrate that has been subjected to oxygen plasma treatment and silanization treatment to produce cells.

[0030] With such a method for producing cells, a large number of cells can be efficiently produced by efficiently controlling the temperature of the culture medium and the substrate. In addition, by subjecting the temperature-responsive culture support to autoclave treatment and repeatedly using it, the environmental load is small, enzyme and chemical treatment are unnecessary, and cells can be non-invasively detached without damaging the cells themselves and proteins around the cells such as the extracellular matrix (ECM), and a large number of cells can be produced at low cost. Moreover, in the culture substrate to be used, the fixed temperature-responsive polymer is difficult to peel off, and the culture substrate appropriately exhibits temperature responsiveness even after autoclave treatment, and a large number of cells can be produced at low cost. In addition, by rapidly changing the temperature of the metal substrate using a Peltier element or the like, it becomes possible to rapidly detach cells, and cells can be efficiently produced.

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

[0032] With such a method for producing cells, a cell sheet can be produced.

[0033] <11>Moreover, the method for producing cells of the present invention preferably includes a cell detachment step of changing the temperature to detach the cells from the temperature-responsive polymer after the cell production step.

[0034] With such a method for producing cells, cells can be recovered without the need for enzyme and chemical treatment and without damaging the cells themselves and proteins around the cells such as the extracellular matrix (ECM).

[0035] <12>Also, in the method for producing cells of the present invention, it is preferable that the temperature-responsive polymer is poly-N-isopropylacrylamide (PIPAAm).

[0036] In such a method for producing cells, since the LCST of PIPAAm is near the body temperature of animals, it is easy to culture and recover cells.

[0037] <13>Also, in the method for producing cells of the present invention, it is preferable that the metal substrate is made of titanium.

[0038] In such a method for producing cells, since titanium, which is lightweight, high-strength, and excellent in corrosion resistance, is used for the metal substrate of the culture substrate, the culture operation is easy, ultrasonic cleaning can be performed, autoclave treatment can be carried out and it is easy to reuse, so it is easy to produce a large amount of cells at low cost.

[0039] <14>Also, in the method for producing cells of the present invention, it is preferable that the temperature-responsive polymer is connected to the metal substrate via a silicon atom.

[0040] In such a method for producing cells, 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 difficult to peel off. Therefore, the culture substrate can be easily autoclaved and reused, and it is easy to produce a large amount of cells at low cost.

[0041] <15>Also, in the method for producing cells of the present invention, the culture substrate may have a substrate having a recess and a lid covering the substrate, and the temperature-responsive polymer may be provided on the bottom surface of the recess in the substrate.

[0042] In such a method for producing cells, general cell culture is easy.

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

[0044] In the case of such a culture substrate for repeated use, even after autoclaving, the temperature-responsive polymer appropriately exhibits temperature responsiveness, can be repeatedly used by autoclaving, has a low environmental impact, does not require enzyme or chemical treatment, and non-invasively detaches cells without damaging the cells themselves and proteins around the cells such as the extracellular matrix (ECM), and can recover a large number of cells at low cost, and can provide a temperature-responsive culture support capable of efficiently controlling the temperature of the culture medium.

[0045] <17> Further, in the culture substrate for repeated use of the present invention, it is preferable that the temperature-responsive polymer is poly-N-isopropylacrylamide (PIPAAm).

[0046] In the case of such a culture substrate for repeated use, since the LCST of PIPAAm is near the body temperature of animals, it is easy to culture and recover cells.

Advantages of the Invention

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

Brief Description of the Drawings

[0048]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5A

Figure 5B

Figure 6A

Figure 6B

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

BEST MODE FOR CARRYING OUT THE INVENTION

[0049] As a base material of a conventionally used temperature-responsive culture support, those using a polymer compound are common. The specifications of a cell culture support using such a polymer compound are usually disposable and discarded once used. Therefore, for example, when producing a three-dimensional tissue from cells, etc., it is necessary to use a very large number of disposable cell culture supports to produce cells or cell sheets that make up the tissue, etc., which results in high costs and is difficult to put into practical use. Also, it becomes a high environmental load. Furthermore, since the polymer compound has a low thermal conductivity, when using a temperature-responsive culture support in which the polymer compound is used, it is difficult to efficiently change the temperature of the culture medium or the base material when detaching cells.

[0050] In order to solve such problems, the present inventors have developed a culture substrate that can be reused by autoclaving even after use and can rapidly change the temperature of the culture medium or the base material with respect to an external temperature change, a method for manufacturing the same, a method for manufacturing 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 having a glass transition temperature exceeding 121°C on a metal substrate that has been subjected to oxygen plasma treatment and silanization treatment. The culture substrate of the present invention can be repeatedly used by autoclaving, has a low environmental load, does not require enzyme / chemical treatment, and can non-invasively detach cells without damaging cells themselves and proteins around cells such as extracellular matrix (ECM), and can recover a large number of cells at low cost, and can provide a temperature-responsive culture support capable of efficiently controlling the temperature of the culture medium.

[0052] The "temperature-responsive polymer" in the present invention is a polymer having a glass transition temperature exceeding 121°C. It is also a temperature-responsive polymer that does not denature under autoclave sterilization conditions. The temperature-responsive polymer is such that even if it is autoclaved while being fixed on a culture substrate, its temperature-responsive function is not impaired. If the culture substrate of the present invention does not have the temperature-responsive polymer, it is impossible to recover cells only by changing the temperature of the culture medium, and it becomes difficult to efficiently recover cells while retaining proteins around the cells such as extracellular matrix (ECM). Furthermore, it becomes difficult to sufficiently secure a cell scaffold (cell adhesion surface) during cell culture.

[0053] The substrate used for the culture substrate of the present invention is a "metal substrate" that has been subjected to oxygen plasma treatment and silanization treatment. The metal substrate that can be used in the present invention is not particularly limited as long as it can bind a temperature-responsive polymer, and an appropriate metal substrate can be used according to the purpose. If the substrate of the culture substrate is not one that does not deform at high temperatures such as metal, but is a polymer compound or the like used in a normal culture substrate, the culture substrate will be greatly deformed under autoclave sterilization conditions.

[0054] Examples of the shape of the culture substrate of the present invention include a dish shape and a film shape. When using a film-shaped substrate, after forming a temperature-responsive polymer layer on the surface of the film-shaped substrate, it can be processed into a shape suitable for cell culture (for example, a dish shape). During processing, a member made of another material can also be used in combination with the substrate as needed. When using a dish-shaped substrate, at least the inner bottom surface portion of the dish that serves as the cell adhesion surface may be coated with a temperature-responsive polymer.

[0055] In addition, as the culture substrate of the present invention, one having a substrate with a recess and a lid covering the substrate, and having the temperature-responsive polymer on the bottom surface of the recess in the substrate can be used. Further, a plate-shaped substrate to which a temperature-responsive polymer is bonded and provided with a wall made of a material such as metal, ceramics, glass, silicone such as polydimethylsiloxane (PDMS), etc. that can be used for the culture substrate of the present invention may be used.

[0056] <Autoclave sterilization conditions> The "autoclave treatment" means sterilization treatment by an autoclave that washes and / or sterilizes an object by increasing the internal pressure in heated water or in the atmosphere of water vapor generated from the heated water. In this specification, "under autoclave sterilization conditions" means "saturated steam conditions of 121°C or higher and 2 atmospheres". The autoclave treatment is generally carried out under the autoclave sterilization conditions for 15 to 20 minutes, but the sterilization time is not limited as long as sufficient sterilization can be achieved, and the sterilization time can be further increased or decreased according to the content and purpose of the object to be sterilized. .

[0057] <Temperature-responsive polymer> The temperature-responsive polymer of the culture substrate of the present invention is a temperature-responsive polymer having a glass transition temperature exceeding 121°C. The glass transition temperature of the temperature-responsive polymer of 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 "temperature responsiveness" of the "temperature-responsive polymer" means that the solubility in water changes dramatically with temperature changes, and it dissolves in water at low temperatures, but becomes insoluble and aggregates and precipitates when the temperature is raised 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 rapidly increases its viscosity in a certain temperature range due to a drop in temperature and almost loses its fluidity and changes 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 autoclave treatment. Therefore, after the autoclave treatment of the culture substrate, the temperature-responsive polymer on the metal substrate may fail to appropriately exhibit temperature responsiveness.

[0059] <<Measurement of Glass Transition Temperature (Tg) of Polymer>> The glass transition temperature of the polymer can be measured by differential scanning calorimetry (DSC). As a method for measuring the glass transition temperature of the polymer, for example, it may be determined under the following measurement conditions. When measuring a highly hygroscopic sample, in order to remove excess moisture, heat treatment may be performed on the measurement sample in the DSC apparatus or the like before measurement, and the temperature of the heat treatment may be 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 Heat 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: Standard aluminum container

[0061] In the glass transition signal of the DSC curve of the measurement sample, when a large endotherm derived from enthalpy relaxation (volume relaxation) overlaps, assuming a baseline for the curve showing the glass transition signal of the DSC curve, the glass transition temperature of the measurement sample can be obtained by 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, at the time of cell recovery after culture, changes the hydrophobic part to hydrophilic and separates the cultured cells from the cell culture substrate, so as to make the cell recovery easier. It preferably shows hydrophobicity at the cell culture temperature (usually about 37°C) and hydrophilicity at the temperature at the time of recovery of the cultured cells. Poly-N-isopropylacrylamide is particularly preferred. The temperature at which the temperature-responsive polymer changes from hydrophobic to hydrophilic (lower critical solution temperature (T) with respect to water) is not particularly limited, but from the viewpoint of ease of cell recovery after culture, it is preferably a temperature lower than the cell culture temperature. In particular, a temperature-responsive polymer that shows hydrophilicity at a temperature lower than a predetermined lower critical solution temperature and shows hydrophobicity at a temperature equal to or higher than the same temperature is preferred. The critical solution temperature in such a temperature-responsive polymer is particularly called the lower critical solution temperature (LCST).

[0063] As the temperature-responsive polymer in the present invention, those having a lower critical solution temperature LCST of 0 to 80°C are preferred, and polymers having an LCST of 0 to 50°C are more preferred. If the LCST is 0°C or higher, generally the cell growth rate extremely decreases or the cells are not easily killed. If the LCST is 80°C or lower, the cells are not easily killed.

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

[0065] Examples of the 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, or vinyl ether derivatives. One or more of these can be used. When a single type of monomer is used alone, the polymer formed on the substrate is a homopolymer. When a plurality of types of monomers are used together, the polymer formed on the substrate is a copolymer. Both forms are included in the present invention. In the case of a copolymer, any two or more of these can be used.

[0066] Also, 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 hydrophilic-hydrophobic balance of the cell support, other monomers other than the above may be further added and copolymerized. Furthermore, a graft, copolymer, or block copolymer of the above polymer used in the present invention and other polymers, or a mixture of the polymer of the present invention and other polymers or copolymers may be used. Also, it is possible to crosslink within a range where the original properties of the polymer are not impaired. At that time, since the material to be cultured and peeled is a cell, separation is performed in the range of 5°C to 50°C. Therefore, as temperature-responsive polymers, for example, poly-N-n-propylacrylamide (LCST of the homopolymer is 21°C), poly-N-n-propylmethacrylamide (27°C), poly-N-isopropylacrylamide (32°C), poly-N-isopropylmethacrylamide (43°C), poly-N-cyclopropylacrylamide (45°C), poly-N-ethoxyethylacrylamide (about 35°C), poly-N-ethoxyethylmethacrylamide (about 45°C), poly-N-tetrahydrofurfurylacrylamide (about 28°C), poly-N-tetrahydrofurfurylmethacrylamide (about 35°C), poly-N,N-ethylmethylacrylamide (56°C), poly-N,N-diethylacrylamide (32°C), etc. can be mentioned.

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

[0068] <<Use of a Temperature-Responsive Polymer for a Reusable Culture Substrate>> The temperature-responsive polymer having a glass transition temperature of more than 121°C used in the present invention can be used for a substrate for repeated use. This is because a reusable culture substrate can be provided by binding the temperature-responsive polymer to a substrate that can be repeatedly used. The temperature-responsive polymer in the reusable 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 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. Also, the temperature-responsive polymer in the reusable culture substrate of the present invention is preferably poly-N-isopropylacrylamide (PIPAAm). Since the LCST of PIPAAm is near the body temperature of animals, by applying PIPAAm to the temperature-responsive polymer in the reusable substrate of the present invention, cell culture is easy, and it is easy to recover a large number of cells at low cost. In addition, the material of the substrate used in the reusable culture substrate of the present invention is not limited as long as it can be autoclaved and repeatedly used, and any material can be used as long as it meets the purpose of the present invention. However, from the viewpoint of repeatedly using it as a culture substrate, it is preferably one that does not deform even after autoclaving. For example, substrates using silicone such as metal, ceramics, glass, and polydimethylsiloxane (PDMS) can be mentioned, and a metal substrate is more preferable.

[0069] <Metal Substrate> The substrate used in the culture substrate of the present invention is a metal substrate, and the metal substrate that can be used in the present invention is not particularly limited as long as it can bind a temperature-responsive polymer, and an appropriate metal substrate can be used according to the purpose. Further, it is not particularly limited as long as it contains a material into which the temperature-responsive polymer can be introduced onto its surface via a covalent bond by electron beam irradiation, radiation irradiation, etc. Only the surface may contain the temperature-responsive polymer and a material that can form a covalent bond by electron beam irradiation, radiation irradiation, etc., or the entire substrate may contain such a material. The metal substrate used in the culture substrate of the present invention can express hydrophilic reactive groups on its surface by oxygen plasma treatment. Examples of the reactive groups expressed by the 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. In this bonding treatment, it is preferable to use a silane agent (silanization treatment). In the present invention, the metal substrate and the temperature-responsive polymer are preferably connected via a silicon atom, and it is preferable that a silicon atom is contained in the bonding portion 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 silanization treatment using a silane agent after oxygen plasma treatment. With such a metal substrate, the temperature-responsive polymer can 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 enables cell culture and can bind to the temperature-responsive polymer via a covalent bond. However, if it can form an oxide film on the surface, since hydroxyl groups serving as reactive groups can be expressed by plasma treatment or the like in the process of fixing the temperature-responsive polymer, it is preferably a metal that can form an oxide film on the 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 containing titanium. As the metal used for the metal substrate in the present invention, for example, pure titanium and titanium alloys are preferable, titanium-based alloys are more preferable, and alloys of titanium, aluminum, and vanadium are even more preferable. Examples of titanium-based 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), Ti-15Mo (Ti15Mo), and the like. Since titanium is excellent in light weight, high strength, and corrosion resistance, the culture substrate of the present invention made of titanium is easy to handle in culture operations, can be ultrasonically cleaned, and is easy to reuse after autoclave treatment. In addition, it can be used as a metal-based biomaterial and is preferable because it does not show toxicity 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 alloy, pure iron, steel, cast iron, nickel alloy, gold, platinum, pure aluminum, aluminum alloy, pure copper, copper alloy (bronze, cupronickel, phosphor bronze, brass), pure cobalt, cobalt alloy, magnesium alloy, silver, tungsten, zinc, zinc alloy, lead, tin, pure tantalum, tantalum alloy, pure niobium, niobium alloy, molybdenum alloy, zirconium (alloy), gold-silver-palladium alloy, and the like. Stainless steel and cobalt-chromium alloy can be used as metal-based biomaterials and are preferable as the metals used in the present invention because they do not show toxicity to cells.

[0073] In addition, as long as the object of the present invention is not hindered, an arbitrary layer may be provided on the surface or intermediate layer of the metal substrate in the present invention, or an arbitrary treatment may be performed. For example, hydrophilic treatment can be performed on the surface of the metal substrate using treatment techniques such as ozone treatment, plasma treatment, and sputtering.

[0074] The culture substrate of the present invention may further have a cell-adhesive substance containing a cell-adhesive natural substance and / or a cell-adhesive synthetic substance. The cell-adhesive substance may be between the temperature-responsive polymer layer and the metal substrate as long as the metal substrate and the temperature-responsive polymer can be sufficiently bonded, or may be on the metal substrate to which the temperature-responsive polymer is bonded. However, from the viewpoint of making it easier to bond when bonding the temperature-responsive polymer to the metal substrate, it is preferably on the metal substrate to which the temperature-responsive polymer is bonded.

[0075] The cell-adhesive substance that can be used for the culture substrate of the present invention is not limited as long as it has affinity / adhesiveness with cells, and any substance can be used as long as it is suitable for the purpose. Examples of the cell-adhesive natural substance include, for example, oligosaccharides, gelatin, collagen, fibronectin, laminin, fibrin, and cell adhesion peptides which are their components. The cell-adhesive synthetic substance can be obtained by homopolymerizing a monomer containing a cell-adhesive group, copolymerizing monomers containing a cell-adhesive group with each other, or copolymerizing a monomer containing a cell-adhesive group and a monomer not containing a cell-adhesive group. Examples of the cell-adhesive group include a carboxylic acid group and its salts, anhydrides, sulfonic acid groups and their salts, sulfonic acid esters, sulfonic acid amides, 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, or phosphobetaine groups and sulfobetaine groups in which these cell-adhesives are combined and contained in the same monomer. In the present invention, these cell-adhesive natural substances or cell-adhesive synthetic substances can be used alone or in combination.

[0076] (Method for manufacturing a culture substrate) The method for manufacturing the culture substrate of the present invention includes a silane agent treatment step of treating the surface of a metal substrate treated with oxygen plasma with a silane agent, and a temperature-responsive polymer binding step of binding a temperature-responsive polymer having a glass transition temperature exceeding 121°C onto the metal substrate. In the method for manufacturing the culture substrate of the present invention, if the step of treating the surface of the metal substrate with a silane agent is not included, in the subsequent temperature-responsive polymer binding step, it becomes difficult to bond the metal substrate and the temperature-responsive polymer via a covalent bond, making it difficult to fix the temperature-responsive polymer on the metal substrate, and there is a risk that it will be easily peeled off after fixation. As a result, the number of times the culture substrate can be autoclaved and reused decreases, and the amount of cultured cells that can be recovered at low cost and non-invasively may decrease.

[0077] In the temperature-responsive polymer binding step, when binding a temperature-responsive polymer onto a metal substrate using monomer units of the temperature-responsive polymer, the temperature-responsive polymer may be provided in any state of gas, liquid, or solid, but it is preferable to provide a solution of the monomer units 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 onto the metal substrate in the temperature-responsive polymer binding step is preferably 5 to 65% by weight, more preferably 10 to 55% by weight, and even more preferably 30 to 50% by weight. 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. When the concentration of the solution of the monomer units of the temperature-responsive polymer is 5% by weight or more, cells are easily detached when the temperature of the medium is changed for cell detachment. When it is 65% by weight or less, the adhesiveness of the cultured cells to the bottom surface of the culture substrate is good. From the viewpoint of efficiency in cell culture, the concentration of the solution of the monomer units of the temperature-responsive polymer is particularly preferably 30 to 50% by weight.

[0079] The temperature-responsive polymer used in the present invention is a polymer having a glass transition temperature of more 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 autoclave treatment. Therefore, after the autoclave treatment of the culture substrate, the temperature-responsive polymer on the metal substrate may fail to appropriately exhibit temperature responsiveness. 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 the culture substrate of the present invention is preferably poly-N-isopropylacrylamide (PIPAAm). Since the LCST of PIPAAm is near the body temperature of animals, a culture substrate suitable for cell culture and recovery can be produced.

[0080] The metal substrate of the culture substrate used in the method for producing the culture substrate of the present invention is preferably made of titanium. Since titanium is a metal with excellent light weight, high strength, and corrosion resistance, the culture operation is easy, ultrasonic cleaning can be performed, and autoclave treatment and reuse are easy. Therefore, a culture substrate capable of producing a large amount of cells at low cost can be produced.

[0081] Hereinafter, an example of the method for producing the culture substrate of the present invention will be specifically described with reference to FIG. 3. As an example, the present invention is mainly produced by the following steps [1] to [5]. [1] A hydrophilic treatment is performed on the surface of the polished substrate to express hydrophilic reactive groups on the substrate surface (in FIG. 3, the surface of the Ti6Al4V substrate is treated with oxygen plasma to express hydroxyl groups). [2] A surface treatment is performed with a silane agent (3-(methacryloyloxy)propyltrimethoxysilane in FIG. 3) under appropriate temperature and time conditions under vacuum (silane agent treatment step). [3] The surface treated with the silane agent is washed and dried under vacuum conditions. [4] After cooling the substrate treated with the silane agent at room temperature (about 25°C), a monomer unit solution of the temperature-responsive polymer is dropped onto the sample. After the solution spreads on the surface, the substrate and the temperature-responsive polymer are bonded by a chemical reaction (in FIG. 3, irradiated with an electron beam for a certain period of time in a nitrogen atmosphere) (temperature-responsive polymer bonding step). [5] After bonding the substrate and the temperature-responsive polymer, the substrate is left standing in cold water, washed, and dried, whereby a substrate surface to which the target temperature-responsive polymer is bonded is obtained.

[0082] Regarding the substrate, metal substrate, and hydrophilic treatment that can be used in the above [1], they are as described above. Note that as long as the object of the present invention is not hindered, the surface of the substrate used in the above [1] may be subjected to blasting treatment, microfabrication, etc. in addition to polishing. By performing microfabrication to change the surface properties, the orientation of the seeded cells can be controlled.

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

[0084] In addition, as the silane agent that can be used in the present invention, specifically, for example, vinyltrimethoxysilane, vinyltriethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-(methacryloyloxy)propyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, N-2-(aminoethyl)-3-aminopropylmethyldimethoxysilane, N-2-(aminoethyl)-3-aminopropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-triethoxysilyl-N-(1,3-dimethyl-butylidene)propylamine, N-phenyl-3-aminopropyltrimethoxysilane, hydrochloride of N-(vinylbenzyl)-2-aminoethyl-3-aminopropyltrimethoxysilane, tris-(trimethoxysilylpropyl)isocyanurate, 3-ureidopropyltrialkoxysilane, 3-mercaptopropylmethyldimethoxysilane, 3-mercaptopropyltrimethoxysilane, 3-isocyanatopropyltriethoxysilane, 3-trimethoxysilylpropylsuccinic anhydride and other compounds can be mentioned. From the viewpoint of immobilizing the polymer, it is preferable to use vinyltrimethoxysilane, vinyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-(methacryloyloxy)propyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-mercaptopropylmethyldimethoxysilane, 3-mercaptopropyltrimethoxysilane.

[0085] When bonding monomer units of a temperature-responsive polymer to the substrate surface, the monomer may be in any state of gas, liquid, or solid. Also, when coating with a homopolymer or copolymer, the polymer may be in either a solution state or a solid state. When bonding these by a chemical reaction, electron beam irradiation (EB), γ-ray irradiation, ultraviolet irradiation, plasma treatment, corona treatment, and when having appropriate reactive functional groups, general organic reactions such as radical reaction, anion reaction, cation reaction, etc. can be used, but it is preferably carried out by electron beam irradiation (EB). In addition, in FIG. 3, the bonding part between the substrate surface and the silane agent is illustrated. Regarding the bonding of Si in this bonding part, it is shown as -O-Si-O(CH2)3-. However, since Si usually has four bonding hands, Si has a bonding that is not illustrated in FIG. 3, but is omitted in FIG. 3 for the convenience of notation.

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

[0087] The bonding 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 by X-ray photoelectron spectroscopy (XPS). In the XPS analysis of the surface of the substrate after immobilization of the temperature-responsive polymer, the C1s spectrum was waveform-separated, and a signal derived from the temperature-responsive polymer (in Fig. 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 Fig. 4)) was confirmed. Thus, it was confirmed that the temperature-responsive polymer was chemically bonded to the metal substrate surface (Fig. 4). Also, the N element derived from the temperature-responsive polymer, which could not be confirmed on the surface of the metal substrate before immobilization of the temperature-responsive polymer, was confirmed on the metal substrate on which the temperature-responsive polymer was immobilized, thereby confirming that the gel layer of the temperature-responsive polymer was immobilized.

[0088] Also, water was dropped onto the surfaces of the metal substrates before and after immobilization of the temperature-responsive polymer, the temperature was changed, the contact angles before and after the temperature change were measured and compared, and the presence or absence of a change in the contact angle of water was confirmed. Thus, it was confirmed that the surface of the metal substrate on which the temperature-responsive polymer was immobilized was a surface with changing hydrophobicity and hydrophilicity (Fig. 5). It shows that the larger the contact angle, the more hydrophobic, and the smaller the contact angle, the more hydrophilic the property is.

[0089] Also, in the same manner as above, when the surface of the temperature-responsive polymer-bonded metal substrate was subjected to XPS analysis after autoclave treatment under autoclave sterilization conditions, carbon atoms and nitrogen atoms derived from the temperature-responsive polymer were confirmed. Thus, even after autoclave treatment, it was confirmed that the culture substrate of the present invention chemically bonded and immobilized the temperature-responsive polymer on the metal substrate.

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

[0091] The thickness of the gel containing the temperature-responsive polymer on the substrate surface to which the temperature-responsive polymer obtained after drying in the above [5] is bound 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 culture medium is changed for cell detachment. If it is 3 nm, 4 nm or more, this tendency becomes stronger. If the thickness of the gel containing the temperature-responsive polymer is 40 nm or less, the adhesion of the cultured cells to the bottom surface of the culture substrate is good. If it is 35 nm, 20 nm or less, this tendency becomes 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] In the method for producing the culture substrate of the present invention, a step of adding a cell-adhesive substance containing a cell-adhesive natural substance and / or a cell-adhesive synthetic substance can be included. By adding a solution obtained by diluting the cell-adhesive substance at an appropriate ratio to the surface of the metal substrate after immobilizing the temperature-responsive polymer, allowing it to stand for 1 to 10 minutes, and then washing it 1 to 3 times with water or a buffer solution, in the present invention, a culture substrate having a cell-adhesive substance can be further obtained. The cell-adhesive substance containing a cell-adhesive natural substance and / or a cell-adhesive synthetic substance that can be used in this step is as described above.

[0093] Regarding the application of the cell adhesion substance to the culture substrate of the present invention, it may be applied in the manufacturing process of the culture substrate of the present invention before autoclaving, or the cell adhesion substance may be applied to the culture substrate of the present invention after autoclaving.

[0094] (Method for manufacturing cells) The method for manufacturing cells of the present invention includes a cell manufacturing step of seeding and culturing cells on the surface of a culture substrate having a temperature-responsive polymer with a glass transition temperature exceeding 121°C on a metal substrate that has been subjected to oxygen plasma treatment and silanization treatment to manufacture cells. In cell manufacturing, when a large number of cells are non-invasively recovered without subjecting the cells themselves and proteins around the cells such as extracellular matrix (ECM) to enzyme or chemical treatment so as not to damage them, if a culture substrate having a temperature-responsive polymer with a glass transition temperature exceeding 121°C on a metal substrate is not used, it is difficult to efficiently control the temperature of the culture substrate, the solutions (culture medium, PBS, physiological saline, serum, etc.) used for cell culture, the temperature-responsive polymer, and the cells to be recovered, and it is also difficult to reuse them after autoclaving. Therefore, a large amount of disposable culture substrates need to be used, resulting in a high environmental burden and making it difficult to efficiently manufacture a large number of cells at low cost. Further, in the method for manufacturing cells of the present invention, it is preferable to further include a cell detachment step of changing the temperature to detach the cells from the temperature-responsive polymer after the cell manufacturing step. When using the method for manufacturing cells of the present invention, since the culture substrate used has a metal substrate, the temperature of the culture medium and the substrate can be efficiently changed, so that the cells can be quickly detached, and more cells can be manufactured more efficiently. Also, by using a Peltier element or the like, the temperature of the metal substrate can be changed more quickly, so that cells can be manufactured more efficiently.

[0095] The temperature-responsive polymer used in the present invention is a polymer with a glass transition temperature exceeding 121°C. If the glass transition temperature of the temperature-responsive polymer used in the present invention is 121°C or lower, there is a risk that the polymer may be denatured by autoclaving, resulting in a possible loss of proper temperature responsiveness. 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 cells of the present invention is preferably poly-N-isopropylacrylamide (PIPAAm). Since the LCST of PIPAAm is near the body temperature of animals, it is suitable for cell culture and recovery.

[0096] The metal substrate of the culture substrate used in the method for producing cells of the present invention is preferably made of titanium. Since titanium is a metal with excellent light weight, high strength, and corrosion resistance, it is easy to perform culture operations, can be ultrasonically cleaned, and can be autoclaved and reused easily, so it is easy to produce a large number of cells at low cost.

[0097] In addition, the surface of the metal substrate of the culture substrate used in the method for producing cells of the present invention is a surface treated with oxygen plasma and a silane agent. By subjecting the surface of the metal substrate to oxygen plasma treatment, it becomes easier to introduce the temperature-responsive polymer onto the surface of the metal substrate via a covalent bond. When the surface of the metal substrate is treated with a silane agent, it is easy to immobilize the temperature-responsive polymer by covalent bonds on the metal substrate of the culture substrate used for cell production, and the immobilized temperature-responsive polymer is less likely to peel off, so the culture substrate can be autoclaved and reused easily, and it is easy to produce a large number of cells at low cost.

[0098] The temperature-responsive polymer is preferably connected to the metal substrate via a silicon atom. In the culture substrate used for cell production, when the temperature-responsive polymer and the metal substrate are joined via a silicon atom, it becomes easier to have a covalent bond in the bond between the metal substrate and the temperature-responsive polymer, and the temperature-responsive polymer fixed on the metal substrate is less likely to peel off. Therefore, the culture substrate can be autoclaved and reused easily, and it becomes easier to produce a large number of cells at low cost.

[0099] In the method for producing cells of the present invention, the culture substrate may have a base body having a recess and a lid portion covering the base body, and the temperature-responsive polymer may be provided on the bottom surface of the recess in the base body. When such a culture substrate is used for cell production, general cell culture becomes easier.

[0100] As an example of the method for producing cells of the present invention, taking poly-N-isopropylacrylamide (PIPAAm) as an example of the temperature-responsive polymer, FIGS. 1 and 2 will be used for explanation. PIPAAm is known as a polymer having a lower critical solution temperature of 32°C. In the free state, dehydration occurs in water at a temperature higher than 32°C, causing the polymer chains to aggregate and precipitate, resulting in turbidity. Conversely, at a temperature lower than 32°C, the polymer chains are hydrated and dissolved in water (upper part of FIG. 2). The culture substrate used in the present invention has a temperature-responsive polymer with a glass transition temperature exceeding 121°C bonded and fixed to the surface of a culture substrate having a metal substrate treated with oxygen plasma and silanization treatment (FIG. 1). Therefore, at a temperature higher than 32°C, the polymer on the substrate surface also undergoes dehydration in the same manner. However, since the polymer chains are coated and fixed on the substrate surface, the substrate surface becomes hydrophobic (lower right part of FIG. 2). Conversely, at a temperature lower than 32°C, the polymer on the substrate surface is hydrated with the water contained in the culture medium. However, since the polymer chains are coated and fixed on the substrate surface, the substrate surface becomes hydrophilic (lower left part of FIG. 2). The hydrophobic surface at this time is an appropriate surface on which cells can adhere and proliferate. Also, the hydrophilic surface becomes a surface on which cells cannot adhere, and the cells and cell sheets during culture can be detached simply by cooling (FIG. 1).

[0101] Hereinafter, the method for producing cells of the present invention will be described more specifically. Note that the temperature-responsive polymer and metal substrate that can be used for the culture substrate in the present invention are as described above.

[0102] <Cells> The cells used in the present invention are not particularly limited in terms of cell type, and cells at 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 evaluate myocardial function, the cells to be used include any one of cardiomyocytes, cardiomyoblasts, myoblasts, mesenchymal stem cells, vascular endothelial cells, vascular endothelial progenitor cells, fibroblasts, bone marrow-derived cells, and adipose-derived cells, or a mixture of two or more types of cells, etc., and the type thereof is not limited at all.

[0103] When the purpose is to regenerate liver tissue, produce an artificial liver simulating liver tissue, or evaluate liver tissue function, etc., for example, the cells to be used include any one of hepatocytes, 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, or a mixture of two or more types of cells, etc., and the type thereof is not limited at all.

[0104] When the purpose is to regenerate kidney tissue, produce an artificial kidney simulating kidney tissue, or evaluate kidney function, for example, the cells to be used include any one of renal 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, or a mixture of two or more types of cells, etc., and the type thereof is not limited at all.

[0105] When the purpose is to regenerate adrenal tissue, produce an artificial adrenal simulating the adrenal gland, or evaluate adrenal function, for example, the cells to be used include any one of adrenal medulla cells, adrenal cortex cells, zona glomerulosa cells, zona fasciculata cells, zona reticularis 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, etc., and the type thereof is not limited at all.

[0106] When the purpose is to evaluate a method for skin regeneration or skin function, for example, as the cells to be used, any one type of 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, mesenchymal stem cells, or a mixture of two or more types of cells, etc. can be mentioned, and the type thereof is not limited at all.

[0107] When the purpose is to evaluate a method for mucosal tissue regeneration or mucosal tissue function, for example, as the cells to be used, cells collected from the tissue constituting the mucosa may be used. Examples of the types of mucosa include buccal mucosa, gastric mucosa, intestinal mucosa, olfactory epithelium, oral mucosa, uterine mucosa, etc. Among the cells collected from the mucosal tissue, any one type or a mixture of two or more types of cells, etc. can be mentioned, and the type thereof is not limited at all.

[0108] The content ratio of the above cells is not particularly limited either. In that case, if vascular endothelial cells, vascular endothelial progenitor cells, etc. are mixed in the cell group, the construction of a vascular network within the cell group is efficiently carried out, which is convenient.

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

[0110] The cells used in the present invention are not particularly limited. For example, the cells may be those fluorescently stained and / or pigment-stained by at least one method such as a reagent, protein, gene, etc. When using cells into which a reporter gene has been introduced, if the fluorescence by the reporter protein produced by the expression of the reporter gene or the fluorescence emitted when the reporter protein reacts with a specific substrate is detected, the activity of the cells, cell sheet, or cell sheet laminate can be known. 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, alkaline phosphatase, etc.

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

[0112] The promoter sequence for regulating the expression of the reporter gene is not particularly limited either, and it may be appropriately selected according to the purpose of detecting reporter gene expression.

[0113] The culture conditions other than temperature may follow a conventional method and are not particularly restricted. For example, regarding the medium to be used, a medium supplemented with a known serum such as fetal bovine serum (FCS) may be used, or a serum-free medium without such serum added may also be used.

[0114] In the method for producing the cells 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 the tissue from which they are derived. Generally, it is 1×10 4 ~10×10 4 cell / cm 2is preferred, 2×10 4 ~8×10 4 cells / cm 2 is more preferred, 3×10 4 ~5×10 4 cells / cm 2 is even more preferred. If the seeding density is 1×10 4 cells / cm 2 or more, generally, cell growth is easy, and the degree of expression of the functions of the obtained cells is not likely to deteriorate. If it is 10×10 4 cells / cm 2 or less, it is preferred because the cells have not reached confluence yet.

[0115] In the present invention, in order to detach and recover the cultured cells from the culture substrate, the temperature of the culture substrate to which the cultured cells are attached can be made lower than the lower critical solution temperature of the temperature-responsive polymer on the metal substrate of the culture substrate, thereby detaching the cells. At this time, it can be carried out in a culture medium or in other isotonic solutions, and can be selected according to the purpose.

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

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

[0118] The cells produced in the present invention are not damaged by proteolytic enzymes and chemical agents typified by dispase, trypsin, etc. when the cultured cells are recovered. Therefore, the cells detached from the substrate have adhesive proteins. As a result, when the cultured cells are applied to a tissue, they can adhere well and can be efficiently engrafted.

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

[0120] For example, dispase, which is a proteolytic enzyme, is known to be able to detach while retaining 10 to 40% of the desmosome structure between cells. However, since it almost destroys the basement membrane-like proteins between cells and the substrate, generally, cell sheets obtained using proteolytic enzymes and chemical agents are weak in strength. On the other hand, the cell sheet produced by the present invention has less destruction of basement membrane-like proteins, desmosome structures, and basement membrane-like proteins between cells and the substrate. When the cultured cell sheet is detached, the desmosome structure between cells can be retained to some extent. Therefore, the cell sheet detached from the substrate has adhesive proteins, and when the cultured cells are applied to a tissue, they can adhere well and can be efficiently engrafted.

[0121] Also, 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. As those materials, usually, materials such as polyvinylidene difluoride (PVDF), silicone, polyvinyl alcohol, urethane, cellulose and its derivatives, chitin, chitosan, collagen, gelatin, and fibrin glue are used, and these materials may be used in contact with the cell sheet in the form of a film, porous film, non-woven fabric, or woven fabric.

[0122] <Stacked Cell Sheets> The method for producing the stacked cell sheets in the present invention is not particularly limited. For example, it can be obtained by detaching cultured cells in the form of a sheet and laminating the cultured cell sheets with each other using a cultured cell transfer jig as necessary. At that time, the temperature of the culture medium is not particularly limited as long as it is higher than the lower critical solution temperature of the temperature-responsive polymer used for the culture substrate.

[0123] Thus, according to the present invention, a thick cell sheet laminate can also be obtained. The lamination method is not particularly limited, but it is preferable to laminate the cell sheets in multiple layers in a plurality of times rather than laminating the cell sheets at once. Also, the number of lamination times may be appropriately adjusted according to the purpose for which the cell sheet laminate is used and is not particularly limited, but preferably 5 layers or more, more preferably 10 layers or more, and even more preferably 15 layers or more. If the thickness of the cell sheet laminate increases, the effects of the present invention can be significantly achieved, and a large number of cells can also be transplanted at the transplantation site, which is convenient.

Examples

[0124] Hereinafter, examples of the present invention will be described, but the present invention is not limited to these examples. Unless otherwise specified, various preparations and evaluations were carried out under the 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 (Differential Scanning Calorimeter: DSC). Since the above measurement sample had high hygroscopicity, the measurement sample was heat-treated in the DSC apparatus at room temperature to 120 ° C before measurement to remove excess moisture, and then the glass transition temperature was measured. In addition, when a large endotherm derived from enthalpy relaxation (volume relaxation) overlaps in the glass transition signal of the DSC curve of the measurement sample, a baseline was assumed for the curve showing the glass transition signal of the DSC curve, and the central temperature of the stepped signal was read and taken as the glass transition temperature of each sample. The measurement method and measurement 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 heat 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: Standard aluminum container

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

[0128] <Changes in the polymer after autoclave treatment> Approximately 5 mg of poly-N-isopropylacrylamide (PIPAAm) was placed into a glass container, and after covering the mouth of the container with aluminum foil, it was subjected to autoclave treatment (121 °C, 2 atm, 15 minutes). After the temperature had sufficiently dropped, each glass container was taken out and each polymer was observed. No change in appearance was observed in poly-N-isopropylacrylamide (PIPAAm) before and after autoclave treatment.

[0129] <Manufacture of the culture substrate> As the titanium metal substrate used for the culture substrate, a round bar with a diameter of 24 mm (manufactured by Daido Steel Co., Ltd.) produced by rolling was cut to a thickness of 4 mm. After polishing the cut surface with emery paper, it was processed into a mirror-like disk-shaped Ti6Al4V (64 titanium: φ24 mm) by buff polishing using a colloidal silica suspension and then used. On the surface of the above disk-shaped Ti6Al4V, oxygen plasma treatment was performed for 3 minutes with an energy of 400 W in an environment where the mixed gas of oxygen and argon was 100 mTorr to hydrophilize the surface of the disk-shaped Ti6Al4V. After that, 3-(methacryloyloxy)propyltrimethoxysilane (3 ml) (manufactured by Tokyo Chemical Industry Co., Ltd.) was used as the silane agent and added to a separable flask. The disk-shaped Ti6Al4V with a hydrophilized surface was placed in the separable flask added with the above silane agent so as not to touch the silane agent solution as much as possible. The separable flask was evacuated and left standing at 70 °C for 3 hours to treat the surface of the hydrophilized disk-shaped Ti6Al4V with the silane agent (Figure 3).

[0130] The silanized surface of the obtained disk-shaped Ti6Al4V was washed in the order of toluene, methanol, and ultrapure water, and then dried under vacuum conditions at 160 °C. After drying, the silanized disk-shaped Ti6Al4V was allowed to stand at room temperature to cool. As monomers of the temperature-responsive polymer, purified N-isopropylacrylamide (manufactured by KJ Chemicals Co., Ltd.) was used to prepare 10 wt%, 30 wt%, 40 wt%, and 50 wt% N-isopropylacrylamide solutions dissolved in 2-propanol. The 10 wt% N-isopropylacrylamide solution was prepared such that 2-propanol was 90 wt%, the 20 wt% N-isopropylacrylamide solution was prepared such that 2-propanol was 80 wt%, the 30 wt% N-isopropylacrylamide solution was prepared such that the 2-propanol concentration was 70 wt%, the 40 wt% N-isopropylacrylamide solution was prepared such that 2-propanol was 60 wt%, and the 50 wt% N-isopropylacrylamide solution was prepared such that 2-propanol was 50 wt%. 20 μl of each concentration of the N-isopropylacrylamide solution was dropped onto the surface of a separate silanized disk-shaped Ti6Al4V.

[0131] After the N-isopropylacrylamide solution spread on the surface of the above-mentioned silanized disk-shaped Ti6Al4V, an electron beam (0.25 MGy) was irradiated onto the surface of Ti6Al4V added with each concentration of the N-isopropylacrylamide solution in a nitrogen atmosphere using an electron beam irradiation device (manufactured by NHV Corporation: Curetron).

[0132] After electron beam irradiation, the disk-shaped Ti6Al4V was placed in cold water and allowed to stand, washed with ultrapure water, and dried at 45 °C to obtain a culture substrate with poly(N-isopropylacrylamide) (PIPAAm) 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 are denoted as "10PIPAAm-Ti6Al4V", "30PIPAAm-Ti6Al4V", "40PIPAAm-Ti6Al4V", and "50PIPAAm-Ti6Al4V". The culture substrates prepared in Example 1 with "10PIPAAm-Ti6Al4V", Example 2 with "30PIPAAm-Ti6Al4V", Example 3 with "40PIPAAm-Ti6Al4V", and Example 4 with "50PIPAAm-Ti6Al4V" were used.

[0134] <Surface Analysis of Temperature-Responsive Polymer-Bonded Metal Substrate> To confirm whether PIPAAm, a temperature-responsive polymer, is bonded to Ti6Al4V, a metal substrate, 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 waveform-separated in the above surface analysis by X-ray photoelectron spectroscopy, a signal (peak IV in Figure 4) derived from the carbonyl group of PIPAAm (the carbon atom of IV in the chemical structural formula of Figure 4) was confirmed. This indicates that PIPAAm is bonded to the Ti6Al4V surface via a covalent bond and is chemically immobilized.

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

[0136] <Changes in commercially available temperature-responsive cell culture substrates before and after autoclave (AC) treatment> A commercially available temperature-responsive cell culture substrate (manufactured by CellSeed Inc., registered trademark: UpCell) and 10PIPAAm-Ti6Al4V prepared in Example 1 were autoclaved (121 °C, 2 atm, 15 minutes). After autoclaving, the commercially available temperature-responsive cell culture substrate (registered trademark: UpCell) was deformed and turbid (Fig. 6B), while 10PIPAAm-Ti6Al4V maintained its original shape without deformation (Fig. 6A).

[0137] <Changes in the elemental composition of the temperature-responsive polymer-bonded metal substrate surface before and after autoclave treatment> The surfaces of Ti6Al4V before the binding treatment of PIPAAm 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 the state without autoclave treatment, N element could not be confirmed on the surface of Ti6Al4V before the binding treatment of PIPAAm (Comparative Example 2), but N atoms derived from PIPAAm could be confirmed on 10PIPAAm-Ti6Al4V with bonded PIPAAm, and it was confirmed that PIPAAm was bound and immobilized on the Ti6Al4V surface. Furthermore, it was confirmed that even when the autoclave treatment of 10PIPAAm-Ti6Al4V was performed one to multiple times, N atoms remained and the PIPAAm gel layer was maintained. Elemental composition analysis of the surface of 10PIPAAm-Ti6Al4V before and after autoclave treatment by X-ray photoelectron spectroscopy (XPS) was used as Example 6, and the results of Example 6 are shown in Table 1. In Table 1, "ND" means "Not Detected".

[0138] [Table 1]

[0139] In the same manner as above, the surface of Ti6Al4V before the bonding treatment of PIPAAm and the surface before and after autoclave treatment of 50PIPAAm-Ti6Al4V prepared in Example 4 were analyzed by X-ray photoelectron spectroscopy (XPS) using an X-ray photoelectron spectrometer (K-Alpha manufactured by ThermoFisher Scientific). N element could not be confirmed on the surface of Ti6Al4V before the bonding treatment of PIPAAm (Comparative Example 3), but N atoms derived from PIPAAm could be confirmed on the surface of 50PIPAAm-Ti6Al4V to which PIPAAm was bonded, and it was confirmed that PIPAAm was bonded and immobilized on the surface of Ti6Al4V. Furthermore, it was confirmed that even when the autoclave treatment of 50PIPAAm-Ti6Al4V was performed one to multiple times, N atoms remained and the PIPAAm gel layer was maintained. Elemental composition analysis of the surface of 50PIPAAm-Ti6Al4V before and after autoclave treatment by X-ray photoelectron spectroscopy (XPS) was used as Example 7, and the results of Example 7 are shown in Table 2. In Table 2, "ND" means "Not Detected".

[0140] [Table 2]

[0141] From the results of Tables 1 and 2, it was shown that PIPAAm-bonded Ti6Al4V still had PIPAAm immobilized on its surface even after multiple autoclave treatments.

[0142] <Confirmation of cell detachability due to temperature change in the culture substrate after autoclave treatment> After subjecting the surface of 50PIPAAm-Ti6Al4V prepared in Example 4 to autoclave treatment (once), mouse-derived myoblast cells (C2C12) pre-labeled with calcein AM (C396-Cellstain (registered trademark)-Calcein-AM solution manufactured by Dojindo Laboratories) were prepared. Also, bovine serum (017-22231 30 w / v% albumin solution, derived from bovine serum, fatty acid-free, manufactured by Wako Pure Chemical Industries, Ltd.) was added to Dulbecco's Modified Eagle Medium (048-29785 D-MEM / Ham's F-12 (containing L-glutamine and phenol red), manufactured by Fujifilm Wako Pure Chemical Corporation) at a concentration of 10%, and penicillin streptomycin (15140-122 PENICILLIN STREPTOMYCIN, manufactured by Life Technologies Corporation) was added as an antibiotic at a concentration of 1% to prepare a cell culture solution. The C2C12 cells labeled with calcein AM were added to the above cell culture solution and suspended, and seeded on the surface of 50PIPAAm-Ti6Al4V to a density of 1.0×10 4 cell / cm 2 and cultured at 37°C for 24 hours in a cell culture incubator (MCO-5AC-PJ, manufactured by Panasonic Corporation). After culturing, the cells on the surface of 50PIPAAm-Ti6Al4V were observed with a fluorescence microscope (ECLIPSE LV100ND manufactured by Nikon Corporation) at a magnification of 5 times, and it was observed that the cells were adhered in a stretched state (left in Fig. 7). Then, the temperature of the culture substrate was lowered to 20 °C by allowing it to stand at 20 °C for 30 minutes in a cell culture incubator (MCO-96 manufactured by Sanyo Electric Co., Ltd. (currently; Panasonic Holdings Corporation)), and the cells on the surface of 50PIPAAm-Ti6Al4V were observed again with a fluorescence microscope. It was confirmed that many cells had a round shape and were adsorbed on the surface of 50PIPAAm-Ti6Al4V or floating in the medium (right in Fig. 7).

[0143] From the above results, it was found that the surface of Ti6Al4V to which PIPAAm was bonded had a temperature-responsive effect on cell detachment even after autoclave treatment. Therefore, it was shown that the temperature-responsive polymer bonded to the metal substrate in the present invention maintains the temperature responsiveness to cell detachment without being denatured even under autoclave sterilization conditions.

[0144] <Recovery of cell sheet cultured on culture substrate after autoclave treatment> After autoclave treatment (once) of each of the disks of 30PIPAAm-Ti6Al4V, 40PIPAAm-Ti6Al4V, and 50PIPAAm-Ti6Al4V prepared in Examples 2 to 4, a silicone rubber ring was adhered, and mouse-derived myoblast cells (C2C12) pre-labeled with calcein AM (C396-Cellstain (registered trademark)-Calcein-AM solution manufactured by Dojindo Laboratories) were suspended in a cell culture solution having the same content as above, 1.5×10 5 cells / cm 2Seeded on each of the above disk surfaces so as to achieve the above, and cultured at 37°C for 48 hours in a cell culture incubator (CO2 incubator (MCO-18AIC(UV)) manufactured by PHC Holdings Corporation). After culturing, when the temperature was changed to 20°C in the same manner as above, it was confirmed that cells could be recovered in a sheet form from the surfaces of 40PIPAAm-Ti6Al4V and 50PIPAAm-Ti6Al4V (Figure 8). It was confirmed that the cell sheet recovered from the surface of 40PIPAAm-Ti6Al4V had no damaged parts and was a single cell sheet in which the cells were continuously joined in a plane (Figure 9).

[0145] From the above results, it was suggested that the efficiency of cell detachment upon temperature change varies depending on the solution concentration of the monomer unit of the temperature-responsive polymer used when binding the temperature-responsive polymer onto the metal substrate in the present invention.

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

[0147] After autoclaving the disk of 50PIPAAm-Ti of Example 8, a silicone rubber ring was adhered, and mouse-derived myoblasts (C2C12) were suspended in the same cell culture medium as above in the ring, 1.0×10 5 cells / cm 2Seeded on the surface of the 50PIPAAm-Ti disk so as to obtain [a certain density], and cultured at 37 °C for 48 hours in a cell culture incubator (CO₂ incubator (MCO-18AIC(UV)) manufactured by PHC Holdings Corporation). Then, when left standing at 20 °C in a cell culture incubator (MCO-96 manufactured by Sanyo Electric Co., Ltd. (currently; Panasonic Holdings Corporation)), within 10 to 20 minutes, the cells peeled off from the 50PIPAAm-Ti surface in a sheet form. It was confirmed that this cell sheet was a single cell sheet in which the cells were continuously bound in a plane (Figure 10).

[0148] Next, after washing this culture substrate by a normal method, performing the second autoclave treatment, adhering a silicone rubber ring, suspending mouse-derived myoblast cells (C2C12) in the ring with the same cell culture medium as above, and seeding them on the surface of the 50PIPAAm-Ti disk so as to obtain 1.0×10 5 cells / cm 2 Seeded on the surface of the 50PIPAAm-Ti disk so as to obtain [a certain density], and cultured at 37 °C for 48 hours in a cell culture incubator (CO₂ incubator (MCO-18AIC(UV)) manufactured by PHC Holdings Corporation). Then, when left standing at 20 °C in a cell culture incubator (MCO-96 manufactured by Sanyo Electric Co., Ltd. (currently; Panasonic Holdings Corporation)), within 10 to 20 minutes, the cells peeled off from the 50PIPAAm-Ti surface in a sheet form. It was confirmed that this cell sheet was a single cell sheet in which the cells were continuously bound in a plane (Figure 11).

[0149] Next, after washing this culture substrate by a normal method, performing the third autoclave treatment, adhering a silicone rubber ring, suspending mouse-derived myoblast cells (C2C12) previously labeled with calcein AM (C396-Cellstain (registered trademark)-Calcein-AM solution manufactured by Dojindo Laboratories) in the ring with the same cell culture medium as above, and seeding them on the surface of the 50PIPAAm-Ti disk so as to obtain 1.0×10 4 cell / cm 2Seeded on the surface of the 50PIPAAm-Ti disk so as to obtain the above, cultured at 37°C for 24 hours in a cell culture incubator (MCO-5AC-PJ manufactured by Panasonic Corporation), and observed the cells on the 50PIPAAm-Ti surface with a fluorescence microscope (ECLIPSE LV100ND manufactured by Nikon Corporation) at a magnification of 5 times. As a result, it was observed that the cells were adhered in a stretched state. Thereafter, the above cultured cells were allowed to stand at 20°C in a cell culture incubator (MCO-96 manufactured by Sanyo Electric Co., Ltd. (currently; Panasonic Holdings Corporation)), and when the cells on the 50PIPAAm-Ti surface were observed again with a fluorescence microscope, it was confirmed that many cells became round within 10 to 20 minutes and adsorbed on the 50PIPAAm-Ti surface or were floating in the medium (Fig. 12).

[0150] Next, after washing this culture substrate by a normal method, after the fourth autoclave treatment, a silicone rubber ring was adhered, and mouse-derived myoblast cells (C2C12) were suspended in the same cell culture solution as above in the ring, and 1.0×10 5 cells / cm 2 Seeded on the surface of the 50PIPAAm-Ti disk so as to obtain the above, cultured at 37°C for 48 hours in a cell culture incubator (CO2 incubator (MCO-18AIC(UV)) manufactured by PHC Holdings Corporation). Thereafter, when allowed to stand at 20°C in a cell culture incubator (MCO-96 manufactured by Sanyo Electric Co., Ltd. (currently; Panasonic Holdings Corporation)), the cells peeled off from the 50PIPAAm-Ti surface in a sheet form within 10 to 20 minutes. It was confirmed that this cell sheet was a single cell sheet in which the cells were continuously connected in a plane (Fig. 13).

[0151] Next, after washing this culture substrate by a normal method, after the fifth autoclave treatment, a silicone rubber ring was adhered, and mouse-derived myoblast cells (C2C12) were suspended in the same cell culture solution as above in the ring, and 1.0×10 5 cells / cm 2Seeded onto the surface of the 50PIPAAm-Ti disk so as to achieve this, and cultured at 37 °C for 48 hours in a cell culture incubator (CO2 incubator (MCO-18AIC(UV)) manufactured by PHC Holdings Corporation). Thereafter, when left standing at 20 °C in a cell culture incubator (MCO-96 manufactured by Sanyo Electric Co., Ltd. (currently; Panasonic Holdings Corporation)), within 10 to 20 minutes, the cells peeled off from the 50PIPAAm-Ti surface in a sheet form. It was confirmed that this cell sheet was a single cell sheet in which the cells were continuously bound in a plane (Figure 14).

[0152] From the above results, it was shown that the culture substrate of the present invention is a reusable culture substrate in which the temperature-responsive polymer appropriately exhibits temperature responsiveness even after being autoclaved multiple times, and cells and cell sheets can be recovered by a temperature change.

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

[0154] Also, examples of the aspects of the present invention are as follows. <1>A culture substrate characterized in that it has a temperature-responsive polymer with a glass transition temperature exceeding 121 °C on a metal substrate that has been subjected to oxygen plasma treatment and silanization treatment. <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>It has a substrate having a concave portion and a lid portion covering the substrate, The culture substrate according to any one of <1> to <4>, wherein the temperature-responsive polymer is provided on the bottom surface of the concave portion in the substrate. <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 exceeding 121 °C onto the metal substrate. A method for producing a culture substrate, characterized by including these steps. <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, characterized by including 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 exceeding 121 °C on an oxygen plasma-treated and silanized metal substrate to produce cells. <10>The method for producing cells according to <9>, wherein in the cell production step, the cells are produced in a sheet form. <11>The method for producing cells according to any one of <9> to <10>, including a cell detachment step of changing the temperature to detach the cells from the temperature-responsive polymer after the cell production step. <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 cells 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 culture substrate includes a substrate having a concave portion and a lid portion covering the substrate, and the temperature-responsive polymer is provided on the bottom surface of the concave portion in the substrate. The method for producing cells according to any one of <9> to <14>. <16>A culture substrate for repeated use, characterized by including a temperature-responsive polymer having a glass transition temperature exceeding 121 °C. <17>The culture substrate for repeated use according to <16>, wherein the temperature-responsive polymer is poly-N-isopropylacrylamide.

[0155] According to the culture substrate described in any one of <1> to <5> above, the method for manufacturing the culture substrate described in any one of <6> to <8> above, the method for manufacturing cells described in any one of <9> to <15> above, and the culture substrate for repeated use described in any one of <16> to <17> above, various problems in the prior art can be solved and the object of the present invention can be achieved.

[0156] This application claims the priority based on Japanese Patent Application No. 2023-012270 filed with the Japan Patent Office on January 30, 2023, and incorporates all the contents described in the above application.

Claims

1. A temperature-responsive polymer layer containing a temperature-responsive polymer with a glass transition temperature exceeding 121°C is provided on a metal substrate that has been treated with oxygen plasma and silanized. The temperature-responsive polymer is bonded to the metal substrate via a covalent bond, The temperature-responsive polymer layer is formed by simultaneously occurring polymerization, cross-linking of monomer units of the temperature-responsive polymer, and immobilization of the temperature-responsive polymer onto the metal substrate. A culture substrate characterized by this.

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. It has a substrate having a recess and a lid covering the substrate, The culture substrate according to any one of Claims 1 to 4, wherein the temperature-responsive polymer is provided on the bottom surface of the recess in the substrate.

6. A silane agent treatment step of treating the surface of a metal substrate treated with oxygen plasma with a silane agent, A temperature-responsive polymer bonding step of bonding a temperature-responsive polymer with a glass transition temperature exceeding 121°C onto the metal substrate via a covalent bond to form a temperature-responsive polymer layer, including, In the temperature-responsive polymer bonding step, the temperature-responsive polymer layer is formed by simultaneously occurring polymerization, cross-linking of monomer units of the temperature-responsive polymer, and immobilization of the temperature-responsive polymer onto the metal substrate. A method for manufacturing a culture substrate characterized by this.

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

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

9. A cell production step of seeding and culturing cells on the surface of a culture substrate having a temperature-responsive polymer layer containing a temperature-responsive polymer with a glass transition temperature exceeding 121°C on a metal substrate that has been treated with oxygen plasma and silanized, and having the temperature-responsive polymer via a covalent bond, to produce cells, including, In the temperature-responsive polymer layer, the temperature-responsive polymer layer is formed by simultaneously occurring polymerization, cross-linking of monomer units of the temperature-responsive polymer, and immobilization of the temperature-responsive polymer onto the metal substrate. A method for producing cells characterized by this.

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

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

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 cells according to claim 9, wherein the temperature-responsive polymer is connected to the metal substrate via a silicon atom.

15. The culture substrate includes a substrate having a recess and a lid covering the substrate, The method for producing cells according to any one of claims 9 to 14, wherein the temperature-responsive polymer is provided on the bottom surface of the recess in the substrate.

16. A temperature-responsive polymer layer containing a temperature-responsive polymer having a glass transition temperature exceeding 121 °C is provided on a metal substrate that has been subjected to oxygen plasma treatment and silanization treatment. The temperature-responsive polymer is bonded to the metal substrate via a covalent bond, The culture substrate for repeated use is characterized in that the temperature-responsive polymer layer is formed by simultaneously performing polymerization, cross-linking of monomer units of the temperature-responsive polymer, and immobilization of the temperature-responsive polymer on the metal substrate.

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

Images