Structure for cells, method for manufacturing structure for cells, and method for culturing cells

Abstract

This structure for cells includes a substrate and a plurality of spots that are disposed at intervals on the surface of the substrate. Each of the plurality of spots includes a cell adhesive substance.

Description

Cell structure, method for producing cell structure, and cell culture method

[0001] The present invention relates to a cell structure, a method for producing a cell structure, and a cell culture method.

[0002] Various materials have been proposed as base film forming agents for forming base films for efficient cell culture. Patent Literature 1 discloses a method for producing a polymer used as a base film for cell culture and a cell culture vessel. Patent Literature 2 discloses a method for producing a cell structure. These base films for cell culture require the use of serum derived from living organisms in the process of producing (culturing) homogeneous cell aggregates to ensure uniform adhesion of cells to the base film, which has problems such as quality variations due to individual differences in proliferation ability, and safety risks such as the occurrence of allergies and viral contamination when serum derived from animals other than humans is used.

[0003] Therefore, the present applicant has proposed a base film for cell culture that can produce homogeneous, high-quality cell aggregates in the mass production of cell aggregates without using serum derived from living organisms, a base film forming agent for forming the same, and a substrate for producing cell aggregates (Patent Document 3).

[0004] International Publication No. 2020 / 040247 Pamphlet Japanese Patent Application Laid-Open No. 2017-143755 Pamphlet International Publication No. 2023 / 282253 Pamphlet

[0005] Stably retaining cells themselves or cell structures at predetermined intervals is useful from the viewpoint of utilizing the cells themselves and producing cell structures of uniform size.

[0006] The present invention aims to provide a cell structure capable of stably retaining cells themselves or cell structures at predetermined intervals, a method for manufacturing the cell structure, and a cell culture method using the cell structure.

[0007] The present inventors have found that the above-mentioned problems can be solved and have completed the present invention.

[0008] That is, the present invention encompasses the following aspects. [1] A cell structure comprising a substrate and a plurality of spots arranged at intervals on the surface of the substrate, wherein each of the plurality of spots comprises a cell adhesive substance. [2] The cell structure according to [1], wherein each of the plurality of spots comprises a cell culture base membrane containing a polymer, and the cell adhesive substance arranged on the cell culture base membrane. [3] The cell structure according to [1] or [2], which is a cell culture vessel. [4] The cell structure according to [2] or [3], wherein the polymer is a polymer (A) comprising a repeating unit represented by the following formula (Ia): (In formula (Ia), U ａ１ and U ａ２ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ａ１ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ａ２ represents a linear or branched alkylene group having 1 to 5 carbon atoms.) [5] The cell structure according to any one of [2] to [4], wherein the polymer (A) includes a crosslinked structure. [6] The cell structure according to [5], wherein the polymer (A) includes at least one of a structure represented by the following formula (IIIa), a structure represented by the following formula (IVa), and a structure represented by the following formula (Va): (In the formula, R ｃ and R ｄ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ｅrepresents a linear or branched alkylene group having 1 to 5 carbon atoms, and n represents a number from 1 to 50.) [7] The cell structure according to any one of [1] to [6], wherein the surface of the substrate has a coating film that inhibits cell adhesion, and the spots are arranged on the coating film. [8] A method for manufacturing a cell structure having a substrate and a plurality of spots arranged at intervals on the surface of the substrate, comprising a step of applying droplets containing a cell adhesive substance to the substrate to arrange the cell adhesive substance in areas that will become the spots. [9] The method for manufacturing a cell structure according to [8], wherein the application of the droplets containing the cell adhesive substance to the substrate is carried out by an inkjet method.

[10] The method for manufacturing a cell structure according to [8] or [9], wherein the surface of the substrate has a coating film that inhibits cell adhesion, and the plurality of spots are arranged on the coating film.

[11] A method for producing a cell structure having a substrate and a plurality of spots arranged at intervals on the surface of the substrate, comprising the steps of: applying droplets of a cell culture base film forming agent containing a polymer and a solvent to the substrate to form a cell culture base film in areas that will become the spots; and applying droplets containing a cell adhesive substance to the cell culture base film to arrange the cell adhesive substance on the cell culture base film.

[12] The method for producing a cell structure according to

[11] , wherein the application of the droplets of the cell culture base film forming agent to the substrate is carried out by an inkjet method.

[13] The method for producing a cell structure according to

[11] or

[12] , wherein the application of the droplets containing the cell adhesive substance to the cell culture base film is carried out by an inkjet method.

[14] The method for producing a cell structure according to any of

[11] to

[13] , wherein the polymer is a polymer containing a repeating unit represented by the following formula (Ia): (In formula (Ia), U ａ１ and U ａ２each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ａ１ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ａ２ represents a linear or branched alkylene group having 1 to 5 carbon atoms.)

[15] In the cell structure to be produced, the surface of the substrate has a coating film that has the ability to inhibit cell adhesion, and the plurality of spots are arranged on the coating film, and the method for producing the cell structure according to any of

[11] to

[14] further comprises the step of applying a coating agent containing a polymer that has the ability to inhibit cell adhesion onto the substrate to form the coating film.

[16] The method for producing a cell structure according to any of

[11] to

[15] , wherein the cell structure to be produced is a cell culture vessel.

[17] A cell culture method comprising the step of seeding cells into the cell structure according to any of [1] to [7].

[18] The cell culture method according to

[17] , for producing a cell structure.

[0009] According to the present invention, it is possible to provide a cell structure capable of stably retaining cells themselves or cell structures at a predetermined interval, a method for manufacturing the cell structure, and a cell culture method using the cell structure.

[0010] FIG. 1A is a perspective view of an example of a cell structure. FIG. 1B is a top view of an example of a cell structure. FIG. 2A is a perspective view of another example of a cell structure. FIG. 2B is a top view of another example of a cell structure. FIG. 3A is a perspective view of another example of a cell structure. FIG. 3B is a top view of another example of a cell structure. FIG. 4A is an observation result after 26 hours of culture in the test of Example 1. FIG. 4B is an observation result after 72 hours of culture in the test of Example 1. FIG. 4C is an observation result (overall photograph) after 72 hours of culture in the test of Example 1. FIG. 5A is an observation result after 26 hours of culture in the test of Comparative Example 1. FIG. 5B is an observation result after 72 hours of culture in the test of Comparative Example 1. FIG. 5C is an observation result (overall photograph) after 72 hours of culture in the test of Comparative Example 1. FIG. 6 is an observation result of Test Example 2. FIG. 7A is an observation result after 3 days of culture in the test of Example 2. FIG. 7B shows the observation results after 6 days of culture in the test of Example 2. FIG. 7C shows the observation results (overall photograph) after 6 days of culture in the test of Example 2. FIG. 8A shows the observation results after 3 days of culture in the test of Example 3. FIG. 8B shows the observation results after 6 days of culture in the test of Example 3. FIG. 8C shows the observation results (overall photograph) after 6 days of culture in the test of Example 3. FIG. 9A shows the observation results after 3 days of culture in the test of Comparative Example 2. FIG. 9B shows the observation results after 6 days of culture in the test of Comparative Example 2. FIG. 9C shows the observation results (overall photograph) after 6 days of culture in the test of Comparative Example 2. FIG. 10A shows the observation results (overall photograph) after 3 days of culture in the test of Example 4. FIG. 10B shows the observation results (overall photograph) after 7 days of culture in the test of Example 4. FIG. 11A shows the observation results (overall photograph) after 3 days of culture in the test of Example 5. FIG. 11B is an observation result (overall photograph) after 7 days of culture in the test of Example 5. FIG. 11C is an observation result (overall photograph) after complete medium exchange in the test of Example 5. FIG. 12A is an observation result (overall photograph) after 3 days of culture in the test of Comparative Example 3. FIG. 12B is an observation result (overall photograph) after 7 days of culture in the test of Comparative Example 3. FIG. 12C is an observation result (overall photograph) after complete medium exchange in the test of Comparative Example 3. FIG. 13A is an observation result after 7 days of culture in the test of Example 6, in which half of the medium was exchanged.Fig. 13B shows the results of observation after 7 days of culture following complete medium exchange in the test of Example 6. Fig. 14A shows the results of observation after 7 days of culture following half-medium exchange in the test of Comparative Example 4. Fig. 14B shows the results of observation after 7 days of culture following complete medium exchange in the test of Comparative Example 4.

[0011] (Cell structure) The cell structure of the present invention comprises a substrate and a plurality of spots. The plurality of spots are arranged at intervals on the surface of the substrate. In one embodiment, the spots comprise a cell adhesive substance. In another embodiment, the spots comprise a cell culture base membrane and a cell adhesive substance arranged on the cell culture base membrane. Here, "surface of the substrate" refers to the surface that comes into contact with contents such as a cell culture medium.

[0012] In one embodiment of the cell structure, multiple cell adhesive materials are arranged at intervals on a substrate, allowing cells or cell structures to be retained at predetermined intervals. In this embodiment, cells can be adhered to the cell adhesive material and cultured for a long period of time. Furthermore, depending on the type of cell, the adhered cells can proliferate and form cell structures (e.g., hemispherical spheroids).

[0013] In another embodiment of the cell structure, multiple cell culture substratums are arranged at intervals on a substrate. In this embodiment, cell structures (e.g., spheroids) can be produced on the cell culture substratum. By aligning the shape and size of the cell culture substratum at each spot, cell structures of uniform size can be produced. Furthermore, the presence of a cell adhesive substance on the cell culture substratum makes the produced cell structures less likely to detach from the cell culture substratum than when the cell adhesive substance is not present on the cell culture substratum. This prevents the detached cell structures from coalescing, resulting in uneven size and a reduction in the number of cell structures.

[0014] The cell structure is preferably a cell culture vessel.

[0015] <Spots> The spots have a cell adhesive substance and are locations on the cell structure where cells are attached or cultured (e.g., structured).

[0016] The number of spots arranged at intervals on the surface of the substrate is not particularly limited. For example, the number of spots may be 1 cm. ２ Preferably, 50 to 5,000 particles per spot are present, and more preferably 100 to 2,000 particles per spot. The distance between adjacent spots is not particularly limited, and is preferably 100 μm to 6,000 μm, more preferably 150 μm to 4,000 μm, and particularly preferably 150 μm to 3,000 μm. Here, the distance refers to the length between the centers of two spots. The size of the spot surface is not particularly limited, and for example, the circle-equivalent diameter is preferably 25 μm to 5,000 μm, and more preferably 50 μm to 3,000 μm. The circle-equivalent diameter refers to the diameter of a perfect circle equivalent to the area of ​​the target surface. When the spot surface is a perfect circle, the circle-equivalent diameter refers to the diameter of that perfect circle. When the spot surface is other than a perfect circle, the circle-equivalent diameter refers to the diameter of a perfect circle having the same area as the area of ​​that surface.

[0017] The area ratio of the spots on the substrate is not particularly limited, but is preferably 30% or more, 40% or more, 50% or more, and is preferably 99% or less.

[0018] The spot preferably has a cell culture base membrane. In this case, the cell adhesive substance is disposed on the cell culture base membrane. In this case, the area occupied by the cell culture base membrane is the spot. Therefore, the surface of the cell culture base membrane corresponds to the spot. The size of the surface of the cell adhesive substance disposed on the cell culture base membrane is not particularly limited as long as it is less than the surface size of the cell culture base membrane, but is preferably 1 / 80 to 1 / 3, and more preferably 1 / 50 to 1 / 4, of the area of ​​the cell culture base membrane.

[0019] <<Cell Adhesive Substance>> The cell adhesive substance is present, for example, at one location per spot, but may also be present at multiple locations per spot.

[0020] Cell adhesive substances promote cell adhesion, spreading, proliferation, and differentiation. Examples of cell adhesive substances that can be used include known substances of biological origin, such as extracellular matrix (ECM) proteins, glycoproteins, and peptides, as well as synthetic compounds (low molecular weight, high molecular weight). However, non-biological compounds, such as synthetic compounds (low molecular weight, high molecular weight), are preferred. A low molecular weight is, for example, a compound with a weight-average molecular weight of 2,000 or less, and a high molecular weight is, for example, a compound with a weight-average molecular weight of 2,000 or more, with an upper limit of, for example, 1,000,000.

[0021] Examples of extracellular matrix (ECM) proteins include collagen (e.g., Merck's type I collagen (product numbers C9791, C7661, C1809, C2249, C2124), type II collagen (product number C9301), type IV collagen (product numbers C0543, C5533), elastin (e.g., Merck's product numbers E1625, E6527), fibronectin (e.g., Merck's product numbers F1141, F0635, F2518, F0895, F4759, F2006), laminin (e.g., Merck's product numbers L6724, L2020, L4544), laminin fragments (e.g., Matricsome's product number 892011), vitronectin (e.g., VTN-N (Gibco)), Vitronectin, Human Recombinant, Animal Free (PeproTech), Merck product numbers: V0132, V9881, V8379, 08-126, SRP3186).

[0022] The cell adhesive substance is preferably a glycoprotein, specifically selected from vitronectin, integrin, cadherin, fibronectin, laminin, tenascin, osteopontin, and bone sialoprotein, and preferably a protein having an RGD amino acid sequence.

[0023] Examples of peptides include ECM peptide (MAPTrix ​​(registered trademark) from Kollodis Bio Sciences) and RGD peptide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: 180-01531).

[0024] Examples of synthetic compounds (polymers) include polylysine (e.g., Merck products: P4707, P4832, P7280, P9155, P6407, P6282, P7405, P5899) and polyornithine (e.g., Merck product number P4975). Examples of synthetic compounds (low molecular weights) include adhesamine (e.g., Nagase & Co.: AD-00000-0201) and synthetic cyclic RGD peptide (e.g., IRIS BIOTECH: LS-3920.0010).

[0025] <<Cell Culture Substrate>> The cell culture substrate contains a polymer. The polymer is a substance different from the cell adhesive substance and is typically a synthetic polymer. The cell culture substrate may further contain a cell adhesive substance.

[0026] The thickness of the cell culture base film is not particularly limited, but is, for example, in the range of 1 to 1000 nm, and preferably in the range of 5 to 500 nm.

[0027] The polymer is not particularly limited as long as it is a polymer that can be used for a base film for cell culture. However, from the viewpoint of suitably obtaining the effects of the present invention, a polymer (A) containing a repeating unit represented by the following formula (Ia) is preferred. (In formula (Ia), U ａ１ and U ａ２ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ａ１ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ａ２ represents a linear or branched alkylene group having 1 to 5 carbon atoms.

[0028] The polymer (A) preferably further contains a repeating unit represented by the following formula (IIa): (In formula (IIb), R ｂ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms.

[0029] Unless otherwise defined, in this specification, examples of a "linear or branched alkyl group having 1 to 5 carbon atoms" include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, an s-butyl group, a t-butyl group, an n-pentyl group, a 1-methylbutyl group, a 2-methylbutyl group, a 3-methylbutyl group, a 1,1-dimethylpropyl group, a 1,2-dimethylpropyl group, a 2,2-dimethylpropyl group, and a 1-ethylpropyl group.

[0030] R ａ１ and R ｂ are preferably each independently selected from a hydrogen atom and a methyl group.

[0031] U ａ１ and U ａ２ are each independently preferably selected from a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, and an n-butyl group, more preferably a methyl group or an ethyl group, and most preferably a methyl group.

[0032] In this specification, unless otherwise defined, examples of a "linear or branched alkylene group having 1 to 5 carbon atoms" include a methylene group, an ethylene group, a propylene group, a trimethylene group, a tetramethylene group, a 1-methylpropylene group, a 2-methylpropylene group, a dimethylethylene group, an ethylethylene group, a pentamethylene group, a 1-methyl-tetramethylene group, a 2-methyl-tetramethylene group, a 1,1-dimethyl-trimethylene group, a 1,2-dimethyl-trimethylene group, a 2,2-dimethyl-trimethylene group, and a 1-ethyl-trimethylene group. ａ２ is preferably selected from an ethylene group and a propylene group.

[0033] Examples of the monomer (cationic monomer) that provides the repeating unit represented by formula (Ia) in the polymer (A) include 2-N,N-dimethylaminoethyl methacrylate and N,N-dimethylaminomethyl methacrylate, and 2-N,N-dimethylaminoethyl methacrylate (2-(dimethylamino)ethyl methacrylate) is preferred.

[0034] Examples of the monomer (anionic monomer) that provides the polymer (A) with the repeating unit represented by formula (IIa) include acrylic acid and methacrylic acid, with methacrylic acid being preferred.

[0035] The molar ratio of the repeating unit (Ia) represented by formula (Ia) to the repeating unit (IIa) represented by formula (IIa) in the polymer (A) [(Ia) / (IIa)] is, for example, 100 / 0 to 50 / 50. It is preferably 98 / 2 to 50 / 50, more preferably 98 / 2 to 60 / 40, and particularly preferably 98 / 2 to 70 / 30. When the molar ratio [(Ia) / (IIa)] is 1 or more, a decrease in cell adhesive strength due to the anionic nature of the polymer can be suppressed.

[0036] The polymer (A) may contain other repeating units. In the polymer (A), the total content of the repeating units represented by formula (Ia) and the repeating units represented by formula (IIa) relative to all repeating units is, for example, 50 mol % or more, preferably 75 mol % or more, more preferably 80 mol % or more, and even more preferably 90 mol % or more.

[0037] From the viewpoint of suitably achieving the effects of the present invention, it is preferable that the polymer (A) further contains a crosslinked structure. The crosslinked structure is obtained by using a monomer having two or more carbon-carbon unsaturated bonds when synthesizing the polymer (A). Specific examples of the monomer having two or more carbon-carbon unsaturated bonds include a monomer having two or more carbon-carbon double bonds, such as a polyfunctional acrylate compound, a polyfunctional acrylamide compound, a polyfunctional polyester, or an isoprene compound.

[0038] The polymer (A) includes, for example, a structure derived from at least one of a polyfunctional acrylate compound, a polyfunctional acrylamide compound, a polyfunctional polyester, and an isoprene compound. The polymer (A) preferably includes at least one of a structure represented by the following formula (IIIa), a structure represented by the following formula (IVa), and a structure represented by the following formula (Va). Among these, the structure represented by formula (IIIa) is preferred. (In the formula, R ｃ and Rｄ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ｅ represents a linear or branched alkylene group having 1 to 5 carbon atoms, and n represents a number from 1 to 50.

[0039] R ｃ and R ｄ are preferably each independently selected from a hydrogen atom and a methyl group. ｅ is preferably selected from a methylene group, an ethylene group, and a propylene group, and more preferably an ethylene group. n is a number from 1 to 50, preferably a number from 1 to 30, and more preferably a number from 1 to 10.

[0040] The molar ratio of the structures represented by formulae (IIIa) to (Va) to the entire polymer (A) is preferably 0 mol % to 50 mol %, more preferably 2 mol % to 25 mol %. When the molar ratio of the structures represented by formulae (IIIa) to (Va) is 50 mol % or less of the entire polymer (A), gelation of the solid content during production due to high molecular weight caused by excessive crosslinking can be suppressed, and production can be facilitated.

[0041] The synthesis method of the polymer (A) is not particularly limited, and examples thereof include known radical polymerization methods. Alternatively, the method described in WO 2020 / 040247 may be used.

[0042] The number average molecular weight (Mn) of the polymer is, for example, 20,000 to 1,000,000, preferably 50,000 to 800,000. The number average molecular weight (Mw) of the polymer is, for example, 50,000 to 2,000,000, preferably 10,000 to 1,200,000. The ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) of the polymer is, for example, 1.01 to 10.00, preferably 1.2 to 8.0, more preferably 1.4 to 6.0, even more preferably 1.5 to 5.0, and particularly preferably 1.6 to 4.5. The number average molecular weight (Mn) and weight average molecular weight (Mw) can be determined, for example, by gel filtration chromatography.

[0043] <Substrate> There are no particular limitations on the material, shape, size, or structure of the substrate. The surface of the substrate may be flat or may have irregularities, but is preferably flat.

[0044] Examples of the substrate material include glass, metal, metal-containing compounds or metalloid-containing compounds, activated carbon, and resin. Examples of metals include typical metals (aluminum group elements: Al, Ga, In; iron group elements: Fe, Co, Ni; chromium group elements: Cr, Mo, W, U; manganese group elements: Mn, Re; and precious metals: Cu, Ag, and Au). Examples of metal-containing compounds or metalloid-containing compounds include ceramics, which are sintered bodies whose basic component is a metal oxide and which are hardened by heat treatment at high temperatures; semiconductors such as silicon; inorganic solid materials such as molded bodies of inorganic compounds such as metal oxides or metalloid oxides (silicon oxide, alumina, etc.), metal carbides or metalloid carbides, metal nitrides or metalloid nitrides (silicon nitride, etc.), and metal borides or metalloid borides; aluminum, nickel titanium, and stainless steel (SUS304, SUS316, SUS316L, etc.).

[0045] The resin may be a natural resin or a derivative thereof, or a synthetic resin. Preferred examples of natural resins or derivatives thereof include cellulose, cellulose triacetate (CTA), nitrocellulose (NC), and cellulose with immobilized dextran sulfate. Preferred examples of synthetic resins include polyacrylonitrile (PAN), polyimide (PI), polyester polymer alloy (PEPA), polystyrene (PS), polysulfone (PSF), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), polyurethane (PU), ethylene vinyl alcohol (EVAL), polyethylene (PE), polyester, polypropylene (PP), polyvinylidene fluoride (PVDF), polyethersulfone (PES), polycarbonate (PC), cycloolefin polymer (COP), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), ultra-high molecular weight polyethylene (UHPE), polydimethylsiloxane (PDMS), acrylonitrile-butadiene-styrene resin (ABS), and Teflon (registered trademark).

[0046] The substrate may be in the form of a plate or a film. The thickness of the substrate is not particularly limited. For example, the substrate may be a film having a thickness of 1 mm or less.

[0047] The substrate may also be a substrate used in a so-called cell culture vessel, and examples thereof include dishes or petri dishes such as Petri dishes, tissue culture dishes, and multi-dishes commonly used for cell culture, flasks such as cell culture flasks, spinner flasks, and multi-tier flasks, bags such as plastic bags, Teflon (registered trademark) bags, and culture bags, plates such as microplates, microwell plates, multi-plates, and multi-well plates, chamber slides, tubes, trays, and bottles such as roller bottles.

[0048] <<Coating Film>> The surface of the substrate may have a coating film capable of inhibiting cell adhesion. In this case, the spots are arranged on the coating film. The coating film can be obtained, for example, by applying a coating agent containing a polymer capable of inhibiting cell adhesion to the substrate. The shape of the coating film on the surface of the substrate is not particularly limited, as long as the spots are arranged thereon. One film may uniformly cover the entire surface of the substrate, one film may be formed within the plane of the surface of the substrate, or multiple films may be scattered on the surface of the substrate.

[0049] Here, having the ability to inhibit cell adhesion means that the relative absorbance (WST O.D. 450 nm) (%) ((absorbance (WST O.D. 450 nm) of the example) / (absorbance (WST O.D. 450 nm) of the comparative example)) when compared with a case without a coating film or without a low cell adsorption treatment, as determined by a fluorescence microscope using the method described in the examples of WO 2016 / 093293, for example, is 50% or less, preferably 30% or less, and more preferably 20% or less.

[0050] The coating film having the ability to inhibit cell adhesion is not particularly limited, but examples thereof include the following coating films.

[0051] <<<Coating Film of First Aspect>>> The coating film of the first aspect includes a copolymer. The copolymer of the first aspect includes a repeating unit containing an organic group represented by the following formula (a) and a repeating unit containing an organic group represented by the following formula (b). The copolymer is, for example, the copolymer described in WO2014 / 196652. The contents of WO2014 / 196652 are incorporated herein by reference to the same extent as if set forth in full.

[0052] (In the formula, U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, and An －represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion.

[0053] The copolymer is not particularly limited as long as it contains a repeating unit containing an organic group represented by the above formula (a) and a repeating unit containing an organic group represented by the above formula (b). The copolymer is preferably one obtained by radical polymerization of a monomer containing an organic group represented by the above formula (a) and a monomer containing an organic group represented by the above formula (b), but one obtained by polycondensation or polyaddition reaction can also be used. Examples of copolymers include vinyl polymers reacted with olefins, polyamides, polyesters, polycarbonates, polyurethanes, etc., among which vinyl polymers reacted with olefins or (meth)acrylic polymers polymerized with (meth)acrylate compounds are particularly preferred. In the present invention, the term "(meth)acrylate compound" refers to both acrylate compounds and methacrylate compounds. For example, "(meth)acrylic acid" refers to acrylic acid and methacrylic acid.

[0054] Preferably, the monomers containing the organic groups represented by the above formulas (a) and (b) are monomers represented by the following formulas (A) and (B), respectively. (In the formula, T ａ , T ｂ , U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; Q ａ and Q ｂ each independently represents a single bond, an ester bond, or an amide bond; R ａ and R ｂ each independently represents a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom; An － represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion, and m represents an integer of 0 to 6.

[0055] Therefore, the repeating units derived from the monomers represented by formulae (A) and (B) are represented by the following formulae (a1) and (b1), respectively. (In the formula, T ａ , T ｂ , U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ , Q ａ and Q ｂ , R ａ and R ｂ , An － and m has the same meaning as above.

[0056] In the present invention, an "ester bond" means -C(=O)-O- or -O-C(=O)-, and an "amide bond" means -NHC(=O)- or -C(=O)NH-.

[0057] In the present invention, the term "a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with one or more halogen atoms" means a linear or branched alkylene group having 1 to 10 carbon atoms, or a linear or branched alkylene group having 1 to 10 carbon atoms which is substituted with one or more halogen atoms. Here, the "straight-chain or branched alkylene group having 1 to 10 carbon atoms" is a divalent organic group in which one hydrogen atom has been further removed from the above-mentioned alkyl group, and examples thereof include a methylene group, an ethylene group, a propylene group, a trimethylene group, a tetramethylene group, a 1-methylpropylene group, a 2-methylpropylene group, a dimethylethylene group, an ethylethylene group, a pentamethylene group, a 1-methyl-tetramethylene group, a 2-methyl-tetramethylene group, a 1,1-dimethyl-trimethylene group, a 1,2-dimethyl-trimethylene group, a 2,2-dimethyl-trimethylene group, a 1-ethyl-trimethylene group, a hexamethylene group, an octamethylene group, and a decamethylene group. Among these, ethylene, propylene, octamethylene and decamethylene are preferred, and linear or branched alkylene groups having 1 to 5 carbon atoms, such as ethylene, propylene, trimethylene and tetramethylene, are more preferred, with ethylene or propylene being particularly preferred. A "linear or branched alkylene group having 1 to 10 carbon atoms substituted with one or more halogen atoms" means such an alkylene group in which any one or more hydrogen atoms have been replaced with halogen atoms, and particularly preferred is an ethylene or propylene group in which some or all of the hydrogen atoms have been replaced with halogen atoms.

[0058] In the present invention, the "halogen atom" includes a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. In the present invention, the "halide ion" means an anion of a halogen atom, including a fluoride ion, a chloride ion, a bromide ion, and an iodide ion, and preferably a chloride ion. In the present invention, the "inorganic acid ion" means a carbonate ion, a sulfate ion, a phosphate ion, a hydrogen phosphate ion, a dihydrogen phosphate ion, a nitrate ion, a perchlorate ion, or a borate ion. －Preferred are halide ions, sulfate ions, phosphate ions, hydroxide ions and isothiocyanate ions, and particularly preferred are halide ions.

[0059] In formulas (A) and (B), T ａ and T ｂ are preferably each independently a hydrogen atom, a methyl group, or an ethyl group, and more preferably each independently a hydrogen atom or a methyl group.

[0060] In formula (a), formula (b), formula (A) and formula (B), U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ are preferably each independently a hydrogen atom, a methyl group, or an ethyl group. ａ１ and U ａ２ is more preferably a hydrogen atom. ｂ１ , U ｂ２ (and U ｂ３ ) is more preferably a methyl group or an ethyl group, and particularly preferably a methyl group.

[0061] In formulas (A) and (B), Q ａ and Q ｂ are preferably each independently an ester bond (-C(=O)-O- or -O-C(=O)-) or an amide bond (-NHC(=O)- or -C(=O)NH-), more preferably each independently -C(=O)-O- or -C(=O)NH-, and particularly preferably -C(=O)-O-.

[0062] In formulas (A) and (B), R ａ and R ｂ are preferably each independently a linear or branched alkylene group having 1 to 3 carbon atoms which may be substituted with a halogen atom, more preferably each independently an ethylene group or a propylene group, or an ethylene group or a propylene group substituted with one chlorine atom, and particularly preferably an ethylene group or a propylene group.

[0063] In formulae (A) and (B), m preferably represents an integer of 0 to 3, more preferably an integer of 1 or 2, and particularly preferably 1.

[0064] Specific examples of the above formula (A) include vinylphosphonic acid, acid phosphooxyethyl (meth)acrylate, 3-chloro-2-acid phosphooxypropyl (meth)acrylate, acid phosphooxypropyl (meth)acrylate, acid phosphooxymethyl (meth)acrylate, acid phosphooxypolyoxyethylene glycol mono(meth)acrylate, and acid phosphooxypolyoxypropylene glycol mono(meth)acrylate, and among these, vinyl phosphonic acid and acid phosphooxyethyl methacrylate (=2-(methacryloyloxy)ethyl phosphate) are preferably used.

[0065] The structural formulas of vinylphosphonic acid, acid phosphooxyethyl methacrylate (=2-(methacryloyloxy)ethyl phosphate), and acid phosphooxypolyoxyethylene glycol monomethacrylate are represented by the following formulas (A-1) to (A-3), respectively.

[0066] In addition to these compounds, a (meth)acrylate compound having two functional groups, such as those represented by general formula (C) or (D) described below, may be used in combination during synthesis.

[0067] Specific examples of the above formula (B) include dimethylaminoethyl (meth)acrylate, diethylaminoethyl (meth)acrylate, dimethylaminopropyl (meth)acrylate, 2-(t-butylamino)ethyl (meth)acrylate, and methacryloylcholine chloride. Among these, dimethylaminoethyl (meth)acrylate, methacryloylcholine chloride, and 2-(t-butylamino)ethyl (meth)acrylate are preferably used.

[0068] The structural formulas of dimethylaminoethyl acrylate (= 2-(dimethylamino)ethyl acrylate), dimethylaminoethyl methacrylate (= 2-(dimethylamino)ethyl methacrylate), methacryloylcholine chloride, and 2-(t-butylamino)ethyl methacrylate (= 2-(t-butylamino)ethyl methacrylate) are represented by the following formulas (B-1) to (B-4), respectively.

[0069]

[0070] The proportion of the repeating units containing the organic group represented by formula (a) (or the repeating units represented by formula (a1)) in the copolymer is 20 mol % to 80 mol %, preferably 30 mol % to 70 mol %, and more preferably 40 mol % to 60 mol %. The copolymer may contain two or more types of repeating units containing the organic group represented by formula (a) (or the repeating units represented by formula (a1)).

[0071] The proportion of the repeating unit containing an organic group represented by formula (b) (or the repeating unit represented by formula (b1)) in the copolymer may be the entire remainder after subtracting the proportion of the above formula (a) (or formula (a1)) from the total copolymer, or may be the remainder after subtracting the total proportion of the above formula (a) (or formula (a1)) and the third component described below. The copolymer may contain two or more types of repeating units containing an organic group represented by formula (b) (or repeating units represented by formula (b1)).

[0072] Furthermore, the copolymer may be copolymerized with an optional third component. For example, a (meth)acrylate compound having two or more functional groups may be copolymerized as the third component, and a part of the polymer may be partially three-dimensionally crosslinked. Examples of such a third component include a bifunctional monomer represented by the following formula (C) or (D):

[0073] (In the formula, T ｃ , T ｄ and U ｄ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ｃ and R ｄeach independently represents a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom.) In other words, the copolymer preferably contains a crosslinked structure derived from such a bifunctional monomer.

[0074] In formulas (C) and (D), T ｃ and T ｄ are preferably each independently a hydrogen atom, a methyl group, or an ethyl group, and more preferably each independently a hydrogen atom or a methyl group.

[0075] In formulas (C) and (D), U ｄ is preferably a hydrogen atom, a methyl group or an ethyl group, and more preferably a hydrogen atom.

[0076] In formulas (C) and (D), R ｃ and R ｄ are preferably each independently a linear or branched alkylene group having 1 to 3 carbon atoms which may be substituted with a halogen atom, more preferably each independently an ethylene group or a propylene group, or an ethylene group or a propylene group substituted with one chlorine atom, and particularly preferably an ethylene group or a propylene group.

[0077] Preferred examples of the bifunctional monomer represented by formula (C) include ethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, propylene glycol di(meth)acrylate, etc. Preferred examples of the bifunctional monomer represented by formula (D) include bis(methacryloyloxymethyl) phosphate, bis[2-(methacryloyloxy)ethyl] phosphate, bis[2-(methacryloyloxy)propyl] phosphate, etc.

[0078] The optional third component may be a trifunctional monomer, such as phosphinylidintris(oxy-2,1-ethanediyl)triacrylate.

[0079] Among these, ethylene glycol di(meth)acrylate represented by the following formula (C-1) and bis[2-(methacryloyloxy)ethyl]phosphate represented by the following formula (D-1) are particularly preferred.

[0080]

[0081] The copolymer may contain one or more of these third components. Among the above, the bifunctional monomer represented by formula (D) is preferred, and the bifunctional monomer represented by formula (D-1) is particularly preferred. The proportion of the third component (for example, a crosslinked structure derived from the bifunctional monomer represented by formula (C) or (D)) in the copolymer is 0 mol % to 50 mol %.

[0082] The method for producing the copolymer is not particularly limited, and examples thereof include the copolymer production method described in WO2014 / 196652 pamphlet.

[0083] <<<Coating film of second aspect>>> The coating film may also be a coating film obtained from a composition for forming a coating film described in WO2021 / 167037 pamphlet. The contents of WO2021 / 167037 pamphlet are incorporated herein by reference to the same extent as if fully set forth herein. Hereinafter, the composition for forming a coating film described in WO2021 / 167037 pamphlet will be described. In this specification, this is referred to as a composition for forming a coating film of the second aspect.

[0084] The composition for forming a coating film of the second embodiment has the following formula (1): (In the formula, R １ represents a hydrogen atom or a methyl group, R ２ represents an alkylene group having 1 to 6 carbon atoms, and n represents an integer of 1 to 30), a compound represented by the following formula (2): (In the formula, R ３ represents a hydrogen atom or a methyl group, R ４ represents a monovalent organic group having cationic properties), and a compound represented by the following formula (3): (In the formula, R ５each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms; R ６ represents a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom, and m represents an integer of 1 to 30), in which the proportion of the compound represented by formula (3) relative to the total mass of the monomer mixture is 2 to 40 mass %, and a solvent is included.

[0085] In the compound of formula (1) which is a monomer component of the copolymer according to the second aspect, R １ represents a hydrogen atom or a methyl group, R ２ represents an alkylene group having 1 to 6 carbon atoms, preferably a linear or branched alkylene group having 1 to 6 carbon atoms, more preferably a linear or branched alkylene group having 2 to 5 carbon atoms, particularly preferably an ethylene group or a propylene group; n represents an integer of 1 to 30, preferably an integer of 1 to 20, more preferably an integer of 2 to 10, particularly preferably an integer of 3 to 6.

[0086] Specific examples of the compound of formula (1) include acid phosphooxyethyl (meth)acrylate, 3-chloro-2-acid phosphooxypropyl (meth)acrylate, acid phosphooxypropyl (meth)acrylate, acid phosphooxymethyl (meth)acrylate, acid phosphooxypolyoxyethylene glycol mono(meth)acrylate, and acid phosphooxypolyoxypropylene glycol mono(meth)acrylate, and among these, acid phosphooxyethyl methacrylate (=2-(methacryloyloxy)ethyl phosphate), acid phosphooxypolyoxyethylene glycol monomethacrylate, and acid phosphooxypolyoxypropylene glycol monomethacrylate are preferably used.

[0087] The structural formulas of acid phosphooxyethyl methacrylate (=2-(methacryloyloxy)ethyl phosphate), acid phosphooxypolyoxyethylene glycol monomethacrylate, and acid phosphooxypolyoxypropylene glycol monomethacrylate are represented by the following formulas (1-1) to (1-3), respectively.

[0088]

[0089] In the compound of formula (2) which is a monomer component of the copolymer according to the second embodiment, R ３ represents a hydrogen atom or a methyl group, R ４ represents a monovalent organic group having cationic properties, and is typically a monovalent group having a primary amine, secondary amine, tertiary amine or quaternary ammonium structure. Examples of the monovalent group having a primary amine, secondary amine, tertiary amine or quaternary ammonium structure include groups represented by the formula: -R ４ａ -NH ２ , -R ４ａ -NHR, -R ４ａ -NRR', -R ４ａ -N ＋ RR'R'' [where R ４ａ represents an alkylene group having 1 to 6 carbon atoms which may be interrupted by an ester bond, an amide bond, an ether bond or a phosphodiester bond, and R, R' and R'' are each independently a linear or branched alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 10 carbon atoms or an aralkyl group having 7 to 16 carbon atoms. In the second aspect, the monovalent group having a primary amine, secondary amine or tertiary amine structure may be quaternized or salified, and similarly, the monovalent group having a quaternary ammonium structure may be salified.

[0090] Specific examples of the compound of the above formula (2) include dimethylaminoethyl (meth)acrylate, diethylaminoethyl (meth)acrylate, dimethylaminopropyl (meth)acrylate, 2-(t-butylamino)ethyl (meth)acrylate, (meth)acryloylcholine chloride, and 2-(meth)acryloyloxyethyl phosphorylcholine (MPC).

[0091] The structural formulas of 2-(dimethylamino)ethyl methacrylate, methacroylcholine chloride, and 2-methacryloyloxyethyl phosphorylcholine (MPC) are represented by the following formulas (2-1) to (2-3), respectively.

[0092]

[0093] The compound of formula (3), which is a monomer component of the copolymer according to the second embodiment, is a bifunctional monomer having crosslinkability. ５ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, preferably a hydrogen atom or a methyl group; R ６ represents a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom, preferably a linear or branched alkylene group having 1 to 6 carbon atoms which may be substituted with a halogen atom, more preferably a linear or branched alkylene group having 1 to 6 carbon atoms which may be substituted with a chlorine atom, particularly preferably an ethylene group, a propylene group, or a trimethylene group, and m represents an integer of 1 to 30, preferably an integer of 1 to 20, more preferably an integer of 1 to 10, even more preferably an integer of 1 to 6, and particularly preferably an integer of 1 to 4.

[0094] Specific examples of the compound of the above formula (3) include poly(ethylene glycol) di(meth)acrylate, poly(trimethylene glycol) di(meth)acrylate, poly(propylene glycol) di(meth)acrylate, and the like.

[0095] The structural formulas of poly(ethylene glycol) dimethacrylate, poly(trimethylene glycol) dimethacrylate, and poly(propylene glycol) dimethacrylate are represented by the following formulas (3-1) to (3-3), respectively.

[0096]

[0097] The copolymer according to the second aspect is obtained by polymerizing a monomer mixture containing the compounds of the above formulas (1), (2), and (3) as monomer components. Such a monomer mixture may optionally contain an ethylenically unsaturated monomer, or a polysaccharide or its derivative as a fourth component, as long as the ability to inhibit adhesion of biological materials is not impaired. Examples of ethylenically unsaturated monomers include one or more ethylenically unsaturated monomers selected from the group consisting of (meth)acrylic acid and its esters; vinyl acetate; vinylpyrrolidone; ethylene; vinyl alcohol; and hydrophilic functional derivatives thereof. Examples of polysaccharides or their derivatives include cellulose-based polymers such as hydroxyalkyl cellulose (e.g., hydroxyethyl cellulose or hydroxypropyl cellulose), starch, dextran, and curdlan. The copolymer according to the second aspect is preferably obtained by polymerizing a monomer mixture containing only the compounds of the above formulas (1), (2), and (3) as monomer components.

[0098] The copolymer according to the second aspect is obtained by polymerizing a monomer mixture containing the compounds of formulas (1), (2), and (3) as monomer components, wherein the proportion of the compound of formula (3) relative to the total mass of the monomer components contained in the monomer mixture is 2 to 40 mass%. By including the bifunctional monomer compound of formula (3) in the range of 2 to 40 mass%, preferably 3 to 35 mass%, and more preferably 5 to 30 mass%, relative to the total mass of the monomer components contained in the monomer mixture, the coating film-forming composition of the second aspect, which includes the copolymer obtained from the monomer mixture, exhibits excellent ability to inhibit the adhesion of biological materials. The monomer mixture may contain two or more compounds of formula (3).

[0099] In the monomer mixture, the proportion of the compound of formula (1) relative to the total mass of the monomer components is 10 to 95 mass%, preferably 10 to 80 mass%, and more preferably 45 to 75 mass%. The monomer mixture may contain two or more types of compounds of formula (1).

[0100] In the monomer mixture, the proportion of the compound of formula (2) relative to the total mass of the monomer components is 3 to 90 mass%, preferably 10 to 70 mass%, and more preferably 15 to 35 mass%. The monomer mixture may contain two or more types of compounds of formula (2).

[0101] The copolymer according to the second aspect can be synthesized by a method such as radical polymerization, anionic polymerization, or cationic polymerization, which is a general method for synthesizing (meth)acrylic polymers. Various methods such as solution polymerization, suspension polymerization, emulsion polymerization, and bulk polymerization are possible. In one embodiment, the copolymer can be prepared by a production method including a step of reacting (polymerizing) a monomer mixture containing the compounds of the above formulas (1), (2), and (3) in a solvent at a total concentration of the monomer components of 0.01 to 20% by mass.

[0102] The solvent used in the polymerization reaction may be water, a phosphate buffer solution, an alcohol such as ethanol, or a mixed solvent of these, but preferably contains water or ethanol. It is more preferable that the solvent contains 10% by mass or more and 100% by mass or less of water or ethanol. It is even more preferable that the solvent contains 50% by mass or more and 100% by mass or less of water or ethanol. It is even more preferable that the solvent contains 80% by mass or more and 100% by mass or less of water or ethanol. It is even more preferable that the solvent contains 90% by mass or more and 100% by mass or less of water or ethanol. Preferably, the total of water and ethanol is 100% by mass.

[0103] In order to efficiently advance the polymerization reaction, it is desirable to use a polymerization initiator. Examples of polymerization initiators include "thermal radical polymerization initiators" and "photoradical polymerization initiators." Examples of "thermal radical polymerization initiators" include 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2-methylbutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile) (Fujifilm Wako Pure Chemical Industries, Ltd. product name: V-65, 10-hour half-life temperature: 51°C), 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile), 1,1'-azobis(cyclohexane-1-carbonitrile), 1-[(1-cyano 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (FUJIFILM Wako Pure Chemical Industries, Ltd.; VA-044, 10-hour half-life temperature; 44°C), 2,2'-azobis[2-(2-imidazolin-2-yl)propane] (FUJIFILM Wako Pure Chemical Industries, Ltd.; VA-061, 10-hour half-life temperature; 61°C), 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azo(2-methyl-N-(2-hydroxyethyl) Propionamide (Fujifilm Wako Pure Chemical Industries, Ltd. product name: VA-086, 10-hour half-life temperature: 86°C), benzoyl peroxide (BPO), 2,2'-azobis(N-(2-carboxyethyl)-2-methylpropionamidine) n-hydrate (Fujifilm Wako Pure Chemical Industries, Ltd. product name: VA-057, 10-hour half-life temperature: 57°C), 4,4'-azobis(4-cyanopentanoic acid) (Fujifilm Wako Pure Chemical Industries, Ltd. product name: VA-501), 2,2'-azobis[2-(2-imidazoline

[0033] 2,2'-azobis(2-amidinopropane) disulfate dihydrate (FUJIFILM Wako Pure Chemical Industries, Ltd. product name: VA-046B, 10-hour half-life temperature: 46°C), 2,2'-azobis(2-amidinopropane) dihydrochloride (FUJIFILM Wako Pure Chemical Industries, Ltd. product name: V-50, 10-hour half-life temperature: 56°C), peroxodisulfuric acid, t-butyl hydroperoxide, dimethyl 1,1'-azobis(1-cyclohexanecarboxylate) (FUJIFILM Wako Pure Chemical Industries, Ltd. product name: VE-073), and the like.

[0104] Examples of the "photoradical polymerization initiator" include acetophenone, chloroacetophenone, hydroxyacetophenone, 2-aminoacetophenone, dialkylaminoacetophenone, 2,2-diethoxyacetophenone, 2,2-dimethoxy-2'-phenylacetophenone (BASF product name: Irgacure 651), 2-hydroxy-2-methyl-1-phenylpropanone (BASF product name: Irgacure 1173), 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxymethyl ... acetophenones such as 2-hydroxy-1-{4-[4-(2-hydroxy-2-methylpropionyl)benzyl]phenyl}-2-methyl-1-propan-1-one (BASF product name: Irgacure 2959), 2-hydroxy-1-{4-[4-(2-hydroxy-2-methylpropionyl)benzyl]phenyl}-2-methyl-1-propan-1-one (BASF product name: Irgacure 127), 2-methyl-1-[4-(methylthio)phenyl]-2-morpholinopropan-1-one (BASF product name: Irgacure 907), and 2-benzyl-2-(dimethylamino)-4-morpholinobutyrophenone (BASF product name: Irgacure 369). benzoins such as benzoin, benzoin methyl ether, benzoin ethyl ether, benzoin isopropyl ether, benzoin isobutyl ether, and 1-hydroxycyclohexyl phenyl ketone (BASF product name: Irgacure 184); benzophenone, benzoylbenzoic acid, methyl benzoylbenzoate, methyl-o-benzoylbenzoate, 4-phenylbenzophenone, hydroxybenzophenone, hydroxypropylbenzophenone, acrylic benzophenone, 4,4'-bis(dimethylamino)benzoate, benzophenone, benzoylbenzoic acid, benzoylbenzoic acid methyl ester, methyl o-benzoylbenzoate, 4-phenylbenzophenone, hydroxybenzophenone, hydroxypropylbenzophenone, acrylic benzophenone, 4,4'-bis(dimethylamino)benzoate, benzophenone, benzoylbenzoic acid methyl ester ... benzophenones such as 4,4'-dichlorobenzophenone and 4,4'-dichlorobenzophenone; thioxanthone, 2-chlorothioxanthone, 2-methylthioxanthone, diethylthioxanthone, dimethylthioxanthone, and the like; acylphosphine oxides such as 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (BASF product name: Irgacure TPO) and bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide (BASF product name: Irgacure 819);Examples of such an oxyphenylacetic acid mixture include 2-[2-oxo-2-phenylacetoxyethoxy]ethyl ester and 2-(2-hydroxyethoxy)ethyl ester (BASF product name: Irgacure 754), methyl benzoylformate (BASF product name: Irgacure MBF), α-acyloxime ester, benzyl-(o-ethoxycarbonyl)-α-monoxime, glyoxyester, 2-ethylanthraquinone, camphorquinone, tetramethylthiuram sulfide, azobisisobutyronitrile, benzoyl peroxide, dialkyl peroxide, and tert-butyl peroxypivalate.

[0105] Taking into consideration solubility in water, ion balance, and interaction with the monomer, it is preferably selected from 2,2'-azo(2-methyl-N-(2-hydroxyethyl)propionamide, 2,2'-azobis(N-(2-carboxyethyl)-2-methylpropionamidine) n-hydrate, 4,4'-azobis(4-cyanopentanoic acid), 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, 2,2'-azobis[2-(2-imidazolin-2-yl)propane]disulfate dihydrate, 2,2'-azobis[2-(2-imidazolin-2-yl)propane], 2,2'-azobis(2-amidinopropane)dihydrochloride, and peroxodisulfuric acid. Considering the solubility in organic solvents, ion balance, and interaction with the monomer, it is desirable to use 2,2'-azobis(2,4-dimethylvaleronitrile) or 2,2'-azobis(isobutyronitrile).

[0106] The amount of the polymerization initiator added is 0.05% by mass to 10% by mass based on the total mass of the monomer components used in the polymerization.

[0107] The reaction conditions are as follows: the reaction vessel is heated to 50 to 200°C in an oil bath or the like, and the mixture is stirred for 1 to 48 hours, more preferably at 80 to 150°C for 5 to 30 hours, whereby the polymerization reaction proceeds and the copolymer of the second embodiment is obtained. The reaction atmosphere is preferably a nitrogen atmosphere.

[0108] As a reaction procedure, all of the reactants may be placed in a reaction solvent at room temperature and then heated to the above temperature to polymerize, or all or a portion of the mixture of reactants may be added dropwise little by little to a preheated solvent.

[0109] The weight-average molecular weight of the copolymer according to the second embodiment may be several thousand to several million, preferably 5,000 to 5,000,000, and more preferably 10,000 to 2,000,000. The copolymer may be a random copolymer, a block copolymer, or a graft copolymer, with a random copolymer being preferred. The copolymer produced in this manner is considered to be a three-dimensional polymer because it contains a bifunctional monomer, and is dissolved or dispersed in a solution containing water or alcohol.

[0110] The coating film-forming composition according to the second embodiment may be prepared by isolating and purifying the copolymer obtained in this manner and then diluting the copolymer with a desired solvent to a predetermined concentration. Furthermore, the coating film-forming composition according to the second embodiment may be prepared from the reaction solution (i.e., copolymer-containing varnish) obtained after the polymerization reaction.

[0111] Examples of the solvent contained in the coating film-forming composition of the second embodiment include water, phosphate buffered saline (PBS), and alcohol. Examples of the alcohol include alcohols having 2 to 6 carbon atoms, such as ethanol, propanol, isopropanol, 1-butanol, 2-butanol, isobutanol, t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-heptanol, 2-heptanol, 2,2-dimethyl-1-propanol (neopentyl alcohol), 2-methyl-1-propanol, 2-methyl-1-butanol, 2-methyl-2-butanol (t-amyl alcohol), 3-methyl-1-butanol, 3-methyl-3-pentanol, cyclopentanol, 1-hexanol, 2-hexanol, 3-hexanol, and 2,3-dimethyl-1-propanol (neopentyl alcohol). Examples of suitable solvents include 2-ethyl-2-butanol, 3,3-dimethyl-1-butanol, 3,3-dimethyl-2-butanol, 2-ethyl-1-butanol, 2-methyl-1-pentanol, 2-methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-1-pentanol, 3-methyl-2-pentanol, 3-methyl-3-pentanol, 4-methyl-1-pentanol, 4-methyl-2-pentanol, 4-methyl-3-pentanol, and cyclohexanol. These solvents may be used alone or in combination, but from the viewpoint of dissolving the copolymer, they are preferably selected from water, PBS, ethanol, and propanol.

[0112] The concentration of solids in the coating film-forming composition according to the second aspect is desirably 0.01 to 50% by mass in order to form a uniform coating film. The concentration of the copolymer in the coating film-forming composition is preferably 0.01 to 5% by mass, more preferably 0.01 to 4% by mass, and particularly preferably 0.01 to 3% by mass. If the concentration of the copolymer is 0.01% by mass or less, the concentration of the copolymer in the resulting coating film-forming composition is too low to form a coating film with a sufficient thickness. If the concentration is 5% by mass or more, the storage stability of the coating film-forming composition deteriorates, and precipitation or gelation of the dissolved material may occur.

[0113] Furthermore, in addition to the copolymer and the solvent, the composition for forming a coating film of the second embodiment may contain other substances as needed within the range that does not impair the performance of the resulting coating film. Examples of other substances include preservatives, surfactants, primers that improve adhesion to the substrate, antifungal agents, and sugars.

[0114] To adjust the ion balance of the copolymer in the coating film-forming composition according to the second aspect, the preparation of the coating film of the second aspect may further include a step of pre-adjusting the pH of the coating film-forming composition. The pH adjustment may be performed, for example, by adding a pH adjuster to a composition containing the copolymer and a solvent to adjust the pH of the composition to 2 to 13.5, preferably 2 to 8.5, more preferably 3 to 8, or preferably 8.5 to 13.5, more preferably 10 to 13.5. The type and amount of pH adjuster to be used are appropriately selected depending on the concentration of the copolymer and the ratio of its anions to cations. Examples of pH adjusters include organic amines such as ammonia, diethanolamine, pyridine, N-methyl-D-glucamine, and tris(hydroxymethyl)aminomethane; alkali metal hydroxides such as potassium hydroxide and sodium hydroxide; alkali metal halides such as potassium chloride and sodium chloride; inorganic acids such as sulfuric acid, phosphoric acid, hydrochloric acid, and carbonic acid, or alkali metal salts thereof; quaternary ammonium cations such as choline, and mixtures thereof (e.g., buffer solutions such as phosphate-buffered saline). Among these, ammonia, diethanolamine, sodium hydroxide, choline, N-methyl-D-glucamine, and tris(hydroxymethyl)aminomethane are preferred, and ammonia, diethanolamine, sodium hydroxide, and choline are particularly preferred.

[0115] <<<Coating Film of Third Aspect>>> The coating film may also be a coating film obtained from a composition for forming a coating film described in WO2022 / 259998. The contents of WO2022 / 259998 are incorporated herein by reference to the same extent as if fully set forth herein. The composition for forming a coating film described in WO2022 / 259998 will be described below. In this specification, this composition is referred to as a coating film-forming composition of the third aspect.

[0116] The coating film-forming composition of the third aspect is used to inhibit adhesion of biological materials. The coating film-forming composition of the third aspect contains at least a copolymer, and further contains other components such as a solvent, as necessary. Note that the coating film-forming composition of the third aspect is capable of forming a coating film that is difficult to dissolve in phosphate-buffered saline, but the use of the coating film-forming composition of the third aspect is not particularly limited as long as it is used to inhibit adhesion of biological materials, and is not limited to the formation of a coating film that comes into contact with phosphate-buffered saline.

[0117] The copolymer according to the third aspect is water-insoluble. Here, "water-soluble" means that it can dissolve 1.0 g or more in 100 g of water at 25°C. "Water-insoluble" means that it does not fall under the category of "water-soluble," i.e., its solubility in 100 g of water at 25°C is less than 1.0 g. The copolymer according to the third aspect has a repeating unit (A) represented by the following formula (A) and a repeating unit (B) represented by the following formula (B). The molar ratio (A:B) of the repeating unit (A) to the repeating unit (B) in the copolymer according to the third aspect is 89:11 to 50:50. (In the formula, R １ ~R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms; X １ and X ２ each independently represents a single bond, an ester bond, an ether bond, an amide bond, or an alkylene group having 1 to 5 carbon atoms which may be interrupted by an oxygen atom.

[0118] The copolymer related to the third aspect may have two or more types of repeating units (A). The copolymer related to the third aspect may have two or more types of repeating units (B). The copolymer related to the third aspect preferably has one type of repeating unit (A) and one type of repeating unit (B).

[0119] Examples of alkyl groups having 1 to 5 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, an s-butyl group, a t-butyl group, an n-pentyl group, a 1-methylbutyl group, a 2-methylbutyl group, a 3-methylbutyl group, a 1,1-dimethylpropyl group, a 1,2-dimethylpropyl group, a 2,2-dimethylpropyl group, and a 1-ethylpropyl group. １ ~R ３ are each independently preferably a hydrogen atom, a methyl group, or an ethyl group.

[0120] The above "ester bond" means -C(=O)-O- or -O-C(=O)-, an "ether bond" means -O-, and an "amide bond" means -NHC(=O)- or -C(=O)NH-.

[0121] The alkylene group having 1 to 5 carbon atoms may be interrupted by an oxygen atom. Examples of the alkylene group having 1 to 5 carbon atoms include a methylene group, an ethylene group, a propylene group, a trimethylene group, a tetramethylene group, a 1-methylpropylene group, a 2-methylpropylene group, a dimethylethylene group, an ethylethylene group, a pentamethylene group, a 1-methyl-tetramethylene group, a 2-methyl-tetramethylene group, a 1,1-dimethyl-trimethylene group, a 1,2-dimethyl-trimethylene group, a 2,2-dimethyl-trimethylene group, and a 1-ethyl-trimethylene group. １ and X ２ is preferably a methylene group, an ethylene group, or a propylene group. The phrase "optionally interrupted by an oxygen atom" means that one or more carbon-carbon bonds in the alkylene group having 1 to 5 carbon atoms are bonded via an ether bond.

[0122] The copolymer according to the third aspect comprises R １ and R ２is a hydrogen atom, and R ３ is a methyl group, and X １ and X ２ A copolymer in which is a single bond is preferred.

[0123] The molar ratio (A:B) of the repeating unit (A) to the repeating unit (B) is 89:11 to 50:50. In the copolymer related to the third aspect, when the total number of moles of the repeating unit (A) and the repeating unit (B) is 100, the molar ratio (A:B) of the repeating unit (A) to the repeating unit (B) can be expressed as (100-m):m. In this case, the range of m is 11 to 50. The lower limit of m may be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. The upper limit of m may be 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 38, 37, 36, or 35. The range of m is, for example, 12 to 49, 12 to 48, 15 to 48, 20 to 49, 20 to 45, 22 to 49, or 22 to 45.

[0124] The total mol % of the repeating unit (A) and the repeating unit (B) in all repeating units in the copolymer related to the third aspect is not particularly limited, but is preferably 90 mol % or more, more preferably 95 mol % or more, even more preferably 99.5 mol % or more, and particularly preferably 100%.

[0125] In the third aspect, the molar ratio of the repeating unit (A) to the repeating unit (B) in the copolymer is set to a specific range in order to obtain a coating film that is difficult to dissolve in phosphate-buffered saline. Therefore, in the third aspect, a coating film that is difficult to dissolve in phosphate-buffered saline can be obtained without crosslinking the copolymer. Therefore, the copolymer does not need to have a photosensitive group for crosslinking the copolymer. That is, the copolymer preferably does not have a photosensitive group. Examples of photosensitive groups include azide groups. In the third aspect, the copolymer does not need to have a photosensitive group for crosslinking the copolymer. Therefore, when forming a coating film, light irradiation for crosslinking the copolymer is not required. This simplifies the process of forming a coating film.

[0126] The viscosity average degree of polymerization of the copolymer according to the third aspect (hereinafter, sometimes simply referred to as "degree of polymerization") is not particularly limited, but from the viewpoint of suitably obtaining the effects of the third aspect, it is preferably 200 to 3,000, more preferably 200 to 2,500, and particularly preferably 200 to 2,000. The viscosity average degree of polymerization is measured when the copolymer is in a fully saponified state. The "viscosity average degree of polymerization" of polyvinyl alcohol obtained by fully saponifying is a value calculated by the following formula from the limiting viscosity [η] (g / dL) measured at 30°C using an Ostwald viscometer with ion-exchanged water as a solvent: log(P)=1.613×log([η]×10 ４ / 8.29) where P represents the viscosity-average degree of polymerization. The viscosity-average degree of polymerization can be determined in accordance with JIS K 6726.

[0127] The method for producing the copolymer related to the third aspect is not particularly limited, but for example, a method can be mentioned in which a compound represented by the following formula (C) is polymerized to produce a homopolymer, and the obtained homopolymer is partially hydrolyzed by a known saponification reaction to obtain the copolymer. (In the formula, R １ , R ３ , and X １ has the same meaning as above.)

[0128] Furthermore, as a method for producing the copolymer related to the third aspect, for example, a method of copolymerizing a compound represented by the following formula (C) and a compound represented by the following formula (D) to obtain the copolymer can be mentioned. (In the formula, R １ ~R ３ , X １ , and X ２ has the same meaning as above.)

[0129] The copolymer according to the third aspect may be a random copolymer or a block copolymer. As the copolymer according to the third aspect, a commercially available product may be used. A specific example of a commercially available copolymer is polyvinyl acetate (manufactured by Nippon Vinyl Acetate & Poval, trade name JMR-10L (registered trademark)).

[0130] The content of the copolymer in the film-forming component in the coating film-forming composition of the third aspect is not particularly limited, but is preferably 80% by mass or more, more preferably 90% by mass or more, and particularly preferably 95% by mass or more. Note that the film-forming component refers to all components of the composition excluding the solvent component.

[0131] The content of the copolymer in the composition for forming a coating film of the third aspect is not particularly limited, but from the viewpoint of easily forming a coating film of a desired thickness, it is preferably 0.1 to 10 mass %, more preferably 0.3 to 8 mass %, and particularly preferably 0.5 to 5 mass %. Furthermore, the content of the copolymer in the composition for forming a coating film may be 0.02 to 2 mass %, or may be 0.05 to 1 mass %.

[0132] Examples of the solvent according to the third embodiment include water, phosphate buffered saline (PBS), alcohol, and water-soluble organic solvents (excluding alcohol).

[0133] Examples of the alcohol include alcohols having 2 to 6 carbon atoms. Examples of the alcohol include ethanol, propanol, isopropanol, 1-butanol, 2-butanol, isobutanol, t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-heptanol, 2-heptanol, 2,2-dimethyl-1-propanol (neopentyl alcohol), 2-methyl-1-propanol, 2-methyl-1-butanol, 2-methyl-2-butanol (t-amyl alcohol), 3-methyl-1-butanol, 3-methyl-3-pentanol, cyclopentanol, 1-heptanol, 2-methyl-2-butanol (t-amyl alcohol), 2-methyl-3-pentanol, ... Examples of the alcohols include cyclohexanol, 2-hexanol, 3-hexanol, 2,3-dimethyl-2-butanol, 3,3-dimethyl-1-butanol, 3,3-dimethyl-2-butanol, 2-ethyl-1-butanol, 2-methyl-1-pentanol, 2-methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-1-pentanol, 3-methyl-2-pentanol, 3-methyl-3-pentanol, 4-methyl-1-pentanol, 4-methyl-2-pentanol, 4-methyl-3-pentanol, and cyclohexanol. These may be used alone or in combination of two or more.

[0134] The water-soluble organic solvent refers to an organic solvent that can be mixed with water and alcohol in any ratio and does not separate after mixing. Examples of water-soluble organic solvents include ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, methyl cellosolve acetate, ethyl cellosolve acetate, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, propylene glycol, propylene glycol monomethyl ether, propylene glycol monoethyl ether, propylene glycol monomethyl ether acetate, and propylene glycol propyl ether acetate. These can be used alone or in combination of two or more.

[0135] In the coating film-forming composition of the third aspect, water, phosphate buffered saline (PBS), alcohol, or water-soluble organic solvent may be used alone as the solvent. In the coating film-forming composition of the third aspect, two or more of water, phosphate buffered saline (PBS), alcohol, and water-soluble organic solvent may be used in combination as the solvent. From the viewpoint of the solubility of the copolymer, the solvent is preferably selected from water, alcohol, water-soluble organic solvent, and a combination of two or more thereof, and more preferably selected from water, ethanol, water-soluble organic solvent, and a combination of two or more thereof.

[0136] The following combinations of solvents are preferred: Water and alcohol; Water, alcohol and a water-soluble organic solvent; Alcohol and a water-soluble organic solvent.

[0137] The following combinations of solvents are more preferred: Water and ethanol Water, ethanol and propylene glycol monomethyl ether Ethanol and propylene glycol monomethyl ether

[0138] The mass ratio of water:alcohol in the composition for forming a coating film of the third aspect is, for example, 1:99 to 70:30, or 1:99 to 50:50. The mass ratio of water:alcohol:water-soluble organic solvent (A:B:C) in the composition for forming a coating film of the third aspect is, for example, 5 to 30:65 to 92:1 to 30 (provided that A+B+C is 100). The mixing ratio (mass ratio) of alcohol:water-soluble organic solvent in the composition for forming a coating film of the third aspect is, for example, 30:70 to 97:3.

[0139] The content of the solvent in the composition for forming a coating film of the third aspect is not particularly limited, but from the viewpoint of easily forming a coating film of a desired thickness, it is preferably 90% by mass or more, more preferably 92% by mass or more, and particularly preferably 95% by mass or more.

[0140] The coating film-forming composition of the third embodiment may contain other components as needed, such as a pH adjuster, a preservative, a surfactant, an anti-mold agent, and a sugar.

[0141] An example of a cell structure of the present invention will be described using the drawings. The cell structure 100 shown in Figures 1A and 1B has a main body 1 and cell adhesive substances 2, which are spots. The main body 1 has an outer peripheral wall 1A, an inner bottom surface 1B, and an opening 1C. The opening 1C is an opening formed by the outer peripheral wall 1A, and the inner bottom surface 1B is located below the opening 1C. A plurality of cell adhesive substances 2 are arranged at intervals on the inner bottom surface 1B.

[0142] The cell structure 100 shown in Figures 2A and 2B has a main body 1, a cell adhesive substance 2, and a cell culture base membrane 3, which is a spot. The main body 1 has an outer peripheral wall 1A, an inner bottom surface 1B, and an opening 1C. The opening 1C is an opening formed by the outer peripheral wall 1A, and the inner bottom surface 1B is located below the opening 1C. Multiple cell culture base membranes 3 are arranged at intervals on the inner bottom surface 1B. The cell adhesive substance 2 is arranged on the cell culture base membrane 3.

[0143] The cell structure 100 shown in Figures 3A and 3B has a main body 1, a cell adhesive substance 2, a cell culture substrate membrane 3 as a spot, and a coating film 4. The main body 1 has an outer peripheral wall 1A, an inner bottom surface 1B, and an opening 1C. The opening 1C is an opening formed by the outer peripheral wall 1A, and the inner bottom surface 1B is located below the opening 1C. The surface of the inner bottom surface 1B has a coating film 4 that has the ability to inhibit cell adhesion. Multiple cell culture substrate membranes 3 are arranged at intervals on the coating film 4. The cell adhesive substance 2 is arranged on the cell culture substrate membrane 3.

[0144] (Method for manufacturing a cell structure) The method for manufacturing a cell structure of the present invention is a method for manufacturing a cell structure having a substrate and a plurality of spots arranged at intervals on the surface of the substrate.

[0145] One embodiment of the method for producing a cell structure includes the following step (A1) on a substrate: Step (A1): A step of applying droplets containing a cell adhesive substance to a substrate to arrange the cell adhesive substance in areas to be spots.

[0146] Another embodiment of the method for producing a cell structure comprises the following steps (A2) and (B2): Step (A2): A step of applying droplets of a cell culture base film forming agent to a substrate to form a cell culture base film in the areas to be spots; Step (B2): A step of applying droplets containing a cell adhesive substance to the cell culture base film to arrange the cell adhesive substance on the cell culture base film.

[0147] In the cell structure to be produced, the surface of the substrate may have a coating film capable of inhibiting cell adhesion, and a plurality of spots may be arranged on the coating film. In this case, the method for producing a cell structure may further include the following step (C): Step (C): Applying a coating agent containing a polymer capable of inhibiting cell adhesion onto the substrate to form a coating film.

[0148] The method for producing a cell structure of the present invention is, for example, a method for producing a cell structure of the present invention.

[0149] <Step (A1)> Step (A1) is a step of applying droplets containing a cell adhesive substance to a substrate to deposit the cell adhesive substance in areas that will become spots.

[0150] Examples of the cell adhesive substance include the cell adhesive substances mentioned in the description of the cell structure of the present invention.

[0151] The droplets containing the cell adhesive substance are formed, for example, from a liquid containing the cell adhesive substance. The liquid containing the cell adhesive substance contains, for example, the cell adhesive substance and a solvent. Examples of the solvent include the solvents listed in the description of the cell culture base film forming agent described below.

[0152] The method for applying droplets containing a cell adhesive substance is not particularly limited, and may be an inkjet method, a screen printing method, a slit coating method, a roll-to-roll method, or the like, but is preferably carried out by a printing technique such as an inkjet method or screen printing.

[0153] <Step (A2)> Step (A2) is a step of applying droplets of a cell culture base film forming agent to a substrate to form a cell culture base film in areas that will become spots.

[0154] Examples of the substrate include the substrates mentioned in the description of the cell structure of the present invention.

[0155] A cell culture base film forming agent (hereinafter sometimes referred to as "base film forming agent") contains a polymer and a solvent, and may further contain other components. Examples of the polymer include the polymers mentioned in the description of the cell structure of the present invention. The solvent is not particularly limited, but is preferably an aqueous solution containing water. Examples of aqueous solutions include water, salt-containing aqueous solutions such as physiological saline or phosphate buffer solution, and mixed solvents combining water or a salt-containing aqueous solution with an alcohol. Examples of alcohols include alcohols having 2 to 6 carbon atoms, such as ethanol, propanol, isopropanol, 1-butanol, 2-butanol, isobutanol, t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-heptanol, 2-heptanol, 2,2-dimethyl-1-propanol (neopentyl alcohol), 2-methyl-1-propanol, 2-methyl-1-butanol, 2-methyl-2-butanol (t-amyl alcohol), 3-methyl-1-butanol, 3-methyl-3-pentanol, cyclopentanol, and 1-hexanol. Examples of suitable solvents include 2-methyl-1-pentanol, 2-methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-1-pentanol, 3-methyl-2-pentanol, 3-methyl-3-pentanol, 4-methyl-1-pentanol, 4-methyl-2-pentanol, 4-methyl-3-pentanol, and cyclohexanol. These may be used alone or in combination. The water content in the aqueous solution is, for example, 50% to 100% by mass, 80% to 100% by mass, or 90% to 100% by mass. In addition to the above-mentioned polymer, cell adhesive substance, and solvent, the primer film-forming agent may also contain other substances as needed within the range that does not impair the performance of the resulting primer film, such as pH adjusters, crosslinking agents, preservatives, surfactants, primers that improve adhesion to the container or substrate, antifungal agents, and sugars.

[0156] The method for applying droplets of the primer film-forming agent to the substrate is not particularly limited, and examples thereof include inkjet printing, screen printing, slit coating, and roll-to-roll printing. Preferably, printing techniques such as inkjet printing or screen printing are used. Other application methods include, for example, immersing a substrate, optionally protecting areas that will not become spots, in the primer film-forming agent, or adding the primer film-forming agent to a substrate (container), optionally protecting areas that will not become spots, and allowing it to stand for a predetermined period of time. In one embodiment, when the cell structure is a cell culture vessel, the primer film-forming agent is added to a container, optionally protecting areas that will not become spots, and allowing it to stand for a predetermined period of time. Addition can be carried out, for example, by adding the primer film-forming agent in an amount that is 0.5 to 1 times the total volume of the container using a syringe or the like. The time and temperature for allowing it to stand are appropriately selected depending on the material of the container or substrate and the type of primer film-forming agent for cell culture. For example, the time and temperature are set at 10 to 80°C for 1 minute to 24 hours, preferably 5 minutes to 3 hours.

[0157] Furthermore, after forming a cell culture base film on the surface of the substrate by such a method, step (B2) may be carried out either directly without a drying step or after washing with water or a medium for the sample to be subjected to cell culture (e.g., water, a buffer solution, a culture medium, etc.).

[0158] <Step (B2)> Step (B2) is a step of applying droplets containing a cell adhesive substance to a cell culture substrate membrane to arrange the cell adhesive substance on the cell culture substrate membrane.

[0159] Examples of the cell adhesive substance include the cell adhesive substances mentioned in the description of the cell structure of the present invention.

[0160] The droplets containing the cell adhesive substance are formed, for example, from a liquid containing the cell adhesive substance. The liquid containing the cell adhesive substance contains, for example, the cell adhesive substance and a solvent. Examples of the solvent include the solvents listed in the description of the cell culture base film forming agent.

[0161] The method for applying droplets containing a cell adhesive substance is not particularly limited, and may be an inkjet method, a screen printing method, a slit coating method, a roll-to-roll method, or the like, but is preferably carried out by a printing technique such as an inkjet method or screen printing.

[0162] <Step (C)> Step (C) is a step of applying a coating agent containing a polymer capable of inhibiting cell adhesion onto a substrate to form a coating film. The coating agent is not particularly limited as long as it contains a polymer capable of inhibiting cell adhesion, but for example, it contains a polymer capable of inhibiting cell adhesion and a solvent. Examples of coating films and coating agents include the coating films and coating agents mentioned in the description of the cell structure of the present invention. The method for applying the coating agent onto the substrate is not particularly limited, but can be an inkjet method, screen printing method, slit coating method, roll-to-roll method, etc., but is preferably performed using a printing technique such as an inkjet method or screen printing.

[0163] (Cell Culture Method) The cell culture method (cell production method) of the present invention includes a step of seeding cells into the cell structure of the present invention. Examples of the cell seeding step include adding a medium in which cells are dispersed to the cell structure of the present invention.

[0164] The cell structure is preferably a cell culture vessel. In the cell structure, each of the plurality of spots preferably has a cell culture base membrane containing a polymer and a cell adhesive substance disposed on the cell culture base membrane.

[0165] The cell concentration in the medium in which the cells are dispersed is not particularly limited.

[0166] A cell is the most basic unit constituting an animal or plant, and its elements include cytoplasm and various organelles within the cell membrane. In this case, the nucleus containing DNA may or may not be contained within the cell. For example, animal-derived cells in the present invention include germ cells such as sperm and eggs, somatic cells that constitute an organism, stem cells (such as pluripotent stem cells), progenitor cells, cancer cells isolated from an organism, cells isolated from an organism that have acquired immortalization and are stably maintained outside the body (cell lines), cells isolated from an organism that have been artificially genetically modified, and cells isolated from an organism that have undergone artificial nucleus replacement. Examples of somatic cells that make up a living organism include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes, osteocytes, bone marrow cells, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatic parenchymal cells, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes, cardiac cells, esophageal cells, muscle cells (e.g., smooth muscle cells or skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic progenitor cells (e.g., CD34-positive cells derived from umbilical cord blood), and mononuclear cells. The somatic cells include cells collected from any tissue, such as skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood (including umbilical cord blood), bone marrow, heart, cardiac muscle, eye, brain, neural tissue, hair, etc. Furthermore, the somatic cells include cells induced to differentiate from stem cells or progenitor cells.

[0167] Stem cells are cells that have the ability to replicate themselves and differentiate into cells of multiple lineages. Examples include, but are not limited to, embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, and hair follicle stem cells. Among the stem cells, pluripotent stem cells include ES cells, embryonic germ stem cells, and iPS cells. Progenitor cells are cells that are in the process of differentiating from the stem cells into specific somatic cells or germ cells. Cancer cells are cells that are derived from somatic cells and have acquired the ability to proliferate indefinitely. Cell lines are cells that have acquired the ability to proliferate indefinitely through artificial manipulation outside of the body. Among these, fibroblasts and stem cells are preferred, and among stem cells, pluripotent stem cells are more preferred.

[0168] The medium can be appropriately selected depending on the type of cells to be used, etc. For example, when the purpose is to culture mammalian cells, a medium generally used for culturing mammalian cells can be used as the medium. Examples of media for mammalian cells include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A medium, Eagle's Minimum Essential Medium (EMEM), αMEM (alpha Modified Eagle's Minimum Essential Medium; αMEM), and MEM (Minimum Essential Medium). Medium), RPMI1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, IPL41 medium, Fischer's medium, StemPro34 (Invitrogen), X-VIVO 10 (Cambrex), X-VIVO 15 (Cambrex), HPGM (Cambrex), StemSpan Examples of such medium include H3000 (manufactured by Stem Cell Technology), StemSpan SFEM (manufactured by Stem Cell Technology), Stemline II (manufactured by Sigma-Aldrich), QBSF-60 (manufactured by Quality Biologicals), StemPro hESCSFM (manufactured by Invitrogen), mTeSR1 or 2 medium (manufactured by Stem Cell Technology), Sf-900II (manufactured by Invitrogen), Opti-Pro (manufactured by Invitrogen), and HuMedia-KG2 (manufactured by Kurabo Industries, Ltd.).

[0169] Those skilled in the art may freely add sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various vitamins, antibiotics, serum, fatty acids, sugars, etc. to the above-mentioned medium depending on the purpose. When culturing mammalian cells, those skilled in the art may also add one or more other chemical or biological components in combination depending on the purpose.

[0170] Components that can be added to culture media for mammalian cells include fetal bovine serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, monothioglycerol, 2-mercaptoethanol, bovine serum albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, various cytokines, various hormones, various growth factors, various extracellular matrices, and various cell adhesion molecules.

[0171] Examples of cytokines that can be added to the culture medium include interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), and interleukin-15 (IL-15). Examples of the immunosuppressant include, but are not limited to, interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), granulocyte colony-stimulating factor (G-CSF), monocyte colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), flk2 / flt3 ligand (FL), leukemia inhibitory factor (LIF), oncostatin M (OM), erythropoietin (EPO), and thrombopoietin (TPO).

[0172] Hormones that may be added to the medium include melatonin, serotonin, thyroxine, triiodothyronine, epinephrine, norepinephrine, dopamine, anti-Mullerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen and angiotensin, antidiuretic hormone, atrial natriuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, erythropoietin, follicle-stimulating hormone, gastrin, ghrelin, glucagon, gonadotropin-releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin, insulin-like growth factor, leptin, luteinizing hormone, melanocyte-stimulating hormone, and the like. Examples of hormones that may be involved in the production of steroid hormones include, but are not limited to, oxytocin, parathyroid hormone, prolactin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone, thyrotropin-releasing hormone, cortisol, aldosterone, testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, estradiol, estrone, estriol, progesterone, calcitriol, calcidiol, prostaglandins, leukotrienes, prostacyclin, thromboxane, prolactin-releasing hormone, lipotropin, brain natriuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptide, renin, and enkephalins.

[0173] Growth factors that can be added to the culture medium include transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), macrophage inflammatory protein-1α (MIP-1α), epidermal growth factor (EGF), fibroblast growth factor-1, 2, 3, 4, 5, 6, 7, 8, or 9 (FGF-1, 2, 3, 4, 5, 6, 7, 8, 9), nerve growth factor (NGF), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), protease inhibitory factor (PI), and phospholipase A (PG). Examples of such proteins include, but are not limited to, protease nexin I, protease nexin II, platelet-derived growth factor (PDGF), cholinergic differentiation factor (CDF), chemokine, Notch ligand (Delta1, etc.), Wnt protein, angiopoietin-like protein 2, 3, 5, or 7 (Angpt2, 3, 5, 7), insulin-like growth factor (IGF), insulin-like growth factor binding protein (IGFBP), and pleiotrophin.

[0174] Furthermore, cytokines and growth factors whose amino acid sequences have been artificially modified by genetic recombination techniques can also be added, such as IL-6 / soluble IL-6 receptor complex and Hyper IL-6 (a fusion protein of IL-6 and soluble IL-6 receptor).

[0175] Examples of various extracellular matrices and various cell adhesion molecules include collagens I to XIX, fibronectin, laminin-1 to -12, nitrogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocollins, desmogleins, various integrins, E-selectin, P-selectin, L-selectin, the immunoglobulin superfamily, Matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, Sepharose, hyaluronic acid, alginate gel, various hydrogels, and cleaved fragments thereof.

[0176] Examples of antibiotics that can be added to the medium include sulfa drugs, penicillin, phenethicillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, cyclacillin, carbenicillin, ticarcillin, piperacillin, azlocillin, mexlocillin, mecillinam, andinocillin, cephalosporin and its derivatives, oxolinic acid, amifloxacin, temafloxacin, nalidixic acid, piromidic acid, ciprofloxacin, cinoxacin, norfloxacin, perfloxacin, rozaxacin, ofloxacin, enoxacin, pipemi penicillanic acid, sulbactam, clavulic acid, β-bromopenicillanic acid, β-chloropenicillanic acid, 6-acetylmethylene-penicillanic acid, cefoxazole, sultampicillin, formaldehyde foudate esters of adinocillin and sulbactam, tazobactam, aztreonam, sulfazetine, isosulfazetine, nocardicin, methyl phenylacetamidophosphonate, chlortetracycline, oxytetracycline, tetracycline, demeclocycline, doxycycline, methacycline, and minocycline.

[0177] As described above, serum and / or serum substitutes may be added to the culture medium. The concentration of serum added to the culture medium can be appropriately determined depending on the cell type, culture conditions, culture purpose, etc., but in one embodiment, the concentration of serum (and / or serum substitute) in the culture medium can be 15% by weight or less, 10% by weight or less, 9% by weight or less, 8% by weight or less, 7% by weight or less, 6% by weight or less, 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, 1% by weight or less, 0.9% by weight or less, 0.8% by weight or less, 0.7% by weight or less, 0.6% by weight or less, 0.5% by weight or less, 0.4% by weight or less, or 0.3% by weight or less. The serum concentration may be kept constant throughout the culture period, or may be increased or decreased as needed, for example, when changing the culture medium.

[0178] An example of the cell culture method of the present invention is a method for producing a cell structure. The method for producing a cell structure includes the following step (1), and may further include the following steps (2) to (4): Step (1): Adding a medium containing dispersed cells to the cell structure of the present invention; Step (2): Culturing the cells to form a cell structure; Step (3): Replacing the medium; Step (4): Maturating the cell structure

[0179] The method for producing a cell structure may further include the following steps (5) and (6): step (5): suspending the cell structure in a medium; and step (6): recovering the cell structure.

[0180] A cell structure is a cell aggregate formed by self-assembly of cells into a three-dimensional aggregate. The cell structure is also called a cell aggregate, spheroid, cell mass, etc. The shape of the cell structure is not particularly limited, and examples thereof include a spherical shape and a ring shape.

[0181] Each step will be explained below. <Step (1)> Step (1) is a step of adding a medium in which cells have been dispersed to the cell structure of the present invention. Step (1) is a step of preparing to culture cells, and for example, the following total number of cells is dispersed in the medium and added to the cell structure. The lower limit of the total number of cells is, for example, equal to or greater than the number of spots (n) present in the cell structure. The medium is adjusted according to the cells to be cultured. The type of cell may be one type, or two or more types of cells may be used.

[0182] <Step (2)> Step (2) is a step of culturing cells to form a cell structure. In step (2), for example, cells are cultured in a cell structure for 12 hours or more to form a cell structure. When medium is added to the cell structure, the cells dispersed in the medium remain in the spots and are cultured in each spot. It is preferable that a certain number of cells remain in each spot, and it is preferable that one cell structure is formed in each spot. In order to culture cell structures at a high density, it is preferable that cell structures are formed in all spots, and therefore it is preferable that at least one cell is present in each spot.

[0183] <Step (3)> Step (3) is a step of exchanging the culture medium. In exchanging the culture medium, a certain amount of the culture medium in the cell structure is aspirated, and then the same amount of fresh culture medium is injected. It is preferable that the culture medium is exchanged at least once during cell culture. The certain amount may be, for example, 20% to 80% by mass, or 20% to 50% by mass of the culture medium. Examples of the culture medium include those described above.

[0184] <Step (4)> Step (4) is a step of maturing the cell structure. In step (4), steps (2) and (3) are repeated multiple times to mature the cell structure. "Maturation" means, for example, enlarging the cell structure to a desired size, forming an organoid structure, or inducing differentiation into a target tissue. When inducing differentiation, it is preferable to form a cell structure in the spot and then exchange the medium for a differentiation-inducing medium to differentiate it.

[0185] <Step (5)> Step (5) is a step of suspending the cell structures in the medium. In step (5), for example, after the cell structures have grown to a desired size, the medium is stirred to suspend the cell structures cultured in each spot in the medium. For example, this is carried out by stirring the medium. Specifically, stirring the medium can be performed by (i) shaking the cell structures to stir the medium, (ii) aspirating and discharging the medium (pipetting), (iii) installing a stirring blade to stir the medium, or (iv) inserting a stirring bar into the cell structures to stir the medium. Two or more of these may be combined.

[0186] <Step (6)> Step (6) is a step of recovering the cell structure. In step (6), for example, the medium in which the cell structure of the cell structure is suspended is sucked up with a suction machine, and the cell structure suspended in the medium is recovered.

[0187] The present invention will be described in more detail below based on synthesis examples, examples, test examples, etc., but the present invention is not limited to these.

[0188] <Method for Measuring Molecular Weight> The weight-average molecular weights shown in the following synthesis examples are the results obtained by gel filtration chromatography (hereinafter abbreviated as GFC). (Measurement conditions) Apparatus: HLC-8320GPC (manufactured by Tosoh Corporation) GFC column: TSKgel G 6000 + 3000 PWXL-CP Flow rate: 1.0 mL / min Eluent: salt-containing water / organic mixed solvent Column temperature: 40°C Detector: RI Injection concentration: polymer solids content 0.05 mass% Injection volume: 100 μL Calibration curve: cubic approximation curve Standard sample: polyethylene oxide (manufactured by Agilent) x 10 types

[0189] Synthesis Example 1 24.00 g of 2-(dimethylamino)ethyl methacrylate (Tokyo Chemical Industry Co., Ltd.), 1.46 g of methacrylic acid (Tokyo Chemical Industry Co., Ltd.), 5.09 g of ethylene glycol dimethacrylate (Tokyo Chemical Industry Co., Ltd.), 0.31 g of dimethyl 1,1'-azobis(1-cyclohexanecarboxylate) (VE-073, Fujifilm Wako Pure Chemical Industries, Ltd.), and 111.09 g of 2-propanol were mixed and polymerized dropwise into 166.62 g of 2-propanol at reflux temperature to synthesize a polymer. The reaction product was reprecipitated in hexane, a poor solvent, and the precipitate was collected by filtration and dried under reduced pressure. The weight average molecular weight of this polymer measured by GFC was 228,000.

[0190] Preparation Example 1 Polyvinyl acetate (JMR-150L (registered trademark) (polymerization degree 1480, saponification degree 22.7%), manufactured by Nippon Vinyl Acetate & Poval Co., Ltd.) was dissolved in ethanol / 1-methoxy-2-propanol (7 / 3 mass ratio) to a concentration of 3.0 mg / g to prepare a coating film-forming composition 1 having cell adhesion inhibitory properties. The resulting composition was transparent and uniform.

[0191] Preparation Example 2 Polyvinyl acetate (JMR-150L (registered trademark) manufactured by Nippon Vinyl Acetate & Poval Co., Ltd. (degree of polymerization: 1480, degree of saponification: 22.7%)) was dissolved in ethanol / 1-methoxy-2-propanol (85 / 15 mass ratio) to a concentration of 20 mg / g to prepare a coating film-forming composition 2 having cell adhesion inhibitory properties. The resulting composition was transparent and uniform.

[0192] Preparation Example 3: 49.95 g of sterilized water was added and thoroughly stirred to 0.05 g of the polymer obtained in Synthesis Example 1. 24.38 g of sterilized water was added to 4.30 g of the resulting solution and thoroughly stirred to prepare primer film-forming agent 1.

[0193] Preparation Example 4: 49.95 g of sterile water was added and thoroughly stirred to 0.05 g of the polymer obtained in Synthesis Example 1. To 4.94 g of the resulting solution, 26.96 g of pure water and 0.98 g of Recombinant Human Vitronectin (manufactured by Peprotech) diluted to 1.0 mg / mL with sterile water were added and thoroughly stirred to prepare primer film-forming agent 2.

[0194] Preparation Example 5: 49.95 g of sterile water was added to 0.05 g of the polymer obtained in Synthesis Example 1 and thoroughly stirred. 41.97 g of pure water and 0.60 g of GRGDS (manufactured by Peptide Institute) diluted to 2.5 mg / mL with sterile water were added to 7.50 g of the resulting solution and thoroughly stirred to prepare primer film-forming agent 3.

[0195] Preparation Example 6 32.82 g of sterile water was added to 0.016 g of cell adhesive peptide PASAP1 (manufactured by Nanomed3D) and the mixture was thoroughly stirred to prepare temporary film-forming agent 1.

[0196] Preparation Example 7 0.5 mg / mL vitronectin VTN-N (Gibco) was used as the temporary film forming agent 2.

[0197] Preparation Example 8 16.41 g of sterile water was added to 0.016 g of the cell adhesive peptide PASAP1 (manufactured by Nanomed3D) and thoroughly stirred. 17.40 g of sterile water and 0.60 g of V-02 (manufactured by Nisshinbo Chemical Inc.) diluted to 5.0 mg / mL with sterile water were added to 6.0 g of the above solution and thoroughly stirred to prepare temporary film-forming agent 3.

[0198] Preparation Example 9 16.41 g of sterile water was added to 0.016 g of the cell adhesive peptide PASAP2 (manufactured by Nanomed3D) and thoroughly stirred. 17.40 g of sterile water and 0.60 g of V-02 (manufactured by Nisshinbo Chemical Inc.) diluted to 5.0 mg / mL with sterile water were added to 6.0 g of the above solution and thoroughly stirred to prepare temporary film-forming agent 4.

[0199] Preparation Example 10: 49.95 g of sterilized water was added and thoroughly stirred to 0.05 g of the polymer obtained in Synthesis Example 1. 48.9 g of pure water was added to 1.00 g of the resulting solution and thoroughly stirred to prepare primer film-forming agent 4.

[0200] Preparation Example 1 (Preparation of a substrate capable of inhibiting cell adhesion by inkjet) Using an inkjet device (Seiko Epson Corporation, R&D inkjet device) and an inkjet head (Seiko Epson Corporation, Precision Core Head S800-A1), an appropriate amount of the coating film-forming composition 1 prepared in Preparation Example 1 was applied to a polystyrene substrate measuring 79 mm x 121 mm in the form of a perfect circle with a diameter of 18 mm. This was dried in an oven at 70°C for 24 hours to prepare a substrate 1 capable of inhibiting cell adhesion.

[0201] Preparation Example 2 (Preparation of a substrate capable of inhibiting cell adhesion using a film applicator) Using a film applicator 100-3 (manufactured by Allgood Co., Ltd.) and a bar (wet film thickness 10 μm, manufactured by Allgood Co., Ltd.), an appropriate amount of the coating film-forming composition 2 prepared in Preparation Example 2 was applied to a polystyrene substrate having a size of 79 mm × 121 mm. The substrate was dried in an oven at 70°C for 24 hours to prepare a substrate 2 capable of inhibiting cell adhesion.

[0202] Example 1 Using an inkjet device (Seiko Epson Corporation, R&D inkjet device) and an inkjet head (Seiko Epson Corporation, Precision Core Head S800-A1), an appropriate amount of base film forming agent 2 was applied to the culture surface of substrate 1 having the ability to inhibit cell adhesion, prepared in Preparation Example 1, to form multiple spots with a diameter of 400 μm. After drying at room temperature, an appropriate amount of temporary film forming agent 2 was applied to the center of the 400 μm diameter spots prepared above using an inkjet device (Microjet Corporation, model number: LaboJet-600) and an inkjet head (model number: IJHBS-300), to form multiple spots with a diameter of 100 to 150 μm. The substrate was dried for one day in a constant temperature dryer at 70°C to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0203] Example 2 Using an inkjet device (Microjet Corporation, model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000), an appropriate amount of base film forming agent 1 was applied to the culture surface of a commercially available low-adhesion cell plate, PrimeSurface 24F (Sumitomo Bakelite Co., Ltd.), forming multiple spots with a diameter of 400 μm. After drying at room temperature, an appropriate amount of temporary film forming agent 2 was applied to the center of the 400 μm diameter spots created above using an inkjet device (Microjet Corporation, model number: LaboJet-600) and an inkjet head (model number: IJHBS-300), forming multiple spots with a diameter of 100 to 150 μm. The plate was dried in a thermostatic oven at 70°C for one day to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0204] Example 3 Using an inkjet device (manufactured by Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000), an appropriate amount of base film forming agent 1 was applied to the culture surface of a commercially available low-adhesion cell plate, PrimeSurface 24F (manufactured by Sumitomo Bakelite Co., Ltd.), forming multiple spots with a diameter of 400 μm. After drying at room temperature, an appropriate amount of temporary film forming agent 1 was applied to the center of the 400 μm diameter spots created above using an inkjet device (manufactured by Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: IJHBS-300), forming multiple spots with a diameter of 100 to 150 μm. The plate was dried for 1 day in a constant temperature dryer at 70°C, preparing a substrate for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0205] Example 4 Using an inkjet device (Seiko Epson Corporation, R&D inkjet device) and an inkjet head (Seiko Epson Corporation, Precision Core Head S800-A1), an appropriate amount of base film forming agent 3 was applied to the culture surface of substrate 2 having the ability to inhibit cell adhesion, prepared in Preparation Example 2, to form a large number of spots with a diameter of 400 μm. After drying at room temperature, an appropriate amount of temporary film forming agent 3 was applied to the center of the 400 μm diameter spots prepared above, to form a large number of spots with a diameter of 100 to 150 μm. The substrate was dried for one day in a constant temperature dryer at 70°C, to prepare a substrate for producing cell aggregates. Sterilization was carried out by irradiation with gamma rays at 25 kGy.

[0206] Example 5 Using an inkjet device (Seiko Epson Corporation, R&D inkjet device) and an inkjet head (Seiko Epson Corporation, Precision Core Head S800-A1), an appropriate amount of base film forming agent 3 was applied to the culture surface of substrate 2 having the ability to inhibit cell adhesion, prepared in Preparation Example 2, to form a large number of spots with a diameter of 400 μm. After drying at room temperature, an appropriate amount of temporary film forming agent 4 was applied to the center of the 400 μm diameter spots prepared above, to form a large number of spots with a diameter of 100 to 150 μm. The substrate was dried in a thermostatic dryer at 70°C for one day to prepare a substrate for producing cell aggregates. Sterilization was carried out by irradiation with gamma rays at 25 kGy.

[0207] Example 6 Using an inkjet device (Seiko Epson Corporation, R&D inkjet device) and an inkjet head (Seiko Epson Corporation, Precision Core Head S800-A1), an appropriate amount of base film forming agent 4 was applied to the culture surface of substrate 2 having the ability to inhibit cell adhesion, prepared in Preparation Example 2, to form a large number of spots with a diameter of 400 μm. After drying at room temperature, an appropriate amount of temporary film forming agent 4 was applied to the center of the 400 μm diameter spots prepared above, to form a large number of spots with a diameter of 100 to 150 μm. The substrate was dried for one day in a constant temperature dryer at 70°C, to prepare a substrate for producing cell aggregates. Sterilization was carried out by irradiation with gamma rays at 25 kGy.

[0208] Comparative Example 1 Using an inkjet device (Seiko Epson Corporation, R&D inkjet device) and an inkjet head (Seiko Epson Corporation, Precision Core Head S800-A1), an appropriate amount of base film forming agent 2 was applied to the culture surface of substrate 1 having the ability to inhibit cell adhesion, prepared in Preparation Example 1, to form a large number of spots with a diameter of 400 μm. The substrate was dried in a thermostatic dryer at 70°C for one day to prepare a substrate for producing cell aggregates. Sterilization was carried out by irradiation with gamma rays at 25 kGy.

[0209] Comparative Example 2 Using an inkjet device (manufactured by Microjet Corporation, model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000), an appropriate amount of base film forming agent 1 was applied to the culture surface of a commercially available low cell adhesion plate, PrimeSurface24F (manufactured by Sumitomo Bakelite Co., Ltd.), to form a large number of spots with a diameter of 400 μm. The plate was dried in a thermostatic oven at 70° C. for one day to prepare a substrate for producing cell aggregates.

[0210] Comparative Example 3 Using an inkjet device (Seiko Epson Corporation, R&D inkjet device) and an inkjet head (Seiko Epson Corporation, Precision Core Head S800-A1), an appropriate amount of base film forming agent 3 was applied to the culture surface of substrate 2 having the ability to inhibit cell adhesion, prepared in Preparation Example 2, to form a large number of spots with a diameter of 400 μm. This was dried in a thermostatic dryer at 70°C for one day to prepare a substrate for producing cell aggregates. Sterilization was carried out by irradiating with gamma rays at 25 kGy.

[0211] Comparative Example 4 Using an inkjet device (Seiko Epson Corporation, R&D inkjet device) and an inkjet head (Seiko Epson Corporation, Precision Core Head S800-A1), an appropriate amount of base film forming agent 4 was applied to the culture surface of substrate 2 having the ability to inhibit cell adhesion, prepared in Preparation Example 2, to form a large number of spots with a diameter of 400 μm. This was dried in a thermostatic dryer at 70°C for one day to prepare a substrate for producing cell aggregates. Sterilization was carried out by irradiating with gamma rays at 25 kGy.

[0212] <Test Example 1: Test to confirm the ability of human adipose tissue-derived mesenchymal stem cells of Example 1 and Comparative Example 1 to inhibit spheroid migration in a serum-free medium> (Cell preparation) Human adipose tissue-derived mesenchymal stem cells (ADSCs: manufactured by Cellsource Co., Ltd.) were used. The cells were cultured in a low-serum medium, Mesenchymal Stem Cell Growth Medium GM2 (manufactured by Takara Bio Inc.). The cells were incubated at 37°C / CO ２The cells were cultured in a 10 cm diameter dish (10 mL of medium) for at least two days in an incubator maintained at 5% carbon dioxide. Subsequently, the cells were washed with 3 mL of PBS solution (Fujifilm Wako Pure Chemical Industries, Ltd.), and then 3 mL of trypsin-EDTA solution (PromoCell) was added and allowed to stand at room temperature for 3 minutes to detach the cells. Cells were recovered by adding 7 mL of serum-free Mesenchymal Stem Cell Growth Medium DXF medium. The suspension was centrifuged (Tomy Seiko Co., Ltd., Model No. LC-230, 200 × g / 3 minutes, room temperature), the supernatant was removed, and the serum-free medium was added to prepare a cell suspension.

[0213] (Test for Confirming Cell Adhesion and Cell Aggregate Formation) A cell suspension was applied to each of the substrates for producing cell aggregates prepared in Example 1 and Comparative Example 1 at a concentration of 1.5 × 10 ５ cells / cm ２ Then, the mixture was incubated at 37°C / CO2 while maintaining a 5% carbon dioxide concentration. ２The wells were then placed in an incubator. After 26 and 74 hours, the cells were observed and photographed using a stereo microscope SZX16 (Olympus Corporation). Photographs of the entire well surface were also taken using a Cell3imager duos (SCREEN Corporation). The results are shown in Figures 4A to 4C and 5A to 5C. Figure 4A shows the results of the experiment in Example 1 after 26 hours of culture. Figure 4B shows the results of the experiment in Example 1 after 72 hours of culture. Figure 4C shows the results of the experiment in Example 1 after 72 hours of culture (overall photograph). Figure 5A shows the results of the experiment in Comparative Example 1 after 26 hours of culture. Figure 5B shows the results of the experiment in Comparative Example 1 after 72 hours of culture. Figure 5C shows the results of the experiment in Comparative Example 1 after 72 hours of culture (overall photograph). After 26 hours, selective cell adhesion to the base film portion of the substrates prepared in both Example 1 and Comparative Example 1 was confirmed. Furthermore, after 72 hours, it was confirmed that the adhered cells had detached from the Petri dish and aggregated, forming uniformly sized cell aggregates (spheroids). In Example 1, the formed cell aggregates were regularly held in the spotted areas. On the other hand, in Comparative Example 1, the formed cell aggregates detached from the spotted areas. In Example 1, the temporary film-forming agent was applied in small amounts to the base film, which is thought to have caused the bottom surfaces of the formed cell aggregates to adhere and retain their spherical cell aggregate shape on the substrate. Furthermore, photographs taken with the Cell3imager duos showed that in Example 1, cell aggregates were regularly arranged on the spots across the entire well, while in Comparative Example 1, the cell aggregates were detached. Furthermore, the detached cell aggregates adhered to each other to form large aggregates. As described above, by applying the temporary film-forming agent to the base film, it is possible to maintain the regularly arranged state of the aggregates on the substrate after cell aggregate formation.

[0214] Test Example 2: Observation of temporary fixing film applied onto base film Using the same method as in Example 1, base film forming agent 2 was applied to the culture surface of substrate 1 having the ability to inhibit cell adhesion, and then temporary fixing film forming agent 2 was applied. The applied film was observed using a CCD camera. As a result, a film of temporary fixing film forming agent 2 was formed in the center of the spot of base film forming agent 2. The diameter of the spot of temporary fixing film forming agent 2 was also approximately 90 μm. The results are shown in Figure 6. In Figure 6, a base film is formed in the area indicated by (1), and a temporary fixing film is formed in the location indicated by (2).

[0215] Test Example 3: Test to confirm the ability of human adipose tissue-derived mesenchymal stem cells of Examples 2 and 3 and Comparative Example 2 to inhibit spheroid migration in serum medium (Cell Preparation) Human adipose tissue-derived mesenchymal stem cells (ADSCs: manufactured by Cellsource Co., Ltd.) were used. The cells were cultured in a low-serum medium, Mesenchymal Stem Cell Growth Medium GM2 (manufactured by Takara Bio Inc.). The cells were incubated at 37°C / CO ２ The cells were cultured for at least two days in a 10 cm diameter Petri dish (10 mL of medium) in an incubator maintained at 5% carbon dioxide. Subsequently, the cells were washed with 3 mL of PBS solution (Fujifilm Wako Pure Chemical Industries, Ltd.), and then 3 mL of trypsin-EDTA solution (PromoCell) was added and allowed to stand at room temperature for 3 minutes to detach the cells. Cells were recovered by adding 7 mL of low-serum Mesenchymal Stem Cell Growth Medium GM2. The suspension was centrifuged (Tomy Seiko Co., Ltd., Model No. LC-230, 200 × g / 3 minutes, room temperature), the supernatant was removed, and the above-mentioned low-serum medium was added to prepare a cell suspension.

[0216] (Test for Confirming Cell Adhesion and Cell Aggregate Formation) A cell suspension was applied to each of the substrates for cell aggregate production prepared in Examples 2 and 3 and Comparative Example 2 at a concentration of 3.0 × 10 ５ cells / cm ２ Then, the mixture was incubated at 37°C / CO2 while maintaining a 5% carbon dioxide concentration. ２The cells were allowed to stand in an incubator. After 3 and 6 days, the cells were observed and photographed using a stereo microscope SZX16 (Olympus Corporation). The results are shown in Figures 7A to 7C, 8A to 8C, and 9A to 9C. Figure 7A shows the results of the experiment in Example 2 after 3 days of culture. Figure 7B shows the results of the experiment in Example 2 after 6 days of culture. Figure 7C shows the results of the experiment in Example 2 after 6 days of culture (overall photograph). Note that there are ink marks in the upper right corner of the photograph, but these can be ignored. Figure 8A shows the results of the experiment in Example 3 after 3 days of culture. Figure 8B shows the results of the experiment in Example 3 after 6 days of culture. Figure 8C shows the results of the experiment in Example 3 after 6 days of culture (overall photograph). Figure 9A shows the results of the experiment in Comparative Example 2 after 3 days of culture. Figure 9B shows the results of the experiment in Comparative Example 2 after 6 days of culture. Figure 9C shows the observation results (overall photograph) after 6 days of culture in the test of Comparative Example 2. In both Examples 2 and 3, the formed cell aggregates were regularly retained in the spotted area after both 3 and 6 days. On the other hand, in Comparative Example 2, the formed cell aggregates had detached from the spotted area after 3 days, and after 6 days, the detached spheroids had moved even further. These results demonstrate that by applying a small amount of the temporary film-forming agent to the base film, the formed cell aggregates remained adhered at the bottom even after 6 days of long-term culture, allowing them to be retained on the substrate while maintaining their spherical cell aggregate morphology.

[0217] Test Example 4: Test to confirm the ability of human adipose tissue-derived mesenchymal stem cells of Examples 4 and 5 and Comparative Example 3 to inhibit spheroid migration in serum-free medium. (Cell preparation) A cell suspension was prepared in the same manner as in Test Example 1.

[0218] (Test for Confirming Cell Adhesion and Cell Aggregate Formation) A cell suspension was applied to each of the substrates for cell aggregate production prepared in Examples 4 and 5 and Comparative Example 3 at a concentration of 1.5 × 10 ５ cells / cm ２ After that, the mixture was incubated at 37°C / CO2 while maintaining a 5% carbon dioxide concentration. ２The wells were left to stand in an incubator. Half of the medium was replaced every two days. Photographs of the entire well surface were taken using a Cell3imager duos (manufactured by SCREEN Corporation) after three and seven days. The results are shown in Figures 10A-10B, 11A-11C, and 12A-12C. Figure 10A shows the observation results (overall photograph) after three days of culture in the test of Example 4. Figure 10B shows the observation results (overall photograph) after seven days of culture in the test of Example 4. Figure 11A shows the observation results (overall photograph) after three days of culture in the test of Example 5. Figure 11B shows the observation results (overall photograph) after seven days of culture in the test of Example 5. Figure 11C shows the observation results (overall photograph) after a complete medium replacement in the test of Example 5. Figure 12A shows the observation results (overall photograph) after three days of culture in the test of Comparative Example 3. Figure 12B shows the observation results (overall photograph) after 7 days of culture in the test of Comparative Example 3. Figure 12C shows the observation results (overall photograph) after a complete medium change in the test of Comparative Example 3. After 3 days, photographs taken with the Cell3imager duos confirmed that uniformly sized cell aggregates (spheroids) had formed in the base film portion of the substrates prepared in Examples 4, 5, and Comparative Example 3. After 7 days, the formed cell aggregates in Examples 4 and 5 were regularly retained in the spotted areas. On the other hand, the formed cell aggregates in Comparative Example 3 detached from the spotted areas. In Examples 4 and 5, the application of a temporary film-forming agent to the base film is thought to have caused the bottom surfaces of the formed cell aggregates to adhere, maintaining their spherical cell aggregate shape and retaining them on the substrate. Furthermore, to confirm resistance to impact, the entire medium was aspirated from Example 5 and Comparative Example 3, and 1 mL of medium was added (complete medium change). As a result, in Example 5, cell aggregates were regularly arranged on spots over the entire surface of the well, despite the culture medium being replaced, whereas almost all of the cell aggregates were detached in Comparative Example 3. As described above, by applying the temporary film-forming agent to the base film, it is possible to stably maintain the state in which the cell aggregates are regularly arranged on the substrate after their formation.

[0219] Test Example 5: Test to confirm the ability to inhibit spheroid migration in serum medium using HepG2 cells of Example 6 and Comparative Example 4 (Cell preparation) A human liver cancer cell line (HepG2: manufactured by ATCC) was used. The cells were cultured in D-MEM (high glucose) (containing L-glutamine, phenol red, and sodium pyruvate) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 10 v / v% fetal bovine serum (D-MEM + 10% FBS). The cells were incubated at 37°C / CO ２ The cells were statically cultured for at least 2 days in a 10 cm diameter dish (10 mL of medium) in an incubator with a 5% carbon dioxide concentration. Subsequently, the cells were washed with 10 mL of PBS solution (Fujifilm Wako Pure Chemical Industries, Ltd.), and then 1 mL of 0.25 w / v% trypsin-1 mmol / L EDTA 4Na solution (containing phenol red) (Fujifilm Wako Pure Chemical Industries, Ltd.) was added and the cells were incubated at 37°C / CO ２ The cells were detached by leaving the cells in an incubator with a 5% carbon dioxide concentration for 3 minutes. 4 mL of the above D-MEM + 10% FBS was added to recover the cells. This suspension was centrifuged (Tomy Seiko Co., Ltd., Model No. LC-200, 200 × g / 3 minutes, room temperature), the supernatant was removed, and the above D-MEM + 10% FBS was added and the cells were suspended. The suspension was passed through a Falcon (registered trademark) 40 μm cell strainer (Corning) to remove unnecessary cell aggregates, and a cell suspension was prepared.

[0220] (Test for Confirming Cell Adhesion and Cell Aggregate Formation) A cell suspension was applied to each of the substrates for producing cell aggregates prepared in Example 6 and Comparative Example 4 at a concentration of 1.9 × 10 ５ cells / cm ２ After that, the mixture was incubated at 37°C / CO2 while maintaining a 5% carbon dioxide concentration. ２The wells were left to stand in an incubator. Half or full medium changes were performed on days 2, 4, and 7. After 7 days, photographs of the entire well were taken using a Cell3imager duos (manufactured by SCREEN Corporation). The results are shown in Figures 13A-13B and 14A-14B. Figure 13A shows the observation results after 7 days of culture following half-medium changes in the test of Example 6. Figure 13B shows the observation results after 7 days of culture following full-medium changes in the test of Example 6. Figure 14A shows the observation results after 7 days of culture following half-medium changes in the test of Comparative Example 4. Figure 14B shows the observation results after 7 days of culture following full-medium changes in the test of Comparative Example 4. Photographs taken with the Cell3imager duos show that in both Example 6 and Comparative Example 4, under conditions of half-medium exchange, uniformly sized cell aggregates (spheroids) formed in the base film portion of the prepared substrate, and the formed cell aggregates were regularly retained in the spotted area. Furthermore, under conditions of full-medium exchange, most of the formed cell aggregates in Example 6 were regularly retained in the spotted area, while most of the formed cell aggregates in Comparative Example 4 detached from the spotted area. In Example 6, the application of a temporary film-forming agent to the base film is thought to have caused the bottom surfaces of the formed cell aggregates to adhere, maintaining their spherical cell aggregate shape and retaining them on the substrate. Even after 7 days of culture and a full-medium exchange, the cell aggregates could still be regularly arranged on the substrate, suggesting that the method may also be applicable to assays involving fixed cells, such as drug discovery screening.

[0221] The present invention enables the formation of uniform cell aggregates and the placement of the aggregates at any desired position on the plate. This allows the plate to be used as a cell aggregate formation plate for drug discovery screening and animal-alternative testing. Furthermore, applications such as forming uniform cell aggregates in regenerative medicine, followed by differentiation and maturation culture while suppressing fusion between the cell aggregates, can be expanded to include transplantation.

[0222] 1 Main body 1A Outer peripheral wall 1B Inner bottom surface 1C Opening 2 Cell adhesive substance 3 Cell culture base membrane 4 Coating membrane 100 Structure for cells

Claims

1. A cell structure comprising: a substrate; and a plurality of spots spaced apart on a surface of the substrate, each of the plurality of spots comprising a cell adhesive substance.

2. The cell structure according to claim 1, wherein each of the plurality of spots comprises a cell culture base film containing a polymer and the cell adhesive substance disposed on the cell culture base film.

3. The cell structure according to claim 2, which is a cell culture vessel.

4. The cell structure according to claim 2, wherein the polymer is a polymer (A) containing a repeating unit represented by the following formula (Ia): (In formula (Ia), U ａ１ and U ａ２ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ａ１ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ａ２ represents a linear or branched alkylene group having 1 to 5 carbon atoms.

5. The cell structure according to claim 4, wherein the polymer (A) includes a crosslinked structure.

6. The cell structure according to claim 5, wherein the polymer (A) comprises at least one of a structure represented by the following formula (IIIa), a structure represented by the following formula (IVa), and a structure represented by the following formula (Va). (In the formula, R ｃ and R ｄ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ｅ represents a linear or branched alkylene group having 1 to 5 carbon atoms, and n represents a number from 1 to 50.

7. The cell structure according to claim 1, wherein the surface of the substrate has a coating film capable of inhibiting cell adhesion, and the spots are arranged on the coating film.

8. A method for manufacturing a cell structure having a substrate and a plurality of spots arranged at intervals on the surface of the substrate, the method comprising the step of applying droplets containing a cell adhesive substance to the substrate to place the cell adhesive substance in the areas that will become the spots.

9. The method for producing a cell structure according to claim 8, wherein the application of droplets containing the cell adhesive substance to the substrate is carried out by an inkjet method.

10. A method for producing a cell structure as described in claim 8, wherein in the cell structure to be produced, the surface of the substrate has a coating film having the ability to inhibit cell adhesion, and the multiple spots are arranged on the coating film, and the method for producing the cell structure further includes a step of applying a coating agent containing a polymer having the ability to inhibit cell adhesion onto the substrate to form the coating film.

11. A method for manufacturing a cell structure having a substrate and a plurality of spots arranged at intervals on a surface of the substrate, comprising the steps of: applying droplets of a cell culture base film forming agent containing a polymer and a solvent to the substrate to form a cell culture base film in the areas that will become the spots; and applying droplets containing a cell adhesive substance to the cell culture base film to arrange the cell adhesive substance on the cell culture base film.

12. The method for producing a cell structure according to claim 11, wherein the application of droplets of the cell culture base film forming agent to the substrate is carried out by an inkjet method.

13. The method for producing a cell structure according to claim 11, wherein the application of droplets containing the cell adhesive substance to the cell culture base membrane is carried out by an inkjet method.

14. The method for producing a cell structure according to claim 11, wherein the polymer is a polymer containing a repeating unit represented by the following formula (Ia): (In formula (Ia), U ａ１ and U ａ２ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ａ１ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; Rａ２ represents a linear or branched alkylene group having 1 to 5 carbon atoms.

15. A method for producing a cell structure as described in claim 11, wherein in the cell structure to be produced, the surface of the substrate has a coating film having the ability to inhibit cell adhesion, and the multiple spots are arranged on the coating film, and the method for producing the cell structure further includes a step of applying a coating agent containing a polymer having the ability to inhibit cell adhesion onto the substrate to form the coating film.

16. The method for producing a cell structure according to claim 11, wherein the cell structure produced is a cell culture vessel.

17. A cell culture method comprising the step of seeding cells onto the cell structure according to any one of claims 1 to 7.

18. The cell culture method according to claim 17, which produces a cell structure.

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