Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F20/00—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
  • C08F20/02—Monocarboxylic acids having less than ten carbon atoms, Derivatives thereof
  • C08F20/10—Esters
  • C08F20/34—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M1/00—Apparatus for enzymology or microbiology
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

• the present invention relates to a cell structure, a method for producing a cell structure, and a cell culture method.
• Patent Literature 1 discloses a method for producing a polymer used as a base membrane for cell culture and a cell culture vessel.
• Patent Literature 2 discloses a method for producing a cell structure. In the process of producing (culturing) homogenous cell aggregates, these cell culture base membranes require the use of serum derived from living organisms to ensure uniform adhesion of cells to the base membrane, which poses 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 using serum derived from animals other than humans.
• the applicant has proposed a base film for cell culture that can produce homogeneous, high-quality cell aggregates without using serum derived from living organisms in the mass production of cell aggregates, a base film forming agent for forming the same, and a substrate for cell aggregate production (Patent Document 3).
• Stably retaining cells themselves or cell structures at a specified distance is useful from the perspective of utilizing the cells themselves and producing cell structures of uniform size.
• the present invention aims to provide a cell structure capable of stably retaining cells themselves or cell structures at a specified interval, a method for manufacturing the cell structure, and a cell culture method using the cell structure.
• the present invention includes the following aspects.
• a substrate a plurality of spaced apart spots on a surface of the substrate;
• a cell structure comprising: A cell structure, wherein each of the plurality of spots has a cell adhesive substance.
• a method for producing a cell structure having a substrate and a plurality of spots arranged at intervals on a surface of the substrate comprising: A method for producing a cell structure, comprising the step of applying droplets containing a cell adhesive substance to the substrate, thereby disposing the cell adhesive substance in the areas that will become the spots. [9] The method for producing a cell structure described in [8], in which the application of droplets containing the cell adhesive substance to the substrate is carried out by an inkjet method.
• the surface of the substrate has a coating film having cell adhesion inhibitory ability, and the plurality of spots are arranged on the coating film;
• the method for producing a cell structure described in [8] or [9] further comprises a step of applying a coating agent containing a polymer capable of inhibiting cell adhesion onto the substrate to form the coating film.
• a method for producing a cell structure having a substrate and a plurality of spots arranged at intervals on a surface of the substrate comprising: 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 area to be the spot; applying droplets containing a cell adhesive substance to the cell culture base membrane to dispose the cell adhesive substance on the cell culture base membrane;
• the method for producing a cell structure includes the steps of: [12] The method for producing a cell structure described in [11], in which the application of droplets of the cell culture base film forming agent to the substrate is carried out by an inkjet method.
• the surface of the substrate has a coating film having cell adhesion inhibitory ability, and the plurality of spots are arranged on the coating film;
• a cell culture method comprising a step of seeding cells into the cell structure according to any one of [1] to [7].
• the cell culture method according to [17] which produces a cell structure.
• the present invention provides 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.
• 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 shows the observation results after 26 hours of culture in the test of Example 1.
• FIG. 4B shows the observation results after 72 hours of culture in the test of Example 1.
• FIG. 4C shows the observation results (overall photograph) after 72 hours of culture in the test of Example 1.
• FIG. 5A shows the observation results after 26 hours of culture in the test of Comparative Example 1.
• FIG. 5B shows the observation results after 72 hours of culture in the test of Comparative Example 1.
• FIG. 5C shows the observation results (overall photograph) after 72 hours of culture in the test of Comparative Example 1.
• FIG. 6 shows the observation results of Test Example 2.
• FIG. 7A shows the observation results 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. 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
• 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 shows the observation results (overall photograph) after 7 days of culture in the test of Example 5.
• FIG. 11C shows the observation results (overall photograph) after complete medium replacement in the test of Example 5.
• FIG. 12A shows the observation results (overall photograph) after 3 days of culture in the test of Comparative Example 3.
• FIG. 12B shows the observation results (overall photograph) after 7 days of culture in the test of Comparative Example 3.
• FIG. 12C shows the observation results (overall photograph) after complete medium replacement in the test of Comparative Example 3.
• FIG. 13A shows the results of observation after 7 days of culture in which half of the medium was exchanged in the test of Example 6.
• FIG. 13B shows the results of observation after 7 days of culture, in which the entire medium was replaced in the test of Example 6.
• FIG. 14A shows the results of observation after 7 days of culture in which half of the medium was exchanged in the test of Comparative Example 4.
• FIG. 14B shows the results of observation after 7 days of culture in which the entire medium was replaced in the test of Comparative Example 4.
• the cell structure of the present invention comprises a substrate and a plurality of spots.
• the plurality of spots are spaced apart on the surface of the substrate.
• the spots in one embodiment, have a cell adhesive substance.
• the spot has a cell culture base membrane and a cell adhesive substance disposed on the cell culture base membrane.
• the "surface of the substrate” refers to the surface that comes into contact with contents such as cell culture medium.
• multiple cell culture base membranes are arranged at intervals on a substrate.
• a cell structure e.g., a spheroid
• the produced cell structures are less likely to detach from the cell culture base membrane compared to when there is no cell adhesive substance on the cell culture base membrane. Therefore, it is possible to prevent unevenness in the size of the cell structures due to the fusion of detached cell structures and a decrease in the number of cell structures due to fusion.
• the cell structure is preferably a cell culture vessel.
• the number of the spots arranged at intervals on the surface of the substrate is not particularly limited, and is preferably, for example, 50 to 5,000 spots per cm2 , and more preferably 100 to 2,000 spots per cm2.
• the distance between adjacent spots is not particularly limited, but is preferably 100 ⁇ m to 6,000 ⁇ m, more preferably 150 ⁇ m to 4,000 ⁇ m, and particularly preferably 150 ⁇ m to 3,000 ⁇ m.
• the distance refers to the length between the centers of the two spots.
• the size of the spot surface is not particularly limited, and for example, the circle equivalent diameter is preferably 25 ⁇ m to 5,000 ⁇ m, and more preferably 50 ⁇ m to 3,000 ⁇ m.
• the equivalent circle diameter is the diameter of a perfect circle that corresponds to the area of the target surface. If the surface of the spot is a perfect circle, the equivalent circle diameter refers to the diameter of the perfect circle, and if the surface of the spot is other than a perfect circle, the equivalent circle diameter refers to the diameter of a perfect circle that has the same area as the area of the target surface.
• the area percentage of the substrate occupied by the spots is not particularly limited, but is preferably 30% or more, 40% or more, 50% or more, and is preferably 99% or less.
• the spot preferably has a cell culture base membrane.
• the cell adhesive substance is disposed on the cell culture base membrane.
• 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 surface size of the cell adhesive material placed 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.
• the cell adhesive substance is present, for example, at one location per spot, but may be present at multiple locations per spot.
• Cell adhesive substances promote cell adhesion, spreading, proliferation and differentiation.
• known substances such as biological substances such as extracellular matrix (ECM) proteins, glycoproteins, peptides, and synthetic compounds (low molecular weight, high molecular weight) can be used, but compounds not of biological origin, such as synthetic compounds (low molecular weight, high molecular weight), are preferred.
• a low molecular weight is, for example, a compound having a weight average molecular weight of 2,000 or less
• a high molecular weight is, for example, a compound having a weight average molecular weight of 2,000 or more, with an upper limit of, for example, 1,000,000.
• 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 product numbers L6724, L2020, L4544), laminin fragments (e.g., Matrixome: 892011), vitronectin (e.g., VTN-N (Gibco), Vitronectin, Human, Recombinant, Animal Free (PeproTech), Merck product numbers V0132, V9881, V8379, 08-126, SRP3186).
• collagen e.g., Mer
• the cell adhesive substance is preferably a glycoprotein. Specifically, it is preferably selected from vitronectin, integrin, cadherin, fibronectin, laminin, tenascin, osteopontin, and bone sialoprotein. It is also preferably a protein having an RGD sequence as an amino acid sequence.
• peptides examples include ECM peptides (MAPTrix (registered trademark) from Kollodis Bio Sciences) and RGD peptides (180-01531 from Fujifilm Wako Pure Chemical Industries, Ltd.).
• ECM peptides MAPTrix (registered trademark) from Kollodis Bio Sciences
• RGD peptides 180-01531 from Fujifilm Wako Pure Chemical Industries, Ltd.
• Examples of synthetic compounds 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 include adhesamine (e.g. Nagase & Co.: AD-00000-0201) and synthetic cyclic RGD peptide (e.g. IRIS BIOTECH: LS-3920.0010).
• the cell culture substrata contain a polymer, which is a material different from the cell adhesive material and is typically a synthetic polymer.
• the cell culture base membrane may further contain a cell adhesive substance.
• 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.
• the polymer is not particularly limited as long as it is a polymer that can be used for a base membrane for cell culture. From the viewpoint of suitably obtaining the effects of the present invention, however, a polymer (A) containing a repeating unit represented by the following formula (Ia) is preferred.
• U a1 and U a2 each independently represent 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.
• the polymer (A) preferably further contains a repeating unit represented by the following formula (IIa).
• R b represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms.
• examples of "straight-chain or branched alkyl groups having 1 to 5 carbon atoms” include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, and 1-ethylpropyl.
• R a1 and R b are each independently selected from a hydrogen atom and a methyl group.
• Each of U a1 and U a2 is preferably independently selected from a hydrogen atom, a methyl group, an ethyl group, a n-propyl group, an isopropyl group, and a n-butyl group, more preferably a methyl group or an ethyl group, and most preferably a methyl group.
• 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, a 1-ethyl-trimethylene group, etc.
• R a2 is preferably selected from an ethylene group and a propylene group.
• Examples of the monomer (cationic monomer) that gives the polymer (A) the repeating unit represented by formula (Ia) include 2-N,N-dimethylaminoethyl methacrylate, N,N-dimethylaminomethyl methacrylate, etc., and 2-N,N-dimethylaminoethyl methacrylate (2-(dimethylamino)ethyl methacrylate) is preferred.
• Examples of the monomer (anionic monomer) that gives the polymer (A) the repeating unit represented by formula (IIa) include acrylic acid and methacrylic acid, with methacrylic acid being preferred.
• 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, preferably 98/2 to 50/50, more preferably 98/2 to 60/40, and particularly preferably 98/2 to 70/30.
• 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.
• the polymer (A) may have other repeating units.
• the total 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.
• the polymer (A) further contains a crosslinked structure.
• the crosslinked structure can be obtained by using a monomer having two or more carbon-carbon unsaturated bonds in synthesizing the polymer (A).
• the monomer having two or more carbon-carbon unsaturated bonds is 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.
• R c and R d each independently represent 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
• n represents a number from 1 to 50.
• R c and R d are each independently selected from a hydrogen atom and a methyl group.
• R 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, n is preferably a number from 1 to 30, and more preferably a number from 1 to 10.
• the molar ratio of the structures represented by formulae (IIIa) to (Va) to the entire polymer (A) is preferably 0 mol % to 50 mol %, and more preferably 2 mol % to 25 mol %.
• the molar ratio of the structures represented by formulae (IIIa) to (Va) is 50% 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.
• the synthesis method of polymer (A) is not particularly limited, and examples thereof include known radical polymerization methods. In addition, the method described in WO2020/040247 may be used.
• the number average molecular weight (Mn) of the polymer is, for example, 20,000 to 1,000,000, and preferably 50,000 to 800,000.
• the number average molecular weight (Mw) of the polymer is, for example, 50,000 to 2,000,000, and 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 the weight average molecular weight (Mw) can be determined, for example, by gel filtration chromatography.
• the material, shape, size and structure of the substrate are not particularly limited.
• the surface of the substrate may be flat or uneven, but is preferably flat.
• the substrate material may be, for example, glass, metal, a metal-containing compound or a metalloid-containing compound, activated carbon, or a resin.
• 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, Au, etc.).
• Metal-containing compounds or metalloid-containing compounds may be, for example, ceramics, which are sintered bodies made by heat treatment at high temperatures and whose basic component is a metal oxide, 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.).
• the resin may be either a natural resin or its derivative, or a synthetic resin.
• natural resins or their derivatives include cellulose, cellulose triacetate (CTA), nitrocellulose (NC), and cellulose immobilized with dextran sulfate.
• synthetic resins that are preferably used 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), polyd
• the substrate may be in the form of a plate or a film, and the thickness of the substrate is not particularly limited.
• the substrate may be, for example, a film having a thickness of 1 mm or less.
• the substrate may also be a substrate used in a so-called cell culture vessel.
• dish or dishes such as Petri dishes, tissue culture dishes, and multi-dishes that are generally 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.
• the surface of the substrate may have a coating film having a cell adhesion suppressing ability, in which 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 onto a substrate.
• the shape of the coating film on the surface of the substrate is not particularly limited, so 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.
• having the ability to inhibit cell adhesion means that, when compared to no coating film or no low cell adhesion treatment when measured using a fluorescence microscope using the method described in the examples of WO 2016/093293, the relative absorbance (WST O.D. 450 nm) (%) ((Absorbance of Example (WST O.D. 450 nm))/(Absorbance of Comparative Example (WST O.D. 450 nm))) is 50% or less, preferably 30% or less, and more preferably 20% or less.
• coating film that has the ability to inhibit cell adhesion
• coating films include the following coating films:
• the coating film of the first embodiment includes a copolymer.
• the copolymer of the first embodiment contains 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 fully set forth herein.
• U a1 , U a2 , U b1 , U b2 and U b3 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms
• An ⁇ represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion and an isothiocyanate ion.
• 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.
• copolymers examples include vinyl polymerization polymers reacted with olefins, polyamides, polyesters, polycarbonates, polyurethanes, etc., and among these, vinyl polymerization polymers reacted with olefins or (meth)acrylic polymers polymerized with (meth)acrylate compounds are particularly preferable.
• a (meth)acrylate compound means both an acrylate compound and a methacrylate compound.
• (meth)acrylic acid means acrylic acid and methacrylic acid.
• 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.
• T a , T b , U a1 , U a2 , U b1 , U b2 and U b3 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms
• Q a and Q b each independently represent a single bond, an ester bond or an amide bond
• R a and R b each independently represent 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
• m represents an integer of 0 to 6.
• the repeating units derived from the monomers represented by formulae (A) and (B) are represented by the following formulae (a1) and (b1), respectively.
• 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 are as defined above.
• a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom 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.
• the "straight-chain or branched alkylene group having 1 to 10 carbon atoms” refers to 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.
• Linear or branched alkylene groups having 1 to 10 carbon atoms substituted with one or more halogen atoms refers to such alkylene groups in which one or more arbitrary hydrogen atoms have been replaced with halogen atoms, and in particular, ethylene or propylene groups in which some or all of the hydrogen atoms have been replaced with halogen atoms are preferred.
• the "halogen atom” includes a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
• the term “halide ion” refers to an anion of a halogen atom, and examples of such an anion include a fluoride ion, a chloride ion, a bromide ion, and an iodide ion, with a chloride ion being preferred.
• the term "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 as the An ⁇ are a halide ion, a sulfate ion, a phosphate ion, a hydroxide ion and an isothiocyanate ion, and particularly preferred is a halide ion.
• 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.
• 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.
• U a1 and U a2 are more preferably a hydrogen atom.
• U b1 , U b2 (and U b3 ) are more preferably a methyl group or an ethyl group, and particularly preferably a methyl group.
• 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.
• m preferably represents an integer of 0 to 3, more preferably an integer of 1 or 2, and particularly preferably 1.
• (meth)acrylate compounds having two functional groups such as those represented by general formula (C) or (D) described below, may be used in combination during synthesis.
• formula (B) examples include dimethylaminoethyl (meth)acrylate, diethylaminoethyl (meth)acrylate, dimethylaminopropyl (meth)acrylate, 2-(t-butylamino)ethyl (meth)acrylate, methacryloylcholine chloride, etc., among which dimethylaminoethyl (meth)acrylate, methacryloylcholine chloride, or 2-(t-butylamino)ethyl (meth)acrylate are preferably used.
• dimethylaminoethyl acrylate 2-(dimethylamino)ethyl acrylate
• dimethylaminoethyl methacrylate 2-(dimethylamino)ethyl methacrylate
• methacryloylcholine chloride 2-(t-butylamino)ethyl methacrylate
• 2-(t-butylamino)ethyl methacrylate 2-(t-butylamino)ethyl methacrylate
• the proportion of the repeating units containing an 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 kinds of repeating units containing an organic group represented by formula (a) (or the repeating units represented by formula (a1)).
• the proportion of the repeating unit containing an organic group represented by formula (b) (or the repeating unit represented by formula (b1)) in the above copolymer may be the entire remainder after subtracting the proportion of the above formula (a) (or formula (a1)) from the total copolymer, or it 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 kinds of repeating units containing an organic group represented by formula (b) (or repeating units represented by formula (b1)).
• the copolymer may be copolymerized with any third component.
• 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 bifunctional monomers represented by the following formula (C) or (D).
• Tc , Td , and Ud each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, and Rc and Rd each independently represent a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom.
• the copolymer preferably contains a crosslinked structure derived from such a bifunctional monomer.
• Tc and Td 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.
• Ud is preferably a hydrogen atom, a methyl group or an ethyl group, and more preferably a hydrogen atom.
• Rc and Rd 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.
• the bifunctional monomer represented by formula (C) is ethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, propylene glycol di(meth)acrylate, etc.
• the bifunctional monomer represented by formula (D) is bis(methacryloyloxymethyl) phosphate, bis[2-(methacryloyloxy)ethyl] phosphate, bis[2-(methacryloyloxy)propyl] phosphate, etc.
• the optional third component may be a trifunctional monomer.
• An example of such a trifunctional monomer as the third component is phosphinylidintris(oxy-2,1-ethanediyl) triacrylate.
• 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.
• the copolymer may contain one or more of these third components.
• the bifunctional monomer represented by formula (D) is preferred, and the bifunctional monomer represented by formula (D-1) is particularly preferred.
• the ratio of the third component (for example, a crosslinked structure derived from a bifunctional monomer represented by the above formula (C) or (D)) in the copolymer is 0 mol % to 50 mol %.
• the method for producing the copolymer is not particularly limited, but may be, for example, the method for producing the copolymer described in WO2014/196652.
• the coating film may be a coating film obtained from a coating film-forming composition described in WO2021/167037.
• the contents of WO2021/167037 are incorporated herein by reference to the same extent as if fully set forth herein.
• the coating film-forming composition described in WO2021/167037 will be described. In this specification, it is referred to as a coating film-forming composition of the second aspect.
• the composition for forming a coating film according to the second embodiment has the following formula (1): (Wherein, 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.
• R5 each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms
• R6 represents a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom
• m represents an integer of 1 to 30.
• the present invention includes a monomer mixture containing a compound represented by formula (3), wherein the ratio of the compound represented by formula (3) to the total mass of the monomer mixture is 2 to 40 mass %, and a solvent.
• 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.
• R3 represents a hydrogen atom or a methyl group
• R4 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 respectively mean groups represented by the formula: -R 4a -NH 2 , -R 4a -NHR, -R 4a -NRR', -R 4a -N + RR'R'' [wherein R 4a is 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].
• the monovalent group having a primary amine, secondary amine or tertiary amine structure in the second aspect may be quaternized or salified, and similarly the monovalent group having a quaternary ammonium structure
• compound of formula (2) above examples include dimethylaminoethyl (meth)acrylate, diethylaminoethyl (meth)acrylate, dimethylaminopropyl (meth)acrylate, 2-(t-butylamino)ethyl (meth)acrylate, (meth)acryloylcholine chloride, 2-(meth)acryloyloxyethyl phosphorylcholine (MPC), etc.
• the compound of formula (3) which is a monomer component of the copolymer according to the second aspect, is a bifunctional monomer having crosslinkability.
• R 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
• 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
• Specific examples of the compound of formula (3) above include poly(ethylene glycol) di(meth)acrylate, poly(trimethylene glycol) di(meth)acrylate, poly(propylene glycol) di(meth)acrylate, etc.
• 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.
• the copolymer according to the second embodiment is obtained by polymerizing a monomer mixture containing the compounds of the above formulae (1), (2) and (3) as monomer components.
• a monomer mixture may contain an ethylenically unsaturated monomer, or a polysaccharide or a derivative thereof as an optional fourth component, as long as the ability to inhibit adhesion of biological materials is not impaired.
• 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.
• polysaccharides or derivatives thereof include cellulose-based polymers such as hydroxyalkylcellulose (e.g., hydroxyethylcellulose or hydroxypropylcellulose), starch, dextran and curdlan.
• cellulose-based polymers such as hydroxyalkylcellulose (e.g., hydroxyethylcellulose or hydroxypropylcellulose), starch, dextran and curdlan.
• the copolymer according to the second embodiment is preferably obtained by polymerizing a monomer mixture containing only the compounds of the above formulae (1), (2) and (3) as monomer components.
• the copolymer according to the second aspect is obtained by polymerizing a monomer mixture containing the compounds of the above formulae (1), (2), and (3) as monomer components, and characterized in that the ratio of the compound of formula (3) to the total mass of the monomer components contained in the monomer mixture is 2 to 40 mass%.
• the compound of formula (3) which is a bifunctional monomer
• the coating film forming composition of the second aspect which contains the copolymer obtained from the monomer mixture, has excellent adhesion suppression ability for biological materials.
• the monomer mixture may contain two or more compounds of formula (3).
• the ratio of the compound of formula (1) 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).
• the ratio of the compound of formula (2) 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).
• 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.
• the form of the copolymer can be various methods such as solution polymerization, suspension polymerization, emulsion polymerization, and bulk polymerization.
• the copolymer can be prepared by a manufacturing 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.
• the solvent 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 more preferable that the solvent contains 50% by mass or more and 100% by mass or less of water or ethanol. It is more preferable that the solvent contains 80% by mass or more and 100% by mass or less of water or ethanol. It is more preferable that the solvent contains 90% by mass or more and 100% by mass or less of water or ethanol. The total of water and ethanol is preferably 100% by mass.
• polymerization initiator In order to efficiently advance the polymerization reaction, it is desirable to use a polymerization initiator.
• the polymerization initiator include a "thermal radical polymerization initiator” or a "photoradical polymerization initiator.”
• thermal radical polymerization initiator examples include 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2-methylbutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile) (FUJIFILM Wako Pure Chemical Industries, Ltd.
• photoradical polymerization initiators 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-hydroxymethylpropanone (BASF product name: Irgacure 2959), 2-hydroxy-1- ⁇ 4-[4-(2-hydroxy-2-methylpropionyl)benzyl]phenyl ⁇ -2 acetophenones such as 2-methyl-1-propan-1-one (BASF product name: Irgacure 127), 2-methyl-1-[4-(methylthio)phenyl]-2-morpholinopropan-1-one (BASF product name: Irg
• the amount of polymerization initiator added is 0.05% to 10% by mass based on the total mass of the monomer components used in the polymerization.
• the reaction conditions are as follows: the reaction vessel is heated to 50-200°C in an oil bath or the like, and stirred for 1-48 hours, more preferably at 80-150°C for 5-30 hours, to allow the polymerization reaction to proceed and produce the copolymer of the second embodiment.
• the reaction atmosphere is preferably a nitrogen atmosphere.
• the reaction procedure can be such that all reactants are placed in a reaction solvent at room temperature and then heated to the above temperature to polymerize, or the entire mixture of reactants or a portion of it can be added dropwise in small amounts to a pre-heated solvent.
• the weight average molecular weight of the copolymer related to the second embodiment may be several thousand to several million, preferably 5,000 to 5,000,000, more preferably 10,000 to 2,000,000.
• the copolymer may be any of a random copolymer, a block copolymer, and a graft copolymer, and is preferably a random copolymer.
• the copolymer produced in this manner is considered to be a three-dimensional polymer since it contains a bifunctional monomer, and is in a dissolved or dispersed state in a solution containing water or alcohol.
• the coating film-forming composition according to the second embodiment may be prepared by isolating and purifying the copolymer thus obtained, 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 a reaction solution (i.e., a copolymer-containing varnish) obtained after the polymerization reaction.
• a reaction solution i.e., a copolymer-containing varnish
• the solvent contained in the coating film forming composition of the second embodiment includes water, phosphate buffered saline (PBS), and alcohol.
• the concentration of solids in the coating film forming composition according to the second embodiment is preferably 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, and if it is 5% by mass or more, the storage stability of the coating film forming composition is deteriorated, and precipitation or gelation of the dissolved material may occur.
• the composition for forming the coating film of the second embodiment can also contain other substances as necessary within a range that does not impair the performance of the resulting coating film.
• other substances include preservatives, surfactants, primers that improve adhesion to the substrate, antifungal agents, and sugars.
• the method for obtaining the coating film according to the second embodiment may further include a step of adjusting the pH of the composition for forming a coating film in advance.
• the pH adjustment may be performed, for example, by adding a pH adjuster to a composition containing the copolymer and a solvent, and adjusting 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 the pH adjuster that can be used are appropriately selected depending on the concentration of the copolymer, the ratio of anions to cations, and the like.
• 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 or alkali metal salts thereof such as sulfuric acid, phosphoric acid, hydrochloric acid, and carbonic acid; quaternary ammonium cations such as choline, or mixtures thereof (e.g., buffer solutions such as phosphate buffered saline).
• 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 or alkali metal salts thereof such as sulfuric
• 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.
• the coating film may 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 expressly set forth in their entirety.
• the composition for forming a coating film described in WO2022/259998 will be described. In this specification, it is referred to as a composition for forming a coating film of the third embodiment.
• the coating film-forming composition of the third embodiment 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, if necessary.
• the coating film-forming composition of the third aspect can form 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.
• the copolymer according to the third embodiment is water-insoluble.
• water-soluble means that it can dissolve at least 1.0 g in 100 g of water at 25° C.
• Water-insoluble means that it does not fall under the category of “water soluble”, that is, the solubility in 100 g of water at 25° C. is less than 1.0 g.
• the copolymer according to the third embodiment 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 related to the third embodiment is from 89:11 to 50:50.
• R 1 to R 3 each independently represent a hydrogen atom or an alkyl group having 1 to 5 carbon atoms
• X 1 and X 2 each independently represent 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.
• the copolymer related to the third embodiment may have two or more kinds of repeating units (A).
• the copolymer related to the third embodiment may have two or more kinds of repeating units (B).
• the copolymer related to the third embodiment preferably has one kind of repeating unit (A) and one kind of repeating unit (B).
• alkyl group having 1 to 5 carbon atoms examples 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. It is preferable that R 1 to R 3 each independently represent a hydrogen atom, a methyl group, or an ethyl group.
• ether bond refers to -O-
• 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.
• X1 and X2 are 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.
• the copolymer according to the third embodiment is preferably a copolymer in which R 1 and R 2 are hydrogen atoms, R 3 is a methyl group, and X 1 and X 2 are single bonds.
• the molar ratio (A:B) of the repeating unit (A) to the repeating unit (B) is from 89:11 to 50:50.
• the molar ratio (A:B) of the repeating unit (A) to the repeating unit (B) can be expressed as (100-m):m.
• 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.
• the total mole percentage of repeating units (A) and repeating units (B) among all repeating units in the copolymer according to the third embodiment is not particularly limited, but is preferably 90 mole% or more, more preferably 95 mole% or more, even more preferably 99.5 mole% or more, and particularly preferably 100%.
• 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 embodiment, a coating film that is difficult to dissolve in phosphate buffered saline is obtained without crosslinking the copolymer. Therefore, the copolymer does not need to have a photosensitive group for crosslinking the copolymer. That is, it is preferable that the copolymer does not have a photosensitive group.
• An example of the photosensitive group is an azide group.
• the copolymer does not need to have a photosensitive group for crosslinking the copolymer. Therefore, when forming a coating film, it is not necessary to perform light irradiation for crosslinking the copolymer. Therefore, the process for forming the coating film can be simplified.
• the viscosity average degree of polymerization of the copolymer related to the third aspect (hereinafter, may be simply referred to as "degree of polymerization”) is not particularly limited, but from the viewpoint of favorably 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 in a completely saponified state of the copolymer.
• P represents the viscosity average degree of polymerization.
• the viscosity average degree of polymerization can be determined in accordance with JIS K6726.
• 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.
• 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.
• R 1 , R 3 , and X 1 are defined as above.
• the copolymer according to the third embodiment may be a random copolymer or a block copolymer.
• a commercially available product may be used as the copolymer according to the third embodiment.
• a specific example of the commercially available copolymer is polyvinyl acetate (manufactured by Nippon Acetic Acid & Poval, trade name JMR-10L (registered trademark)).
• the content of the copolymer in the film-forming component in the coating film-forming composition of the third embodiment 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.
• the film-forming component refers to all the components of the composition excluding the solvent component.
• the content of the copolymer in the coating film-forming composition of the third embodiment is not particularly limited, but from the viewpoint of easily forming a coating film of the 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%.
• the content of the copolymer in the coating film-forming composition may be 0.02 to 2 mass%, or 0.05 to 1 mass%.
• Examples of the solvent in the third aspect include water, phosphate buffered saline (PBS), alcohol, and water-soluble organic solvents (excluding alcohol).
• PBS phosphate buffered saline
• alcohol excluding alcohol
• the alcohol includes alcohols having 2 to 6 carbon atoms.
• alcohols 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,
• the cyclohexanol include cyclohexanol, 2-hexanol, 3-hexanol, 2,3-dimethyl-2-butanol, 3,3-dimethyl-1-butanol, 3,3-dimethyl-2-but
• 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.
• the water-soluble organic solvent 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, and propylene glycol propyl ether acetate. These can be used alone or in combination of two or more.
• the solvent is preferably selected from water, alcohol, a water-soluble organic solvent, and a combination of two or more thereof, and more preferably selected from water, ethanol, a water-soluble organic solvent, and a combination of two or more thereof.
• the mass ratio of water:alcohol 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) 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 is, for example, 30:70 to 97:3.
• the content of the solvent in the coating film-forming composition of the third embodiment is not particularly limited, but from the viewpoint of easily forming a coating film of the 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.
• the coating film-forming composition of the third embodiment may contain other components as necessary.
• other components include pH adjusters, preservatives, surfactants, antifungal agents, and sugars.
• the cell structure 100 shown in FIGS. 1A and 1B has a main body 1 and a cell adhesive substance 2 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 exists below the opening 1C.
• a plurality of cell adhesive substances 2 are arranged at intervals on the inner bottom surface 1B.
• the cell structure 100 shown in FIGS. 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 exists below the opening 1C.
• a plurality of cell culture base membranes 3 are arranged at intervals on the inner bottom surface 1B.
• a cell adhesive substance 2 is disposed on a cell culture base membrane 3 .
• the cell structure 100 shown in FIGS. 3A and 3B has a main body 1, a cell adhesive substance 2, a cell culture base film 3 which is 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 exists below the opening 1C.
• the surface of the inner bottom surface 1B has a coating film 4 having the ability to inhibit cell adhesion.
• a plurality of cell culture base films 3 are arranged on the coating film 4 at intervals.
• a cell adhesive substance 2 is disposed on a cell culture base membrane 3 .
• the method for producing a cell structure of the present invention is a method for producing a cell structure having a substrate and a plurality of spots arranged at intervals on the surface of the substrate.
• Step (A1) A step of applying droplets containing a cell adhesive substance to a substrate to place the cell adhesive substance in areas to be spots.
• 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 area to be a spot.
• 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.
• 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.
• the method for producing a cell structure may further include the following step (C).
• 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.
• Step (A1) is a step of applying droplets containing a cell adhesive substance to a substrate to place the cell adhesive substance in areas that will become spots.
• cell adhesive substances examples include the cell adhesive substances mentioned in the description of the cell structure of the present invention.
• 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.
• the solvent include the solvents mentioned in the description of the cell culture base film forming agent described below.
• the method for applying the droplets containing the cell adhesive substance is not particularly limited, but may be an inkjet method, a screen printing method, a slit coating method, a roll-to-roll method, or the like, but is preferably performed using a printing technique such as an inkjet method or screen printing.
• 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.
• the substrate may be any of the substrates mentioned in the description of the cell structure of the present invention.
• the 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.
• 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.
• the aqueous solution may be water, a salt-containing aqueous solution such as physiological saline or a phosphate buffer solution, or a mixed solvent of water or a salt-containing aqueous solution with an alcohol.
• suitable solvents 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 solvents may be used alone or in combination.
• the water content in the aqueous solution is, for example, 50% by mass to 100% by mass, 80% by mass to 100% by mass, or 90% by mass to 100% by mass.
• the undercoat film forming agent may also contain other substances as necessary within the range that does not impair the performance of the obtained undercoat film. Examples of other substances include pH adjusters, crosslinking agents, preservatives, surfactants, primers that improve adhesion to containers or substrates, antifungal agents, and sugars.
• the method for applying droplets of the undercoat film forming agent to the substrate 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.
• the method is performed by a printing technique such as an inkjet method or screen printing.
• a method of immersing a substrate with a portion that is not to be a spot protected in the above-mentioned primer film forming agent a method of adding a primer film forming agent to a substrate (container) with a portion that is not to be a spot protected and leaving it to stand for a predetermined time, etc. are used.
• the primer film forming agent is added to a container with a portion that is not to be a spot protected and left to stand for a predetermined time.
• the addition can be performed, for example, by adding the primer film forming agent in an amount of 0.5 to 1 times the total volume of the container using a syringe or the like.
• the leaving is performed by appropriately selecting the time and temperature depending on the material of the container or substrate and the type of primer film forming agent for cell culture, and is performed, for example, for 1 minute to 24 hours, preferably 5 minutes to 3 hours, at 10 to 80°C.
• step (B2) may be performed directly without a drying step, or after washing with water or the medium of the sample to be subjected to cell culture (e.g., water, buffer solution, culture medium, etc.).
• water or the medium of the sample to be subjected to cell culture e.g., water, buffer solution, culture medium, etc.
• Step (B2) is a step of providing droplets containing a cell adhesive substance onto the cell culture base membrane, thereby disposing the cell adhesive substance on the cell culture base membrane.
• cell adhesive substances examples include the cell adhesive substances mentioned in the description of the cell structure of the present invention.
• 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.
• the solvent include the solvents mentioned in the description of the cell culture base film forming agent.
• the method for applying the droplets containing the cell adhesive substance is not particularly limited, but may be an inkjet method, a screen printing method, a slit coating method, a roll-to-roll method, or the like, but is preferably performed using a printing technique such as an inkjet method or screen printing.
• 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, and for example, contains a polymer capable of inhibiting cell adhesion and a solvent.
• the coating film and coating agent 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, and may be an inkjet method, a screen printing method, a slit coating method, a roll-to-roll method, or the like.
• the coating agent is applied by a printing technique such as an inkjet method or screen printing.
• the cell culture method (cell production method) of the present invention comprises the step of seeding cells into the cell structure of the present invention.
• An example of the step of seeding cells is adding a medium in which cells are dispersed to the cell structure of the present invention.
• the cell structure is preferably a cell culture vessel.
• 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.
• the cell concentration in the medium in which the cells are dispersed is not particularly limited.
• a cell is the most basic unit constituting an animal or a plant, and has cytoplasm and various organelles inside the cell membrane as its elements.
• the nucleus containing DNA may or may not be contained inside the cell.
• the animal-derived cells in the present invention include germ cells such as sperm and eggs, somatic cells constituting an organism, stem cells (pluripotent stem cells, etc.), progenitor cells, cancer cells isolated from an organism, cells (cell lines) isolated from an organism that have acquired immortalization ability and are stably maintained outside the body, cells isolated from an organism that have been artificially genetically modified, cells isolated from an organism that have been artificially replaced with a nucleus, etc.
• somatic cells that constitute a living body include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, red blood cells, platelets, macrophages, monocytes, bone cells, 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.
• Stem cells are cells that have both the ability to replicate themselves and the ability to differentiate into cells of multiple lineages, and examples of such cells 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, hair follicle stem cells, etc.
• pluripotent stem cells include ES cells, embryonic germ stem cells, and iPS cells.
• a precursor cell is a cell that is at an intermediate stage in the differentiation of the stem cell into a specific somatic cell or germ cell.
• Cancer cells are cells that derive from somatic cells and have acquired the ability to proliferate indefinitely.
• a cell line is a type of cell that has acquired the ability to proliferate indefinitely through artificial manipulation outside of a living organism.
• fibroblasts, stem cells, and among stem cells, pluripotent stem cells are more preferable.
• the medium can be appropriately selected depending on the type of cells used, etc.
• 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), alpha Modified Eagle's Minimum Essential Medium ( ⁇ MEM), and MEM medium (Minimum Essential Medium (MEM)).
• 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 Co., Ltd.), StemSpan SFEM (manufactured by Stem Cell Technology Co., Ltd.), Stemline II (manufactured by Sigma-Aldrich Co., Ltd.), QBSF-60 (manufactured by Quality Biological Co., Ltd.), StemPro hESCSFM (manufactured by Invitrogen Co., Ltd.), mTeSR1 or 2 medium (manufactured by Stem Cell Technology Co., Ltd.), Sf-900II (manufactured by Invitrogen Co
• 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 according to the purpose.
• those skilled in the art may also add one or more combinations of other chemical components or biological components according to the purpose.
• 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.
• cytokines examples 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).
• IL-1 interleukin-1
• IL-2 interleukin-2
• IL-3 interleukin-3
• interleukin-4 IL-4
• interleukin-5 IL-5
• interleukin-6 interleukin-6
• IL-7 interleukin-7
• IL-8 interleukin-8
• interleukin-9 interleukin-9
• interleukin-10 interleukin-10
• IL-11
• interleukin-15 interleukin-18
• interleukin-21 interleukin-21
• IFN- ⁇ interferon- ⁇
• IFN- ⁇ interferon- ⁇
• IFN- ⁇ interferon- ⁇
• G-CSF granulocyte colony-stimulating factor
• M-CSF monocyte colony-stimulating factor
• GM-CSF granulocyte-macrophage colony-stimulating factor
• SCF stem cell factor
• FL leukemia cell inhibitory factor
• LIF leukemia cell inhibitory factor
• OM oncostatin M
• EPO erythropoietin
• TPO thrombopoietin
• Hormones that can 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, These include, but are not limited to, mon
• Growth factors that can be added to the medium include transforming growth factor- ⁇ (TGF- ⁇ ), transforming growth factor- ⁇ (TGF- ⁇ ), macrophage inflammatory protein-1 ⁇ (MIP-1 ⁇ ), 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 inhibitor factor (PI), and erythrocyte growth factor (ERF).
• TGF- ⁇ transforming growth factor- ⁇
• TGF- ⁇ transforming growth factor- ⁇
• MIP-1 ⁇ macrophage inflammatory protein-1 ⁇
• EGF epidermal growth factor
• FGF-1 fibroblast growth factor-1, 2, 3, 4, 5, 6, 7, 8, 9
• NGF nerve growth factor
• HGF hepatocyte growth factor
• LIF leukemia inhibitory factor
• PI protease inhibitor factor
• ERP erythrocyte growth factor
• protease nexin I protease nexin II
• 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), pleiotrophin, etc.
• cytokines and growth factors whose amino acid sequences have been artificially altered using recombinant gene technology.
• examples include the IL-6/soluble IL-6 receptor complex and Hyper IL-6 (a fusion protein of IL-6 and the soluble IL-6 receptor).
• Examples of various extracellular matrices and cell adhesion molecules include collagen 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 of these.
• antibiotics examples include sulfa preparations, penicillin, phenethicillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, cyclacillin, carbenicillin, ticarcillin, piperacillin, azlocillin, mexocillin, mecillinam, anginocillin, cephalosporin and its derivatives, oxolinic acid, amifloxacin, temafloxacin, nalidixic acid, piromidic acid, ciprofloxacin, cinoxacin, norfloxacin, perfloxacin, rozaxacin, ofloxacin, and the like.
• serum and/or serum substitutes may be added to the medium.
• concentration of serum added to the medium may be appropriately set depending on the type of cells, culture conditions, culture purpose, etc., but in one embodiment, the concentration of serum (and/or serum substitute) in the 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 during the culture period, or may be increased or decreased as necessary when changing the medium, etc.
• 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).
• the method for producing a cell structure may further include the following steps (5) and (6).
• Step (5) A step of suspending the cell structure in a medium.
• Step (6) A step of recovering the cell structure.
• 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, a spheroid, a cell cluster, etc.
• the shape of the cell structure is not particularly limited, and examples include a sphere and a ring shape.
• Step (1) is a step of adding a medium in which cells are dispersed to the cell structure of the present invention.
• Step (1) is a step of preparing for cell culture. For example, the following total number of cells are dispersed in a 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 (n) of spots present in the cell structure.
• the medium is adjusted depending on the type of cells to be cultured.
• the type of cells may be one, or two or more types of cells may be used.
• Step (2) is a step of culturing cells to form a cell structure.
• step (2) for example, cells are cultured in the cell structure for 12 hours or more to form a cell structure.
• a 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.
• cell structures are formed in all spots, and therefore it is preferable that at least one cell is present in each spot.
• Step (3) is a step of replacing the medium.
• a certain amount of the medium in the cell structure is aspirated, and then the same amount of fresh medium is poured in.
• the medium exchange is preferably carried out 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 medium.
• the medium may be any of the above-mentioned media.
• Step (4) is a step of maturing the cell structure.
• steps (2) and (3) are repeated multiple times to mature the cell structure.
• “Maturation” refers to, for example, enlarging the cell structure to a desired size, forming an organoid structure, or inducing differentiation into a target tissue.
• the medium is replaced with a differentiation-inducing medium to induce differentiation.
• Step (5) is a step of suspending the cell structure in a medium.
• the medium is stirred to suspend the cell structures cultured in each spot in the medium.
• this is performed by stirring the medium.
• the stirring of the medium can be performed by (i) shaking the cell structure to stir the medium, (ii) aspirating and discharging the medium (pipetting operation) to stir the medium, (iii) installing a stirring blade to stir the medium, or (iv) inserting a stirrer into the cell structure to stir the medium. Two or more of these may be combined.
• Step (6) is a step of recovering the cell structure.
• step (6) for example, the medium in which the cell structures of the cell structure are suspended is sucked up with an aspirator, and the cell structures suspended in the medium are collected.
• GRGDS manufactured by Peptide Institute, Inc.
• ⁇ Preparation Example 9> 16.41 g of sterile water was added to 0.016 g of 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 Co., Ltd.) 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.
• ⁇ Preparation Example 1> (Preparation of a substrate capable of inhibiting cell adhesion by inkjet printing) Using an inkjet device (Seiko Epson Corporation, inkjet device for R&D) and an inkjet head (Seiko Epson Corporation, Precision Core Head S800-A1), an appropriate amount of the composition 1 for forming a coating film prepared in Preparation Example 1 was applied to a polystyrene substrate having a size of 79 mm x 121 mm in a perfect circle having a diameter of 18 mm. The substrate was dried in an oven at 70°C for 24 hours to prepare a substrate 1 having cell adhesion inhibitory ability.
• ⁇ 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 ⁇ m, manufactured by Allgood Co., Ltd.), an appropriate amount of the composition 2 for forming a coating film 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 having the ability to inhibit cell adhesion.
• Example 1 Using an inkjet device (Seiko Epson Corporation, inkjet device for R&D) 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 the substrate 1 having cell adhesion suppression ability prepared in Preparation Example 1, and a large number of spots with a diameter of 400 ⁇ m were formed.
• an inkjet device Seiko Epson Corporation, inkjet device for R&D
• an inkjet head Seiko Epson Corporation, Precision Core Head S800-A1
• an appropriate amount of temporary fixing film forming agent 2 was applied to the center of the 400 ⁇ m diameter spot prepared above, using an inkjet device (Microjet Corporation, model number: LaboJet-600) and an inkjet head (model number: IJHBS-300), and a large number of spots with a diameter of 100 to 150 ⁇ m were formed.
• the substrate was dried for 1 day in a thermostatic dryer at 70° C. to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.
• Example 2 Using an inkjet device (Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000), an appropriate amount of the base film forming agent 1 was applied to the culture surface of a commercially available low cell adhesion plate PrimeSurface24F (Sumitomo Bakelite Co., Ltd.), forming a large number of spots with a diameter of 400 ⁇ m.
• PrimeSurface24F Suditomo Bakelite Co., Ltd.
• an appropriate amount of the temporary fixing film forming agent 2 was applied to the center of the 400 ⁇ m diameter spot created above using an inkjet device (Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: IJHBS-300), forming a large number of spots with a diameter of 100 to 150 ⁇ m.
• the substrate was dried for one day in a thermostatic dryer at 70° C. to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.
• Example 3 Using an inkjet device (Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000), an appropriate amount of the base film forming agent 1 was applied to the culture surface of a commercially available low cell adhesion plate PrimeSurface24F (Sumitomo Bakelite Co., Ltd.), forming a large number of spots with a diameter of 400 ⁇ m.
• PrimeSurface24F Suditomo Bakelite Co., Ltd.
• an appropriate amount of the temporary fixing film forming agent 1 was applied to the center of the 400 ⁇ m diameter spot created above, forming a large number of spots with a diameter of 100 to 150 ⁇ m, using an inkjet device (Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: IJHBS-300).
• the plate was dried for one day in a thermostatic dryer at 70° C. to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.
• Example 4 Using an inkjet device (Seiko Epson Corporation, inkjet device for R&D) 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 the substrate 2 having cell adhesion suppression ability prepared in Preparation Example 2, forming a large number of spots with a diameter of 400 ⁇ m. After drying at room temperature, an appropriate amount of temporary fixing film forming agent 3 was applied to the center of the 400 ⁇ m diameter spots prepared above, forming a large number of spots with a diameter of 100 to 150 ⁇ m. The substrate was dried for one day in a thermostatic dryer at 70° C. to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.
• Example 5 Using an inkjet device (Seiko Epson Corporation, inkjet device for R&D) 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 the substrate 2 having the ability to inhibit cell adhesion prepared in Preparation Example 2, forming a large number of spots with a diameter of 400 ⁇ m. After drying at room temperature, an appropriate amount of temporary fixing film forming agent 4 was applied to the center of the 400 ⁇ m diameter spots prepared above, forming a large number of spots with a diameter of 100 to 150 ⁇ m. The substrate was dried for one day in a thermostatic dryer at 70° C. to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.
• Example 6 Using an inkjet device (Seiko Epson Corporation, inkjet device for R&D) 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 the substrate 2 having cell adhesion suppression ability prepared in Preparation Example 2, forming a large number of spots with a diameter of 400 ⁇ m. After drying at room temperature, an appropriate amount of temporary fixing film forming agent 4 was applied to the center of the 400 ⁇ m diameter spots prepared above, forming a large number of spots with a diameter of 100 to 150 ⁇ m. The substrate was dried for one day in a thermostatic dryer at 70° C. to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.
• ⁇ Comparative Example 1> Using an inkjet device (Seiko Epson Corporation, inkjet device for R&D) 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 cell adhesion inhibitory ability prepared in Preparation Example 1, forming a large number of spots with a diameter of 400 ⁇ m. The substrate was dried for one day in a thermostatic dryer at 70° C. to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.
• ⁇ Comparative Example 4> Using an inkjet device (Seiko Epson Corporation, inkjet device for R&D) 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 cell adhesion inhibitory ability prepared in Preparation Example 2, forming a large number of spots with a diameter of 400 ⁇ m. The substrate was dried for one day in a thermostatic dryer at 70° C. to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.
• Example 1 Confirmation test of the ability to inhibit spheroid migration in serum-free medium using human adipose tissue-derived mesenchymal stem cells of Example 1 and Comparative Example 1> (Cell Preparation) Human adipose tissue-derived mesenchymal stem cells (ADSC: manufactured by Cellsource Co., Ltd.) were used for cell culture. A low serum medium, Mesenchymal Stem Cell Growth Medium GM2 (manufactured by Takara Bio Co., Ltd.), was used for cell culture. The cells were cultured for 2 days or more in a 10 cm diameter petri dish (10 mL of medium) at 37°C/ CO2 incubator with 5% carbon dioxide concentration.
• ADSC Human adipose tissue-derived mesenchymal stem cells
• a low serum medium Mesenchymal Stem Cell Growth Medium GM2 (manufactured by Takara Bio Co., Ltd.) was used for cell culture. The cells were cultured for 2 days or more in a 10 cm diameter petri dish (10
• the cells were subsequently washed with 3 mL of PBS solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and then 3 mL of trypsin-EDTA solution (manufactured by PromoCell Co., Ltd.) was added and the cells were left to stand at room temperature for 3 minutes to detach the cells. 7 mL of serum-free medium, Mesenchymal Stem Cell Growth Medium DXF medium, was added to recover the cells. This suspension was centrifuged (Tomy Seiko Co., Ltd., Model No. LC-230, 200 ⁇ g/3 min, room temperature), the supernatant was removed, and the above serum-free medium was added to prepare a cell suspension.
• PBS solution manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
• trypsin-EDTA solution manufactured by PromoCell Co., Ltd.
• Cell adhesion and cell aggregate formation confirmation test 0.5 mL of cell suspension was added to each cell aggregate production substrate prepared in Example 1 and Comparative Example 1 so that the cell suspension was 1.5 x 105 cells/ cm2 . Then, the substrate was left to stand in a 37°C/ CO2 incubator with a 5% carbon dioxide concentration. After 26 hours and 74 hours, the state of the cells was observed and photographed using a stereo microscope SZX16 (manufactured by Olympus Corporation). In addition, a Cell3imager duos (manufactured by SCREEN Corporation) was used to photograph the entire surface of the well. The results are shown in Figures 4A to 4C and Figures 5A to 5C. FIG.
• FIG. 4A shows the observation results after 26 hours of culture in the test of Example 1.
• FIG. 4B shows the observation results after 72 hours of culture in the test of Example 1.
• FIG. 4C shows the observation results (overall photograph) after 72 hours of culture in the test of Example 1.
• FIG. 5A shows the observation results after 26 hours of culture in the test of Comparative Example 1.
• FIG. 5B shows the observation results after 72 hours of culture in the test of Comparative Example 1.
• FIG. 5C shows the observation results (overall photograph) after 72 hours of culture in the test of Comparative Example 1. After 26 hours, selective adhesion of cells to the base film portion on the substrate prepared in both Example 1 and Comparative Example 1 was confirmed.
• Example 1 the adhered cells peeled off from the petri dish and aggregated to form cell aggregates (spheroids) of uniform size.
• Example 1 the formed cell aggregates were regularly held on the spot portion.
• Comparative Example 1 the formed cell aggregates were detached from the spot portion.
• the temporary fixing film forming agent was applied to the base film in small amounts, and it is considered that the bottom surface of the formed cell aggregates was adhered and the cell aggregates were held on the substrate while maintaining the spherical shape of the cell aggregates.
• the cell aggregates were regularly arranged on the spots over the entire surface of the well in Example 1, while the cell aggregates were detached in Comparative Example 1. Furthermore, the detached cell aggregates were adhered to each other to form huge aggregates.
• the temporary fixing film forming agent to the base film, the state in which the aggregates are regularly arranged on the substrate after the formation of the cell aggregates can be maintained.
• Example 2 Observation when a temporary fixing film is applied onto a base film>
• a base film forming agent 2 was applied to the culture surface of a substrate 1 having the ability to inhibit cell adhesion, and then a temporary fixing film forming agent 2 was applied.
• the applied film was observed using a CCD camera.
• a film of the temporary fixing film forming agent 2 was formed in the center of the spot of the base film forming agent 2.
• the diameter of the spot of the temporary fixing film forming agent 2 was approximately 90 ⁇ m.
• Figure 6 a base film is formed in the area indicated by (1), and a temporary fixing film is formed in the area indicated by (2).
• ⁇ Test Example 3 Confirmation test of the ability to inhibit spheroid migration in serum medium using human adipose tissue-derived mesenchymal stem cells of Examples 2 and 3 and Comparative Example 2> (Cell Preparation) Human adipose tissue-derived mesenchymal stem cells (ADSC: manufactured by Cellsource Co., Ltd.) were used for cell culture. Low serum medium Mesenchymal Stem Cell Growth Medium GM2 (manufactured by Takara Bio Co., Ltd.) was used for cell culture. The cells were cultured for 2 days or more in a 10 cm diameter petri dish (10 mL of medium) at 37 ° C. / CO2 incubator with 5% carbon dioxide concentration.
• ADSC Human adipose tissue-derived mesenchymal stem cells
• GM2 manufactured by Takara Bio Co., Ltd.
• the cells were cultured for 2 days or more in a 10 cm diameter petri dish (10 mL of medium) at 37 ° C. / CO2 incubator with
• the cells were washed with 3 mL of PBS solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and then 3 mL of trypsin-EDTA solution (manufactured by PromoCell Co., Ltd.) was added and left at room temperature for 3 minutes to detach the cells. 7 mL of low serum medium Mesenchymal Stem Cell Growth Medium GM2 was added to recover the cells. This suspension was centrifuged (Tomy Seiko Co., Ltd., Model No. LC-230, 200 ⁇ g/3 min, room temperature), the supernatant was removed, and the above-mentioned low serum medium was added to prepare a cell suspension.
• PBS solution manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
• trypsin-EDTA solution manufactured by PromoCell Co., Ltd.
• FIG. 7A shows the observation results 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 is an observation result (overall photograph) after 6 days of culture in the test of Example 2. Note that there is a marking ink mark in the upper right corner of the photograph, but this can be ignored.
• 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.
• ⁇ Test Example 4 Confirmation test of the ability to inhibit spheroid migration in serum-free medium using human adipose tissue-derived mesenchymal stem cells of Examples 4 and 5 and Comparative Example 3> (Cell Preparation) A cell suspension was prepared in the same manner as in Test Example 1.
• 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 shows the observation results (overall photograph) after 7 days of culture in the test of Example 5.
• FIG. 11C shows the observation results (overall photograph) after complete medium replacement in the test of Example 5.
• FIG. 12A shows the observation results (overall photograph) after 3 days of culture in the test of Comparative Example 3.
• FIG. 12B shows the observation results (overall photograph) after 7 days of culture in the test of Comparative Example 3.
• FIG. 12C shows the observation results (overall photograph) after complete medium replacement in the test of Comparative Example 3.
• Example 5 the entire amount of medium was aspirated for Example 5 and Comparative Example 3, and 1 mL of medium was added (total medium exchange).
• total medium exchange 1 mL of medium was added (total medium exchange).
• Example 5 cell aggregates were regularly arranged on the spots over the entire surface of the well despite the medium exchange, while in Comparative Example 3, almost all of the cell aggregates were detached.
• the temporary fixing film forming agent onto the base film, the state in which the cell aggregates are regularly arranged on the substrate after formation of the aggregates can be stably maintained.
• Example 5 Confirmation test of spheroid migration inhibition ability in serum medium using HepG2 cells of Example 6 and Comparative Example 4> (Cell Preparation)
• the cells used were a human hepatoma cell line (HepG2: manufactured by ATCC).
• D-MEM high glucose
• D-MEM + 10% FBS 10 v/v% fetal bovine serum
• the cells were statically cultured for 2 days or more in a 10 cm diameter petri dish (10 mL of medium) in a 37°C/ CO2 incubator with a carbon dioxide concentration of 5%.
• the cells were subsequently washed with 10 mL of PBS solution (manufactured by 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) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the cells were left to stand for 3 minutes in a 37°C/ CO2 incubator with a 5% carbon dioxide concentration to detach the cells.
• Cell adhesion and cell aggregate formation confirmation test 1.0 mL of cell suspension was added to each cell aggregate production substrate prepared in Example 6 and Comparative Example 4 to give a cell density of 1.9 x 105 cells/ cm2 . After that, the substrate was left in a 37°C/ CO2 incubator with a 5% carbon dioxide concentration. Half or full medium replacement was performed on the 2nd, 4th, and 7th days. After 7 days, a photograph of the entire well surface was taken using a Cell3imager duos (manufactured by SCREEN Co., Ltd.). The results are shown in Figures 13A to 13B and Figures 14A to 14B.
• FIG. 13A to 13B The results are shown in Figures 13A to 13B and Figures 14A to 14B.
• FIG. 13A shows the results of observation after 7 days of culture in which half of the medium was exchanged in the test of Example 6.
• FIG. 13B shows the results of observation after 7 days of culture, in which the entire medium was replaced in the test of Example 6.
• FIG. 14A shows the results of observation after 7 days of culture in which half of the medium was exchanged in the test of Comparative Example 4.
• FIG. 14B shows the results of observation after 7 days of culture in which the entire medium was replaced in the test of Comparative Example 4.
• Example 6 Under the condition of full-volume medium exchange, most of the formed cell aggregates in Example 6 were regularly held in the spot portion, while most of the formed cell aggregates in Comparative Example 4 were detached from the spot portion.
• Example 6 it is considered that the bottom surface of the formed cell aggregates was adhered and the cell aggregates were held on the substrate while maintaining the spherical cell aggregate shape by applying a temporary fixing film forming agent to the base film in minute amounts. Since the cell aggregates can be regularly arranged on the substrate even after 7 days of culture and full-volume medium exchange, it is considered that it can also be applied to assays in which cells are fixed, such as drug discovery screening.
• the present invention makes it possible to form uniform cell aggregates and position the aggregates at any position on the plate. This allows the plate to be used for forming cell aggregates in drug discovery screening and animal alternative testing. It can also be used in applications such as regenerative medicine, where uniform cell aggregates are formed, and then differentiated and matured while suppressing fusion between the cell aggregates, before being used for transplantation.

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