WO2023027079A1 - 細胞構造体製造装置 - Google Patents

細胞構造体製造装置 Download PDF

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Publication number
WO2023027079A1
WO2023027079A1 PCT/JP2022/031747 JP2022031747W WO2023027079A1 WO 2023027079 A1 WO2023027079 A1 WO 2023027079A1 JP 2022031747 W JP2022031747 W JP 2022031747W WO 2023027079 A1 WO2023027079 A1 WO 2023027079A1
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Prior art keywords
substrate
base film
cell
cell structure
cells
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Ceased
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PCT/JP2022/031747
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English (en)
French (fr)
Japanese (ja)
Inventor
佳臣 広井
康平 鈴木
祐揮 上田
美耶 廣飯
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Nissan Chemical Corp
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Nissan Chemical Corp
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Priority to JP2023543936A priority Critical patent/JPWO2023027079A1/ja
Publication of WO2023027079A1 publication Critical patent/WO2023027079A1/ja
Anticipated expiration legal-status Critical
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    • 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
    • C08F218/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an acyloxy radical of a saturated carboxylic acid, of carbonic acid or of a haloformic acid
    • C08F218/02—Esters of monocarboxylic acids
    • C08F218/04—Vinyl esters
    • 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
    • C08F220/00—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
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/04—Acids; Metal salts or ammonium salts thereof
    • C08F220/06—Acrylic acid; Methacrylic acid; Metal salts or ammonium salts thereof
    • 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
    • C08F220/00—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
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/10—Esters
    • C08F220/34—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

Definitions

  • the present invention relates to a cell structure manufacturing apparatus for manufacturing cell structures such as spheroids.
  • Cell structures such as cell aggregates, cell aggregates, or spheroids are believed to be available for drug discovery research, cell therapy, and regenerative therapy.
  • Patent Document 1 discloses a containing plate in which a large number of containing recesses for containing one cell mass are formed, and a needle that pierces and penetrates the cell mass.
  • a support having a plurality of shaped bodies, a suction nozzle connected to a negative pressure generating means for sucking and holding the cell mass, and a moving means for moving and lifting the suction nozzle between the containing plate and the support. and control means for controlling the suction operation of the suction nozzle and the operation of the moving means.
  • Patent Document 2 a plurality of needles with long and narrow pointed tips, a puncturing section that punctures and penetrates a cell mass with the tips of the needles and raises the needle after puncturing, a base section, and a control section.
  • a cell structure manufacturing apparatus comprising:
  • Patent Documents 3 and 4 disclose a storage container for housing a cell mass, a support having a plurality of needle-shaped bodies that pierce and penetrate the cell mass, a suction nozzle that adsorbs and holds the cell mass, A three-dimensional cell structure manufacturing apparatus is described that includes nozzle moving means for moving the suction nozzle, and control means for controlling the suction operation of the suction nozzle and the operation of the nozzle moving means.
  • Patent Document 5 a plurality of cell clusters are arranged on a mounting surface in a culture vessel in a planar manner, and the cell clusters are cultured and mutually fused to produce a cell cluster sheet manufacturing method. A device is described.
  • Patent Document 6 discloses a method for producing a polymer used as a base film for cell culture and a cell culture vessel.
  • Patent Document 7 describes a coating film of an ion complex material that has the ability to suppress adhesion of biological substances.
  • a cell structure is an assembly of cells in which cells self-assemble and aggregate.
  • cell structures are generally referred to as cell aggregates, cell aggregates, spheroids, spheres, or organoids in some cases. It has been reported that since the cell structure has a biological-like structure, the cell function of the cell structure can be maintained for a long period of time, and the physiological function is improved. Therefore, there are growing expectations for the use of cell structures in drug discovery research, cell therapy, and regenerative therapy.
  • the cell structure is a spherical spheroid
  • the diameter of the spheroid is too large, some of the cells in the spherical spheroid may die.
  • the diameter of the spheroids is too small, the spheroids are less effective, such as therapeutic effects. Therefore, appropriately controlling the dimensions of cell structures such as spheroids is an important technique for improving the yield of cell structure production.
  • the spheroid diameter error can be as high as 40%. In order to improve the production yield of cell structures, it is required that the error in spheroid diameter is preferably within 20%.
  • an object of the present invention is to provide a cell structure manufacturing apparatus capable of appropriately controlling the dimensions of the cell structure when manufacturing the cell structure.
  • the present invention enables the production of homogeneous and high-quality cell constructs without using serum derived from a living body, is excellent in operability and mass productivity, and is capable of appropriately controlling the dimensions of cell constructs. It is an object of the present invention to provide a cell structure manufacturing apparatus capable of
  • the present invention has the following configuration.
  • Configuration 1 of the present invention is a cell structure manufacturing apparatus for manufacturing a cell structure, a substrate supply unit that supplies a substrate, wherein at least one surface of the substrate has an ability to suppress adhesion of cells; a base film forming unit for forming a base film on the substrate, the base film forming unit including a base film forming application mechanism for applying a base film forming composition on the substrate; a base film-forming part, wherein the base film has cell adhesiveness; and a seeding section for seeding cells onto the substrate including the base film.
  • the substrate supply unit further includes a coating film forming unit that forms a coating film having an ability to suppress adhesion of cells on at least a portion of at least one surface of the source substrate,
  • the coating film forming unit includes a coating film forming application mechanism for applying a coating film forming composition to the surface of the substrate,
  • the base film-forming application mechanism applies the base film-forming composition to at least a part of the surface of the coating film of the substrate.
  • composition 3 In configuration 3 of the present invention, the composition for forming a coating film is a copolymer having a repeating unit (A) represented by the following formula (A) and a repeating unit (B) represented by the following formula (B).
  • Fig. 2 shows a cell structure manufacturing apparatus of configuration 2, including coalescence.
  • 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 represents an alkylene group having 1 to 5 carbon atoms which may be interrupted by a bond, an amide bond or an oxygen atom.
  • composition 4 At least one selected from the coating mechanism for forming the base film and the coating mechanism for forming the coating film is a stippling coating mechanism. It is a device.
  • Configuration 5 of the present invention is the cell structure manufacturing apparatus according to any one of Configurations 1 to 4, wherein the substrate has a substantially smooth surface.
  • Configuration 6 of the present invention is the cell structure manufacturing apparatus according to any one of Configurations 1 to 4, wherein the surface of the substrate has unevenness.
  • the substrate or the material substrate is flexible
  • the substrate supply unit includes a take-up type substrate cassette or the material substrate cassette, and the substrate is supplied from the substrate cassette, or 7.
  • the cell structure manufacturing apparatus according to any one of configurations 1 to 6, wherein the raw material substrate is supplied from the raw material substrate cassette.
  • the cell structure manufacturing apparatus further includes an agglutination culturing section for culturing cells adhered onto the substrate including the base film. It is a device.
  • Configuration 9 of the present invention is the cell structure manufacturing apparatus according to any one of Configurations 1 to 8, wherein the size error of the cell structure is within 20%.
  • the cell structure manufacturing apparatus has an airtight mechanism capable of making the inside of the cell structure manufacturing apparatus an airtight closed space, and the airtight mechanism is the inside of the closed space. can be maintained in an aseptic environment.
  • composition 11 In Configuration 11 of the present invention, the composition for forming a base film has the following formula (I): [In the formula, Ua1 and Ua2 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, and Ra1 represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms. , Ra2 represents a linear or branched alkylene group having 1 to 5 carbon atoms] and a repeating unit derived from a monomer represented by the following formula (II): [In the formula, Rb represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms]. It is a structure manufacturing device.
  • Composition 12 of the present invention is the cell structure manufacturing apparatus of configuration 11, wherein the base film-forming composition further contains a cell adhesive substance.
  • the present invention it is possible to provide a cell structure manufacturing apparatus capable of appropriately controlling the dimensions of the cell structure when manufacturing the cell structure.
  • FIG. 3 is a schematic plan view showing an example of another aspect of the cell structure manufacturing apparatus of the present embodiment; It is a photograph of the microscopic observation result of the cell adhesion test of Experimental Example A1. It is a photograph of the microscopic observation result of the cell adhesion test of Comparative Experimental Example A3.
  • Fig. 10 is a stereomicroscopic photograph of the cell aggregate-producing substrates produced in Experimental Examples B1 to B3 and Comparative Experimental Examples B1 and 2, which were subjected to the cell adhesion confirmation test of Experimental Example B1.
  • Fig. 10 is a stereomicroscopic photograph of the cell aggregate-producing substrate prepared in Experimental Example B5 and Comparative Experimental Example B3, which was subjected to the cell adhesion confirmation test of Experimental Example B3.
  • Fig. 10 is a stereomicroscopic photograph of the state of the cell aggregate-producing substrate produced in Experimental Example B7, which was subjected to the cell adhesion confirmation test of Experimental Example B4.
  • FIG. 10 is a stereoscopic microscope photograph of the formation of cell aggregates (spheroids) using the cell aggregate-producing substrates of Experimental Examples B8 to B12, taken after 2 hours and 3 days, respectively.
  • FIG. 10 is a diagram showing the size evaluation results of spheroids formed using substrates for producing cell aggregates of Experimental Examples B8 to B10.
  • FIG. 10 is a table showing the relationship between the coating diameter of spheroids formed using the cell aggregate-producing substrates of Experimental Examples B8 to B12, the average spheroid diameter, and the spheroid error.
  • FIG. 10 is a diagram showing the relationship between the coating area and the spheroid diameter when spheroids were formed using the cell aggregate-producing substrates of Experimental Examples B8 to B12.
  • FIG. 10 is a stereoscopic microscope photograph of the formation of cell aggregates (spheroids) using the cell aggregate-producing substrates of Experimental Examples B8 to B12, taken after 2 hours and 3 days, respectively.
  • FIG. 10
  • FIG. 10 is a diagram showing the relationship between the coating area and the spheroid volume when spheroids were formed using the cell aggregate-producing substrates of Experimental Examples B8 to B12.
  • Fig. 10 is stereomicroscopic photographs of the state of the cell aggregate-producing substrate prepared in Comparative Experimental Example B4, which was subjected to the cell adhesion/cell aggregate formation confirmation test of Test Example B6, taken 2 hours and 2 days later.
  • Fig. 10 is stereomicroscopic photographs of the state of the cell aggregate-producing substrate prepared in Comparative Experimental Example B5, which was subjected to the cell adhesion/cell aggregate formation confirmation test of Test Example B7, taken 2 hours and 2 days later.
  • a substrate or film includes not only the case of direct contact with the upper surface of the substrate or film, but also the case of not directly contacting the upper surface of the substrate or film.
  • forming the film A on the substrate means forming the film A directly on the surface of the substrate, or forming the film A on the surface of another film formed on the surface of the substrate. including doing That is, “on” a substrate or film includes the case where another film exists between the substrate or film and the object (film). Also, “above” does not necessarily mean only the upper side in the vertical direction. “Above” simply indicates the relative positional relationship between the substrate and the film.
  • a cell structure manufacturing apparatus 10 of this embodiment will be described with reference to FIG.
  • This embodiment is a cell structure manufacturing apparatus 10 for manufacturing a cell structure 1.
  • the cell structure manufacturing apparatus 10 of this embodiment includes a substrate supply section 20 , a base film forming section 40 and a seeding section 50 .
  • the substrate supply unit 20 of the cell structure manufacturing apparatus 10 of this embodiment can further include a coating film forming unit 30 .
  • the substrate supply section 20 supplies the substrate 80 .
  • the substrate 80 is a planar (plate-like or film-like) structure whose surface has the ability to suppress adhesion of the cells 56 .
  • a planar (plate-like or film-like) structure whose surface does not have the ability to suppress adhesion of cells 56 is referred to as raw material substrate 82 .
  • a substrate 80 can be obtained by forming a coating film 84 having an ability to suppress adhesion of cells 56 on the surface of a raw material substrate 82 .
  • a surface having the ability to suppress the adhesion of cells 56 means that no adhesion or spreading of cells 56 is observed on the surface by microscopic observation, and cell structures 1 such as spheroids are formed on portions other than the surface. means that
  • having the ability to suppress the adhesion of cells 56 means that the luminescence intensity (%) (luminescence intensity of adherent cells on coating film 84) / (luminescence intensity of adherent cells on wells without coating) when compared with no coating with ATPassay emission intensity) is 50% or less, preferably 30% or less, more preferably 10% or less.
  • At least one surface of the substrate 80 has the ability to suppress cell 56 adhesion. As a result, it is possible to prevent the cells 56 from adhering to the exposed portion of the substrate 80 (or the coating film 84 of the substrate 80).
  • the substrate 80 or raw material substrate 82 used in the cell structure manufacturing apparatus 10 of this embodiment preferably has flexibility. Further, in the cell structure manufacturing apparatus 10 of the present embodiment, the substrate supply unit 20 preferably includes a take-up type substrate cassette or raw material substrate cassette 22 .
  • the raw material substrate 82 is flexible and is supplied from the raw material substrate 82 cassette 22 of the winding type.
  • the substrate supply unit 20 can include a take-up type substrate cassette. By using the take-up type substrate cassette or raw material substrate cassette 22, the cell structure 1 can be manufactured continuously.
  • the shape of the source substrate 82 (or substrate 80) can be rectangular with predetermined dimensions.
  • a raw material substrate 82 having a predetermined shape is placed in a batch-type coating film forming unit 30 , a coating film 84 is formed on the surface of the raw material substrate 82 , and the raw material substrate 82 is taken out from the coating film forming unit 30 .
  • a rectangular substrate 80 having a coating film 84 having an ability to suppress adhesion of cells 56 can be obtained.
  • the substrate 80 having a predetermined size can be subjected to predetermined processing in the base film forming section 40 and the seeding section 50 .
  • the raw material substrate 82 or the substrate 80 preferably does not have flexibility in terms of facilitating transportation of the raw material substrate 82 or the substrate 80 .
  • the substrate 80 (or raw material substrate 82) used in the cell structure manufacturing apparatus 10 of this embodiment preferably has a substantially smooth surface.
  • a base film 90 which will be described later, can be formed at an arbitrary location and with an arbitrary size.
  • the dimensions of the resulting cell structure 1 can be controlled.
  • the surface of the substrate 80 (or raw material substrate 82) used in the cell structure manufacturing apparatus 10 of this embodiment can have unevenness.
  • the surface of the substrate 80 (or the source substrate 82) has suitable unevenness, it is possible to specify the place where the base film 90, which will be described later, is to be formed. It is possible to easily determine the place where the base film 90 is to be formed.
  • the base film 90 can be formed in concave portions among the unevenness of the surface of the substrate 80 (or the source substrate 82). By controlling the dimensions of the recess in which the base film 90 is formed, the dimensions of the obtained cell structure 1 can be made appropriate.
  • the surface of the substrate 80 (or the raw material substrate 82) has appropriate unevenness, and the base film 90 is formed in the concave portion, so that the obtained cell structure 1 can have an appropriate size. can. If the surface of substrate 80 (or source substrate 82) has suitable unevenness, source substrate 82 or substrate 80 may not have flexibility.
  • Examples of materials for the raw material substrate 82 include glass, metal, compounds containing metals or metalloids, activated carbon, and resins.
  • metals typical metals: (alkali metals: Li, Na, K, Rb, Cs; alkaline earth metals: Ca, Sr, Ba, Ra), magnesium group elements: Be, Mg, Zn, Cd, Hg; aluminum Group elements: Al, Ga, In; Rare earth elements: Y, La, Ce, Pr, Nd, Sm, Eu; Tin group elements: Ti, Zr, Sn, Hf, Pb, Th; Iron group elements: Fe, Co, Ni; earth-acid elements: V, Nb, Ta; chromium group elements: Cr, Mo, W, U; manganese group elements: Mn, Re; noble metals: Cu, Ag, Au; platinum group elements: Ru, Rh, Pd, Os, Ir, Pt, and the like.
  • metal-containing compounds or semi-metal-containing compounds include ceramics, which are sintered bodies whose basic component is a metal oxide and is sintered by heat treatment at high temperature, semiconductors such as silicon, metal oxides or semi-metal oxides ( inorganic solid materials such as moldings of inorganic compounds such as silicon oxides, alumina, etc.), metal carbides or semi-metal carbides, metal nitrides or semi-metal nitrides (silicon nitrides, etc.), metal borides or semi-metal borides, etc. , aluminum, nickel titanium, and stainless steel (SUS304, SUS316, SUS316L, etc.).
  • ceramics which are sintered bodies whose basic component is a metal oxide and is sintered by heat treatment at high temperature
  • semiconductors such as silicon, metal oxides or semi-metal oxides ( inorganic solid materials such as moldings of inorganic compounds such as silicon oxides, alumina, etc.), metal carbides or semi-metal carbides, metal n
  • the resin that can be used as the material of the raw material substrate 82 may be either a natural resin or a derivative thereof, or a synthetic resin.
  • Natural resins or derivatives thereof include cellulose, cellulose triacetate (CTA), nitrocellulose (NC), Dextran sulfate immobilized cellulose, synthetic resins such as 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 poly
  • the material of the raw material substrate 82 may be of one type or a combination of two or more types.
  • a material having flexibility (flexibility) that can be wound up like a belt conveyor (roll system) is used as the material of the raw material substrate 82.
  • Materials for the substrate 80 used in the roll method include synthetic resins and natural polymers.
  • the raw material substrate 82 may be a substrate 80 used in a so-called cell culture vessel.
  • Petri dishes commonly used for culturing cells 56 tissue culture dishes, petri dishes or dishes such as multi-dishes, cell culture flasks, spinner flasks, flasks such as multi-layered flasks, plastic bags, Teflon (registered trademark) bags, culture Bags such as bags, plates such as microplates, microwell plates, multiplates, multiwell plates, chamber slides, tubes, trays, bottles such as roller bottles, and the like.
  • the substrate supply section 20 can have a coating film formation section 30 .
  • the coating film forming unit 30 is configured to form a coating film 84 having the ability to suppress adhesion of the cells 56 on at least a portion of at least one surface of the raw material substrate 82 .
  • the raw material substrate 82 generally does not have the ability to suppress cell 56 adhesion. Therefore, in the coating film forming section 30, by forming the coating film 84 having the ability to suppress adhesion of the cells 56 on at least one surface of the source substrate 82, the substrate 80 having the ability to suppress adhesion can be obtained.
  • the coating film forming unit 30 includes a coating mechanism 32 for forming a coating film.
  • the coating method by the coating film forming coating mechanism 32 includes, for example, a spin coating method, an inkjet method, a screen printing method, a flexographic printing method, a gravure printing method, an offset printing method, a bar coating method, a slit coating method, and a roll-to-roll method. , dip coating, solvent casting, pad printing, spraying, and the like.
  • the coating film forming application mechanism 32 can apply the coating film forming composition 38 to at least part of the surface of the substrate 80 in a predetermined pattern.
  • the coating film forming composition 38 can be applied to the entire surface of the raw material substrate 82 by the coating film forming application mechanism 32 .
  • the coating film-forming composition 38 can be applied in a predetermined pattern so that the application of the base film 90 and the formation of the cell structure 1 with a predetermined size, which will be described later, are performed appropriately.
  • the size of the base film 90 is important.
  • the coating film forming application mechanism 32 preferably applies the coating film forming composition 38 to the entire surface of the source substrate 82 .
  • the coating can be applied in a predetermined pattern so as not to form the coating film 84 on the unnecessary portions. Thereby, the usage amount of the coating film forming composition 38 can be reduced.
  • the coating film forming application mechanism 32 is preferably a stippling-type application mechanism, for example, an inkjet method using an inkjet printer.
  • a stippling-type application mechanism for applying the coating film-forming composition 38
  • the coating film-forming composition 38 can be applied in a predetermined pattern shape.
  • the film thickness of the coating film 84 can be set to a desired film thickness by controlling the scanning speed of the nozzle of the stippling-type coating mechanism and/or the number of repetitions of stippling.
  • the coating film forming composition 38 is supplied from the coating film forming composition tank 34 to the coating film forming application mechanism 32 (for example, an inkjet printer).
  • the coating film-forming composition 38 is injected from the coating film-forming coating mechanism 32 onto the entire surface of the substrate 80 (or in a predetermined pattern).
  • the coating film forming application mechanism 32 can have a nozzle drive function so that the nozzle of the coating film forming application mechanism 32 can move in a predetermined manner.
  • the cell structure manufacturing apparatus 10 of this embodiment can include a coating film drying mechanism 36 as necessary.
  • the coating film forming composition 38 contains a solvent
  • the coating film drying mechanism 36 evaporates the solvent to form a coating film 84 of the coating film forming composition 38. can be formed.
  • the coating film drying mechanism 36 can be, for example, a heater and/or a blower.
  • Drying can be performed at a temperature within the range of -200°C to 200°C, for example, in the atmosphere or under vacuum.
  • the coating film 84 can also be formed by drying at room temperature (10° C. to 35° C., eg 25° C.), for example. In order to form the coating film 84 more quickly, it may be dried at, for example, 40.degree. C. to 100.degree.
  • the drying temperature is not particularly limited.
  • the drying temperature is preferably lower than the glass transition point of the material substrate 82, for example, 10°C to 180°C, more preferably 20°C to 100°C.
  • the drying time is not particularly limited, but is, for example, 1 minute to 24 hours.
  • the cell structure manufacturing apparatus 10 of this embodiment has a base film forming section 40 for forming a base film 90 .
  • the base film forming section 40 includes a base film forming coating mechanism 42 that applies a base film forming composition 48 onto the substrate 80 . Since the base film 90 has cell adhesiveness, the cells 56 can adhere to the surface of the base film 90 .
  • At least part of the surface of the substrate 80 has the ability to suppress cell 56 adhesion.
  • a base film 90 can be formed on the surface capable of suppressing adhesion of cells 56 so that a part of the surface capable of suppressing adhesion of cells 56 is exposed.
  • the cells 56 can be repelled from the surface of the substrate 80 that has the ability to suppress adhesion of the cells 56, and the cells 56 can be gathered in the portion where the base film 90 is formed.
  • the cell structure 1 can be manufactured.
  • the base film forming unit 40 includes a base film forming coating mechanism 42 .
  • the coating method by the base film forming coating mechanism 42 includes, for example, a spin coating method, an inkjet method, a screen printing method, a slit coating method, a roll-to-roll method, a dip coating method, a solvent casting method, a pad printing method, and a spray. It can be selected from laws, etc.
  • the base film forming composition 48 can be applied to at least a part of the surface of the substrate 80 (or the coating film 84) in a predetermined pattern by the base film forming application mechanism 42 . In this specification, a pattern having a predetermined shape formed by applying the base film forming composition 48 is referred to as "base film pattern 90a".
  • the coating mechanism for base film formation is preferably a stippling coating mechanism, for example, an inkjet method using an inkjet printer.
  • the base film is formed into a predetermined shape of the base film pattern 90a by using a stippling system or a continuous stippling-type line drawing or plane drawing coating mechanism for applying the base film forming composition 48.
  • a forming composition 48 may be applied.
  • the film thickness of the base film pattern 90a can be set to a desired film thickness.
  • the base film forming composition 48 is supplied from the base film forming composition tank 44 to the base film forming coating mechanism 42 (for example, an inkjet printer). .
  • the base film forming composition 48 is injected from the base film forming application mechanism 42 so as to form a predetermined shape of the base film pattern 90a.
  • the base film forming coating mechanism 42 can have a nozzle driving function so that the nozzle of the base film forming coating mechanism 42 can move in a predetermined manner.
  • the substrate 80 optionally protecting the non-formation portion of the base film pattern 90a can be immersed in the base film forming composition 48 .
  • a method of adding the base film forming composition 48 to the substrate 80 (container) in which the portion where the base film pattern 90a is not formed is optionally protected, and allowing it to stand still for a predetermined time can be used.
  • the substrate 80 is a cell culture vessel
  • the base film forming composition 48 may optionally be added to a container in which the non-formation portion of the base film pattern 90a is protected and allowed to stand for a predetermined period of time. can be done.
  • the addition can be performed, for example, by adding the base film forming composition 48 in an amount 0.5 to 1 times the total volume of the container using a syringe or the like.
  • the standing time and temperature are appropriately selected according to the material of the container or substrate 80 and the type of base film-forming agent for cell culture.
  • the standing time can be 1 minute to 24 hours, preferably 5 minutes to 3 hours, and the standing temperature can be 10-80°C.
  • the base film pattern 90a can be appropriately formed on the substrate 80.
  • the area to which the base film forming composition 48 is applied is important.
  • the shape of the pattern of the base film forming composition 48 is arbitrary.
  • the shape of the underlying layer pattern 90a may be a circle, a polygon such as a triangle and a square, a star, a cross, or the like.
  • the base film pattern 90a is preferably circular (dot pattern or spot) in order to facilitate aggregation of the cells 56 to form the cell structure 1 .
  • the ratio of the total area of the dot pattern of the base film 90, the diameter of each dot pattern, and the interval between the dot patterns may vary depending on the cell used. It can be appropriately selected from a predetermined range according to the types of 56 and substrate 80, the desired size of cell aggregates, and the like.
  • the ratio of the total area of the dot pattern of the base film 90 to the surface area of the substrate 80 is preferably 30% or more, 40% or more, 50% or more, and preferably 99% or less.
  • the diameter of each dot pattern of the base film 90 may be, for example, 50-5000 ⁇ m, and may be 300-3000 ⁇ m in some cases.
  • the center-to-center spacing of each dot pattern of the base film 90 may be, for example, 100 to 6000 ⁇ m, and may be 150 to 4000 ⁇ m or 150 to 300 ⁇ m as required.
  • independent micro-sized regions (dot patterns) to which the cells 56 can adhere are arranged at high density, preferably regularly, on the substrate 80 having the ability to suppress adhesion of the cells 56.
  • a plurality of spheroids of uniform size can be formed on one substrate 80 (container) at one time.
  • the film thickness of the base film 90 is, for example, 1 to 1000 nm, preferably 5 to 500 nm, preferably 5 to 300 nm, preferably 5 to 200 nm, preferably 5 to 150 nm, preferably 10 to 150 nm.
  • the base film pattern 90a on the surface of the substrate 80 obtained by the above-described method may be used as it is without a drying process, or may be dried in water or a sample medium to be subjected to cell culture (for example, water, buffer solution, medium, etc.). After washing with , it can be used as a substrate 80 with a base film 90 for the production of the cell structure 1 .
  • Sample medium e.g., water, buffer, medium, etc., particularly preferably medium (e.g., DMEM medium (Dulbecco's modified Eagle medium)) subjected to water or cell culture as it is without drying within 1 hour ), it can be used as a substrate 80 with a base film 90 for the production of the cell structure 1 .
  • DMEM medium Dulbecco's modified Eagle medium
  • the cell structure manufacturing apparatus 10 of this embodiment can include a base film drying mechanism 46 as necessary.
  • the base film forming composition 48 contains a solvent
  • the solvent can be vaporized by the base film drying mechanism 46 after the base film pattern 90a is formed.
  • the undercoat drying mechanism 46 can be, for example, a heater and/or a blower.
  • the drying of the base film 90 can be performed under the same conditions as the drying of the coating film 84 described above.
  • the drying process of the substrate 80 having the base film 90 can be performed in the atmosphere or under vacuum, preferably within the temperature range of -200°C to 200°C. By removing the solvent in the base film forming composition 48 by the drying process, it can be completely fixed to the surface of the substrate 80 or the coating film 84 .
  • the base film pattern 90a can also be formed, for example, by drying at room temperature (10° C. to 35° C., preferably 20° C. to 30° C., eg 25° C.). For faster spot formation, drying may be performed at, for example, 40°C to 80°C. If the drying temperature is less than ⁇ 200° C., an uncommon refrigerant must be used, which lacks versatility, and drying takes a long time due to solvent sublimation, resulting in poor efficiency. If the drying temperature is above 200°C, thermal decomposition of the polymer will occur. Also, the drying temperature is preferably lower than the glass transition point of the raw material substrate 82, more preferably 10°C to 180°C, and more preferably 20°C to 100°C.
  • the substrate 80 having the base film pattern 90a for manufacturing the cell structure of the present embodiment is manufactured through the simple steps described above.
  • a step of washing with at least one solvent selected from aqueous solutions containing water and electrolytes may be performed. Cleaning is preferably performed with running water, ultrasonic cleaning, or the like.
  • the aqueous solution containing the water and the electrolyte may be heated, for example, in the range of 40.degree. C. to 95.degree.
  • Aqueous solutions containing electrolytes are preferably PBS, physiological saline (containing only sodium chloride), Dulbecco's phosphate-buffered saline, Tris-buffered physiological saline, HEPES-buffered physiological saline, and Veronal-buffered physiological saline, and PBS is preferred. Especially preferred.
  • the coating film 84 is not eluted even if it is washed with water, PBS, alcohol, or the like, and remains firmly fixed to the substrate.
  • the film thickness of the base film 90 (or base film pattern 90a) of the present embodiment has a maximum film thickness and a minimum film thickness in the range of 1 to 1000 nm, preferably in the range of 5 to 500 nm.
  • the cell structure manufacturing apparatus 10 of this embodiment has a seeding section 50 for seeding cells 56 on a substrate 80 including a base film 90 .
  • Seeding section 50 includes cell seeding mechanism 52 for seeding cells 56 onto basement membrane 90 .
  • a coating film 84 having an ability to suppress the adhesion of cells 56 is formed on a portion of the surface of the substrate 80 where the underlying film pattern 90a is not formed.
  • the surface of the substrate 80 with the ability is exposed.
  • the cells 56 gather on the base film pattern 90a and the cell structure 1 can be obtained.
  • the cells 56 can also be seeded on a portion of the coating film 84 (or the substrate 80) adjacent to the base film pattern 90a.
  • the surface of the coating film 84 (or the substrate 80) has the ability to suppress the adhesion of the cells 56, the cells 56 seeded on the portion of the coating film 84 (or the substrate 80) gather in the adjacent underlying film pattern 90a, forming a cell structure. It can be part of the body 1.
  • the seeding unit 50 includes a cell seeding mechanism 52.
  • the cell-seeding mechanism 52 can be selected from, for example, a device including a drop-type nozzle, a stippling-type application mechanism such as printing by an inkjet printer, a screen printing device, a pad printing device, a spray device, and the like.
  • the cell seeding mechanism 52 is preferably a device including a drip nozzle.
  • Cells 56 can be seeded on the base film pattern 90a (and the portion of the coating film 84 or the substrate 80 adjacent to the base film pattern 90a) by the cell seeding mechanism 52 .
  • cells 56 are supplied from the cell tank 54 to the cell seeding mechanism 52 (for example, a device including a drip nozzle). Cells 56 are dropped from the cell seeding mechanism 52 onto the base film pattern 90a (and the portion of the coating film 84 or the substrate 80 forming the base film pattern 90a).
  • the cell seeding mechanism 52 can have a nozzle driving function so that the nozzle of the cell seeding mechanism 52 can move in a predetermined manner.
  • the seeded cells 56 gather over a predetermined period of time and can form a cell structure 1 of a predetermined shape, such as a spheroid, on the base film pattern 90a.
  • the cell 56 in this embodiment is the most basic unit that constitutes an animal or plant, and has cytoplasm and various cell organelles inside the cell membrane as its elements.
  • the nucleus containing the DNA may or may not be included inside the cell 56 .
  • the animal-derived cells 56 in the present embodiment include germ cells such as sperm and ovum, somatic cells constituting a living body, stem cells (such as pluripotent stem cells), progenitor cells, cancer cells separated from a living body, and living body cells.
  • Cells (cell lines) that are isolated from living organisms and acquire immortal ability and are stably maintained outside the body, cells that are isolated from living organisms and are artificially genetically modified, and cells that are isolated from living organisms and have their nuclei artificially replaced.
  • somatic cells that make up living organisms include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes, and bones.
  • myeloid cells myeloid cells, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatocytes, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes, cardiac cells, esophageal cells, muscle cells (e.g. smooth or skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic progenitor cells (e.g. umbilical cord blood-derived CD34-positive cells), and mononuclear cells.
  • myeloid cells pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells
  • epithelial cells epidermal cells
  • endothelial cells vascular endothelial cells
  • the somatic cells are, for example, 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, blood vessels. Included are cells harvested from any tissue such as tissue, blood (including cord blood), bone marrow, heart, myocardium, eye, brain or nerve tissue. 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 other cells of multiple lineages, examples of which 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, liver stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, and hair follicle stem cells.
  • Pluripotent stem cells include ES cells, embryonic germ stem cells, and iPS cells among the above stem cells.
  • Progenitor cells are cells that are in the process of differentiating from the stem cells to specific somatic cells or germ cells.
  • Cancer cells are cells that are derived from somatic cells and have acquired unlimited proliferative potential.
  • a cell line is a cell that has acquired unlimited proliferative capacity through artificial manipulation in vitro.
  • fibroblasts and stem cells are more preferable as the cells 56 in this embodiment.
  • pluripotent stem cells are more preferable.
  • the cell structure manufacturing apparatus 10 of the present embodiment includes an agglutination culture section for aggregating and/or culturing cells 56 adhering to a substrate 80 including a base film 90 as necessary. 60 can also be included. A desired cell structure 1 can be formed by aggregation of the cells 56 .
  • the aggregation culture section 60 preferably contains a predetermined culture solution. Since the agglutination culture section 60 contains a predetermined culture medium, the cells 56 can be cultured in the culture medium. A known medium can be used as the culture medium for the cells 56 . Since the cell structure manufacturing apparatus 10 of the present embodiment includes the aggregation culture section 60, the seeded cells 56 can form the cell structures 1 in a stable environment for the cells. Note that the agglutination culture unit 60 can be a culture solution tank containing a culture solution. In the cell structure manufacturing apparatus 10 of the present embodiment, the method of advancing the flexible substrate 80 seeded with the cells 56 is changed downward or obliquely downward so that the substrate 80 is immersed in the culture solution bath containing the culture solution. Can be configured.
  • the cell structure manufacturing apparatus 10 of the present embodiment can further include a cell structure collecting section 70 for collecting the obtained cell structures 1, if necessary.
  • the cell structure 1 is separated from the substrate 80 including the base film 90 and collected by the cell structure collection mechanism 72 of the cell structure collection unit 70 . It should be noted that the separation of the cell structure 1 from the substrate 80 is preferably performed in a solution that is harmless to the cell structure 1, such as a culture solution.
  • the cell structure manufacturing apparatus 10 does not have the cell structure collecting unit 70, and the cell structure 1 is placed on the substrate 80 including the base film 90. A product as the cell structure 1 is possible.
  • the cell structure manufacturing apparatus 10 of the present embodiment preferably has an airtight mechanism capable of making the inside of the cell structure manufacturing apparatus 10 an airtight closed space.
  • the airtight mechanism of the cell structure manufacturing apparatus 10 can maintain the inside of the closed space in a sterile environment.
  • the predetermined airtight mechanism of the cell structure manufacturing apparatus 10 can maintain the inside of the closed space in a sterile environment, so that the cleaning process and the sterilization process can be made unnecessary.
  • ⁇ Washing process and sterilization process> If the cell structure manufacturing apparatus 10 does not have an airtight mechanism, there is a possibility that contamination with germs or the like will occur in each step. In that case, after the completion of each process, for example, after taking out the raw material substrate 82 from the raw material substrate cassette 22, after forming the coating film 84, and/or after forming the base film 90, a cleaning step using a cleaning device and/or Alternatively, a sterilization step is preferably performed. By performing the cleaning process after forming the coating film 84 and/or the base film 90, an excess film can be removed from the formed coating film 84 and/or the base film 90. FIG. Moreover, by performing a sterilization process, adhering germs can be removed.
  • the cleaning process is not particularly limited as long as it is a process in which the coating film 84 and/or the base film 90 are cleaned.
  • the cleaning process is performed, for example, to remove impurities, unreacted monomers, etc. remaining in the coating film 84 and/or the base film 90 from the coating film 84 .
  • Solvents used for washing include water, an aqueous solution containing an electrolyte, and the like. Solvents are generally used at room temperature (eg, 10-35° C.). The solvent may be heated, for example, in the range of 40°C to 95°C.
  • Aqueous solutions containing electrolytes are preferably PBS, physiological saline (containing only sodium chloride), Dulbecco's phosphate-buffered saline, Tris-buffered physiological saline, HEPES-buffered physiological saline, and Veronal-buffered physiological saline, and PBS is preferred. Especially preferred.
  • the coating film 84 of the present embodiment remains firmly adhered to the base material without being eluted even when washed with water, PBS, alcohol, or the like.
  • the coating film 84 and/or the base film 90 may be washed using a sample medium (for example, water, buffer solution, medium, etc.) used for cell culture.
  • a preferred medium is a culture medium, and more preferred mediums are BME medium (Eagle's basal medium) and DMEM medium (Dulbecco's Modified Eagle's Medium).
  • the coating film 84 and/or the base film 90 are preferably subjected to a sterilization process by irradiation.
  • the sterilization step is typically performed at ambient temperature (eg, about 0°C to about 40°C, preferably about 10°C to about 30°C, more preferably about 25°C).
  • Radiation to be irradiated is not limited as long as sterilization can be performed, but ⁇ -ray, X-ray or electron beam irradiation is preferable. ⁇ -rays or electron beams are more preferred, and ⁇ -rays are even more preferred.
  • the irradiation dose of ⁇ -rays may be, for example, the dose adopted in a normal sterilization process. For example, irradiation of about 5 to 40 kGy is sufficient, preferably 10 to 25 kGy.
  • the size error (dimensional error) of the cell structure 1 is preferably within 20%, more preferably within 15%, and preferably within 10%. More preferred.
  • the size error of the cell structure 1 means the value obtained by dividing the standard deviation of the dimensions when manufacturing 100 or more cell structures 1 by the average dimension. If the cell structure manufacturing apparatus 10 of this embodiment is used, the base film pattern 90a can be formed with a predetermined size, so the size of the cell structure 1 can be easily controlled. Therefore, when the cell structure 1 is manufactured using the cell structure manufacturing apparatus 10 of this embodiment, the size error of the cell structure 1 can be reduced as compared with the conventional method.
  • the cell structure 1 is a spherical spheroid
  • the diameter of the spheroid is too large, some of the cells 56 in the spherical spheroid may die.
  • the diameter of the spheroids is too small, the spheroids are less effective, such as therapeutic effects. Therefore, by appropriately controlling the dimensions of cell structures 1 such as spheroids, the production yield of cell structures 1 can be improved. Therefore, mass productivity of the cell structure 1 can be improved.
  • FIG. 3 shows a schematic plan view of another aspect of the cell structure manufacturing apparatus 10 of the present embodiment.
  • the cell structure manufacturing apparatus 10 shown in FIG. 3 is a schematic plan view showing the layout of each part when a batch-type manufacturing method is employed.
  • the raw material substrate 82 (or substrate 80) has a rectangular shape with predetermined dimensions.
  • the raw material substrate 82 (or substrate 80 ) is placed on the substrate supply section 20 .
  • the raw material substrate 82 is taken out from the substrate supply section 20 by the moving mechanism 12 such as a robot.
  • the orientation of the raw material substrate 82 (or the substrate 80) needs to be changed, it is rotated by the rotating mechanism 14.
  • FIG. The source substrate 82 (or the substrate 80) is subjected to predetermined processing in the coating film forming section 30 and the base film forming section 40, and then in the seeding section 50, in the same manner as described in the embodiment shown in FIG. Seeded.
  • the cell structure 1 After being cultured in the agglutination culture section 60 as necessary, the cell structure 1 is collected in the cell structure collection section 70 . At this time, the cell structure 1 can be collected while being placed on the substrate 80 .
  • the cells 56 are seeded onto the substrate 80 on which the base film 90 (base film pattern 90a) is arranged. including the step of The method for producing the cell structure 1 further includes other steps as necessary.
  • the adhesion and spreading proliferation of the cells 56 are suppressed by the cell adhesion suppression function of the coating film 84.
  • a good cell structure 1 for example, a spheroid such as a three-dimensional cell aggregate
  • the substrate 80 has a cell seeding surface having a coating film 84 capable of suppressing cell adhesion and a dot-pattern base film 90 (base film pattern 90a) having cell adhesiveness disposed on the coating film 84. be able to.
  • a cell seeding surface having a coating film 84 capable of suppressing cell adhesion and a dot-pattern base film 90 (base film pattern 90a) having cell adhesiveness disposed on the coating film 84.
  • the cells 56 are selectively cultured on the base film pattern 90a of the dot pattern.
  • a cell structure 1 for example, a spheroid such as a three-dimensional cell aggregate
  • a predetermined size can be obtained.
  • the coating film-forming composition 38 that can be used in the cell structure manufacturing apparatus 10 of the present embodiment will be described.
  • it is preferable to use the following coating film-forming composition 38 (sometimes referred to as “the coating film-forming composition of the present embodiment”).
  • the coating film 84 that can suppress the adhesion of biological substances and is difficult to dissolve in phosphate-buffered saline, and the adhesion of biological substances can be suppressed. It is possible to form a coating film 84 that is difficult to dissolve in phosphate buffered saline.
  • the coating film-forming composition of the present embodiment contains a copolymer having a repeating unit (A) represented by the following formula (A) and a repeating unit (B) represented by the following formula (B). is preferred.
  • 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 represents an alkylene group having 1 to 5 carbon atoms which may be interrupted by a bond, an amide bond or an oxygen atom.
  • the coating film-forming composition of the present embodiment is as follows.
  • the copolymer has a repeating unit (A) represented by the formula (A) and a repeating unit (B) represented by the formula (B),
  • the molar ratio (A:B) of the repeating unit (A) and the repeating unit (B) in the copolymer is 89:11 to 50:50,
  • a composition for forming a coating film is 89:11 to 50:50.
  • composition for forming a coating film according to any one of [1] to [5], containing a solvent.
  • a coating film capable of suppressing adhesion of biological substances and being difficult to dissolve in phosphate-buffered saline and a coating film capable of suppressing adhesion of biological substances and dissolving in phosphate-buffered saline It is possible to provide a coating film-forming composition capable of forming a coating film that is difficult to form.
  • the composition for forming a coating film of the present embodiment is used for suppressing adhesion of biological substances.
  • the composition for forming a coating film contains at least a copolymer and, if necessary, other components such as a solvent.
  • the coating film-forming composition of the present embodiment can form a coating film that is difficult to dissolve in phosphate-buffered saline.
  • the application of the composition for forming a coating film of the present embodiment is not particularly limited as long as it is used for suppressing the adhesion of biological substances, and is not limited to the formation of a coating film in contact with phosphate-buffered saline.
  • the copolymer is water insoluble.
  • water-soluble means that 1.0 g or more can be dissolved in 100 g of water at 25°C.
  • Water-insoluble means that it does not correspond to “water-soluble”, that is, the solubility in 100 g of water at 25°C is less than 1.0 g.
  • the copolymer has a repeating unit (A) represented by formula (A) below and a repeating unit (B) represented by formula (B) below.
  • the molar ratio (A:B) of repeating units (A) and repeating units (B) in the copolymer 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 represents an alkylene group having 1 to 5 carbon atoms which may be interrupted by a bond, an amide bond or an oxygen atom.
  • the copolymer may have two or more repeating units (A).
  • the copolymer may have two or more repeating units (B).
  • the copolymer preferably has one type of repeating unit (A) and one type of repeating unit (B).
  • alkyl groups having 1 to 5 carbon atoms include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, n-pentyl group, 1 -methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, and 1-ethylpropyl group.
  • R 1 to R 3 are each independently preferably a hydrogen atom, a methyl group, or an ethyl group.
  • ether bond means -O-
  • An alkylene group having 1 to 5 carbon atoms may be interrupted by an oxygen atom.
  • the alkylene group having 1 to 5 carbon atoms includes methylene, ethylene, propylene, trimethylene, tetramethylene, 1-methylpropylene, 2-methylpropylene, dimethylethylene, ethylethylene and pentamethylene.
  • 1-methyl-tetramethylene group, 2-methyl-tetramethylene group, 1,1-dimethyl-trimethylene group, 1,2-dimethyl-trimethylene group, 2,2-dimethyl-trimethylene group, and 1-ethyl- A trimethylene group is mentioned.
  • X 1 and X 2 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 an alkylene group having 1 to 5 carbon atoms are bonded through an ether bond.
  • the copolymer is preferably, for example, 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 repeating units (A) and repeating units (B) is 89:11 to 50:50.
  • the molar ratio (A:B) of the repeating unit (A) and the repeating unit (B) is (100-m): can be expressed in m.
  • the range of m is 11-50. and 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.
  • An upper limit for 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, 12-49, 12-48, 15-48, 20-49, 20-45, 22-49, or 22-45.
  • the total mol % of the repeating units (A) and the repeating units (B) in all repeating units in the copolymer is not particularly limited, but is preferably 90 mol % or more, more preferably 95 mol % or more, and 99. 5 mol % or more is more preferable, and 100% is particularly preferable.
  • the molar ratio of repeating units (A) and repeating units (B) in the copolymer is within a specific range in order to obtain a coating film that is difficult to dissolve in phosphate-buffered saline. Therefore, in the present embodiment, a coating film that is difficult to dissolve in a phosphate-buffered saline can be obtained without cross-linking the copolymer.
  • the copolymer need not have photosensitive groups to crosslink the copolymer. That is, it is preferred that the copolymer does not have a photosensitive group. Photosensitive groups include, for example, an azide group.
  • the copolymer need not have photosensitive groups for cross-linking the copolymer. Therefore, when forming the coating film, it is not necessary to perform light irradiation for cross-linking the copolymer. Therefore, the process for forming the coating film can be simplified.
  • the viscosity average degree of polymerization of the copolymer (hereinafter sometimes referred to as “degree of polymerization”) is not particularly limited, but from the viewpoint of suitably obtaining the effects of the present embodiment, it is preferably 200 to 3,000, and 200 to 2 ,500 is more preferred, and 200 to 2,000 is particularly preferred.
  • the viscosity-average degree of polymerization is measured in a completely saponified state of the copolymer.
  • P indicates the viscosity average degree of polymerization.
  • the viscosity average degree of polymerization can be obtained according to JIS K 6726.
  • the method for producing the copolymer is not particularly limited, but for example, a compound represented by the following formula (C) is polymerized to produce a homopolymer, and the resulting homopolymer is partially hydrated by a known saponification reaction.
  • a method of obtaining a copolymer by decomposing is mentioned. (Wherein, R 1 , R 3 and X 1 are as defined above.)
  • a method for producing a copolymer for example, a method of obtaining a copolymer by copolymerizing a compound represented by the following formula (C) and a compound represented by the following formula (D) can be mentioned. be done. (Wherein, R 1 to R 3 , X 1 and X 2 are as defined above.)
  • the copolymer may be a random copolymer or a block copolymer.
  • a commercially available product may be used as the copolymer.
  • Commercially available copolymers include polyvinyl acetate (manufactured by Nippon Acetate & Poval, trade name JMR-10L (registered trademark)).
  • the content of the copolymer in the film-forming component in the coating film-forming composition 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 a component excluding the solvent component from all components of the composition.
  • the content of the copolymer in the coating film-forming composition is not particularly limited, but from the viewpoint of facilitating the formation of a coating film having a desired thickness, it is preferably 0.1 to 10% by mass, and 0.1 to 8% by mass. % by mass is more preferred, and 0.1 to 5% by mass is particularly preferred.
  • solvents examples include water, phosphate buffered saline (PBS), alcohol, and water-soluble organic solvents (excluding alcohol).
  • Alcohols include alcohols with 2 to 6 carbon atoms.
  • Examples of 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-hexanol, 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
  • a water-soluble organic solvent is an organic solvent that can be mixed with water and alcohol in any ratio and that does not separate after mixing.
  • water-soluble organic solvents include ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, methyl cellosolve acetate, ethyl cellosolve acetate, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, propylene glycol, propylene glycol monomethyl ether, and propylene glycol monoethyl ether. , propylene glycol monomethyl ether acetate, and propylene glycol propyl ether acetate. These can be used individually by 1 type or in combination of 2 or more types.
  • water, phosphate-buffered saline (PBS), alcohol, or a water-soluble organic solvent may be used alone as a solvent.
  • Two or more of water, phosphate-buffered saline (PBS), alcohol, and water-soluble organic solvent may be used in combination as a solvent for the composition for forming a coating film.
  • the solvent is preferably selected from water, alcohol, water-soluble organic solvents and combinations of two or more thereof, and water, ethanol, water-soluble organic solvents and two or more thereof. more preferably selected from a combination of
  • the mixing ratio (mass ratio) of water:alcohol in the coating film-forming composition is, for example, 1:99 to 70:30 and 1:99 to 50:50.
  • the mixing ratio of water:alcohol:water-soluble organic solvent (mass ratio (A:B:C)) in the coating film-forming composition is, for example, 5 to 30:65 to 92:1 to 30 (where A + B + C is 100).
  • the mixing ratio (mass ratio) of alcohol:water-soluble organic solvent in the coating film-forming composition is, for example, 1:99 to 97:3.
  • the content of the solvent in the coating film-forming composition is not particularly limited, but is preferably 90% by mass or more, more preferably 92% by mass or more, more preferably 95% by mass, from the viewpoint of facilitating the formation of a coating film having a desired thickness. % or more is particularly preferred.
  • the coating film-forming composition can also contain other components, if necessary.
  • Other components include, for example, pH adjusters, preservatives, surfactants, antifungal agents, sugars, and the like.
  • Having the ability to suppress the adhesion of cells 56 means that cell aggregates (spheroids) are formed without adhesion or spreading of cells 56 observed under a microscope.
  • having the ability to suppress the adhesion of cells 56 means that the luminescence intensity (%) (luminescence intensity of adherent cells on the coating film) / (luminescence of adherent cells on wells without coating) when compared with no coating with ATPassay strength) is 50% or less, preferably 30% or less, more preferably 10% or less.
  • the coating film 84 of the coating film forming composition of the present embodiment (sometimes referred to as “coating film of the present embodiment”) is obtained by applying the coating film forming composition of the present embodiment described above.
  • the coating film of the present embodiment is a coating film of the coating film-forming composition of the present embodiment.
  • the coating film of the present embodiment is used for suppressing adhesion of biological substances.
  • the coating film of the present embodiment is characterized by being difficult to dissolve in phosphate-buffered saline.
  • the application of the composition for forming a coating film of the present embodiment is not particularly limited as long as it is used for suppressing adhesion of biological substances, and is not limited to application in contact with phosphate-buffered saline.
  • the molar ratio of the repeating unit (A) and the repeating unit (B) in the copolymer is set within a specific range. ing. Therefore, in the coating film of the present embodiment, the copolymer is not crosslinked, and a coating film that is difficult to dissolve in a phosphate-buffered saline can be obtained. Therefore, in the coating film of this embodiment, the copolymer may not be crosslinked. In addition, the process for forming the coating film of this embodiment can be simplified.
  • the content of the copolymer in the coating film of the present 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 thickness of the coating film of the present embodiment is not particularly limited, but is, for example, 1 to 10000 nm, preferably 5 to 1000 nm, more preferably 10 to 500 nm, even more preferably 20 to 300 nm, and 50 to 250 nm. Even more preferred, 100 to 250 nm is particularly preferred. This film thickness can also be applied to other types of coating films.
  • a composition for forming a coating film described in International Publication No. 2014/196650 can be used.
  • a copolymer (P) containing 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) [In the formula, U a11 , U a12 , U b11 , U b12 and U b13 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, An ⁇ is a halide ion, an inorganic acid Represents an anion selected from the group consisting of ion, hydroxide ion and isothiocyanate ion] and a solvent, and a step of applying the composition for forming a coating film onto the surface of the container or the substrate and drying the composition.
  • the coating film may be included on at least
  • the ability to suppress the adhesion of cells 56 means that cell aggregates (spheroids) are formed without adhesion or spreading of cells 56 observed under a microscope.
  • the luminescence intensity (%) (luminescence intensity of adherent cells on coating membrane) / (luminescence intensity of adherent cells on uncoated wells) when compared with no coating with ATPassay is 50% or less, preferably 30%.
  • it means 10% or less.
  • a copolymer of an ethylenically unsaturated monomer, a polysaccharide, or a derivative thereof may be used as another coating film of the present embodiment.
  • ethylenically unsaturated monomers include one or two selected from the group consisting of (meth)acrylic acid and its esters; vinyl acetate; vinylpyrrolidone; ethylene; vinyl alcohol; and hydrophilic functional derivatives thereof. More than one species of ethylenically unsaturated monomers may be mentioned.
  • polysaccharides or derivatives thereof include cellulosic polymers such as hydroxyalkylcellulose (eg, hydroxyethylcellulose or hydroxypropylcellulose), starch, dextran, and curdlan.
  • hydrophilic functional derivative refers to an ethylenically unsaturated monomer having a hydrophilic functional group or structure.
  • hydrophilic functional groups or structures include betaine structures; amide structures; alkylene glycol residues; amino groups;
  • a betaine structure means a monovalent or divalent group of compounds having an amphoteric center with a quaternary ammonium type cationic structure and an acidic anionic structure, such as the phosphorylcholine group: can be mentioned.
  • ethylenically unsaturated monomers having such a structure include 2-methacryloyloxyethylphosphorylcholine (MPC).
  • the amide structure has the formula: [Here, R 16 , R 17 and R 18 are each independently a hydrogen atom or an organic group (e.g., a methyl group, a hydroxymethyl group, a hydroxyethyl group, etc.)] means a group represented by Examples of ethylenically unsaturated monomers having such structures include (meth)acrylamide, N-(hydroxymethyl)(meth)acrylamide, N-isopropyl(meth)acrylamide and the like. Furthermore, monomers or polymers having such structures are disclosed in, for example, JP-A-2010-169604.
  • An alkylene glycol residue is an alkylene glycol (HO-Alk-OH; where Alk is an alkylene group having 1 to 10 carbon atoms).
  • the hydroxyl groups at one or both terminals of the alkylene glycol remain after the condensation reaction with other compounds. It means an alkyleneoxy group (--Alk--O--) and also includes a poly(alkyleneoxy) group in which the alkyleneoxy unit is repeated.
  • Examples of ethylenically unsaturated monomers having such structures include 2-hydroxyethyl (meth)acrylate, methoxypolyethylene glycol (meth)acrylate, and the like.
  • monomers or polymers having such structures are disclosed in, for example, JP-A-2008-533489.
  • An amino group has the formula: —NH 2 , —NHR 19 or —NR 20 R 21 [wherein R 19 , R 20 and R 21 are each independently an organic group (for example, linear or branched alkyl group, etc.)].
  • Amino groups in the present invention include quaternary or salified amino groups. Examples of ethylenically unsaturated monomers having such a structure include dimethylaminoethyl (meth)acrylate, 2-(t-butylamino)ethyl (meth)acrylate, methacryloylcholine chloride, and the like.
  • a sulfinyl group has the formula: [Here, R 22 is an organic group (eg, an organic group having 1 to 10 carbon atoms, preferably an alkyl group having 1 to 10 carbon atoms and having one or more hydroxy groups, etc.)] means a group represented by Examples of polymers having such a structure include copolymers disclosed in Japanese Unexamined Patent Application Publication No. 2014-48278.
  • a coating film capable of suppressing adhesion of biological substances and hardly soluble in phosphate-buffered saline and a coating film capable of suppressing adhesion of biological substances and phosphate-buffered saline It is possible to form a coating film that is difficult to dissolve in.
  • the coating film of the embodiment can be preferably used as the coating film 84 used in the cell structure manufacturing device 10 of the present embodiment.
  • Base film forming composition 48 that can be used in the cell structure manufacturing apparatus 10 of the present embodiment will be described.
  • the base film-forming composition 48 (sometimes referred to as "underlying film forming composition of the present embodiment").
  • the base film-forming composition of the present embodiment it is possible to achieve uniform adhesion of the cells 56 to the base film 90 under animal-derived serum-free culture conditions, so that a high-quality cell structure 1 can be produced. be able to.
  • the base film-forming composition of the present embodiment it is possible to achieve mass production of homogeneous and high-quality cell structures 1 used in the field of regenerative medicine.
  • the composition for forming a base film of the present embodiment has the following formula (I): [In the formula, Ua1 and Ua2 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, and Ra1 represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms. , Ra2 represents a linear or branched alkylene group having 1 to 5 carbon atoms] and a repeating unit derived from a monomer represented by the following formula (II): [In the formula, Rb represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms].
  • composition for forming a base film of this embodiment is as follows.
  • the polymer is a repeat derived from a monomer represented by the above formula (II) [wherein R b represents a hydrogen atom or a straight or branched alkyl group having 1 to 5 carbon atoms]
  • the composition for forming an undercoat film of [1] which contains units.
  • composition for forming a base film according to any one of [1] to [3], wherein the cell adhesive substance contains a glycoprotein.
  • the composition for forming a base film of the present embodiment has the following formula (I): [In the formula, U a1 and U a2 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, and R a1 is a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms and R a2 represents a linear or branched alkylene group having 1 to 5 carbon atoms].
  • the polymer contained in the base film-forming composition of the present embodiment is a polymer containing a repeating unit derived from the monomer represented by formula (I) above.
  • the polymer is represented by the following formula (II) together with the cationic monomer represented by the above formula (I): [In the formula, R b represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms].
  • the "linear or branched alkyl group having 1 to 5 carbon atoms” includes, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, n-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2 , 2-dimethylpropyl group or 1-ethylpropyl group.
  • R a1 and R b are each independently selected from a hydrogen atom and a methyl group.
  • U a1 and U a2 are preferably each independently selected from hydrogen atom, methyl group, ethyl group, n-propyl group, isopropyl group and n-butyl group, but are preferably methyl group or ethyl group. Preferred are methyl groups.
  • the "linear or branched alkylene group having 1 to 5 carbon atoms” includes, for example, methylene group, ethylene group, propylene group, trimethylene group, tetramethylene group, 1-methyl propylene group, 2-methylpropylene group, dimethylethylene group, ethylethylene group, pentamethylene group, 1-methyl-tetramethylene group, 2-methyl-tetramethylene group, 1,1-dimethyl-trimethylene group, 1,2- Dimethyl-trimethylene group, 2,2-dimethyl-trimethylene group, 1-ethyl-trimethylene group and the like.
  • R a2 is preferably selected from an ethylene group and a propylene group.
  • cationic monomers represented by formula (I) above include 2-N,N-dimethylaminoethyl methacrylate and N,N-dimethylaminomethyl methacrylate. Ethyl methacrylate is preferred.
  • anionic monomer represented by formula (II) include acrylic acid and methacrylic acid, with methacrylic acid being preferred.
  • the molar ratio of units derived from the monomer represented by formula (I)/units derived from the monomer represented by formula (II) in the polymer is 100/0 to 50/50. It is preferably 98/2 to 50/50. More preferably 98/2 to 60/40, particularly preferably 98/2 to 70/30. When the molar ratio of the formula (II) is 50 or less, it is possible to suppress a decrease in the adhesive strength of the cells 56 due to the anionicity of the polymer.
  • the polymer may be a polymer obtained by polymerizing a monomer represented by Formula (I)/Formula (II) and a monomer having two or more carbon-carbon unsaturated bonds.
  • a monomer having two or more carbon-carbon unsaturated bonds is specifically a monomer having two or more carbon-carbon double bonds, such as polyfunctional acrylate compounds, polyfunctional acrylamide compounds, polyfunctional A polyester or an isoprene compound may be used.
  • Preferred specific examples include monomers represented by the following formulas (III) to (V).
  • 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 is a linear or branched alkylene group having 1 to 5 carbon atoms. group
  • n represents a number from 1 to 50.
  • the monomer represented by Formula (III) is preferred.
  • the molar ratio of the monomers represented by formulas (III)-(V) to the total polymer is preferably 0-50%, more preferably 2-25%.
  • the molar ratio of the formulas (III) to (V) is 50% or less, gelation of the solid content during production due to high molecular weight due to excessive cross-linking can be suppressed, and production can be facilitated.
  • 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, most preferably an ethylene group.
  • n is a number from 1 to 50. n is preferably a number from 1 to 30, and n is preferably a number from 1 to 10.
  • Difference between the mol% value of the monomer represented by the formula (II) with respect to the entire polymer and the mol% value of the monomer represented by the formula (II) with respect to the total amount of monomer charged during the preparation step is 0 to 10 mol %.
  • the polymer of the present embodiment has a small difference between the charged monomer ratio and the measured value of the polymer produced by the production method described later, and is 0 to 10 mol %, more preferably 0 to 8 mol %.
  • the number average molecular weight (Mn) of the polymer is 20,000 to 1,000,000, more preferably 50,000 to 800,000.
  • the ratio (Mw/Mn) between the weight average molecular weight (Mw) and the number average molecular weight (Mn) of the polymer is 1.01 to 10.00, preferably 1.2 to 8.0, It is preferably 1.4 to 6.0, preferably 1.5 to 5.0, preferably 1.6 to 4.5.
  • the number average molecular weight (Mn) and the number average molecular weight (Mn) can be determined, for example, by Gel Filtration Chromatography described in Examples.
  • the polymer of the present embodiment As the base film 90 for cell culture, it is possible to form the cell structure 1 (spheroids such as cell aggregates) by detaching the cells 56 after adhering them.
  • the cell structure 1 indicates a structure formed as a result of aggregation of the cells 56, and its shape is not limited to a spherical shape, a ring shape, or the like.
  • size adjustment of the cell structure 1 by defining the adhesion area (cell aggregates of any size can be produced), etc.
  • the composition for forming a base film of the present embodiment preferably further contains a cell adhesive substance.
  • a cell-adhesive substance in the base film-forming composition 48, adhesion, spreading, proliferation and differentiation of the cells 56 can be promoted. As a result, formation of the desired cell structure 1 can be facilitated.
  • cell adhesive substances known substances such as extracellular matrix (ECM) proteins, glycoproteins, peptides, and other biologically-derived substances, and synthetic compounds (low-molecular-weight, high-molecular-weight) can be used.
  • the cell adhesive substance is preferably a non-biological compound, such as a synthetic compound (low molecular weight, high molecular weight).
  • a low molecular weight compound is, for example, a compound having a weight average molecular weight of 2,000 or less
  • a high molecular weight compound is, for example, a weight average molecular weight of 2,000 or more
  • the upper limit is, for example, 1,000,000.
  • extracellular matrix (ECM) proteins examples include collagen (e.g. Merck 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 product numbers: E1625, E6527), fibronectin (e.g. Merck product numbers F1141, F0635, F2518, F0895, F4759, F2006), laminin (e.g.
  • Merck product numbers L6724, L2020, L4544), laminin fragments ( For example, Matrixome: 892011), and vitronectin (for example, VTN-N (Gibco), Vitronectin, Human, Recombinant, Animal Free (PeproTech), Merck product numbers: V0132, V9881, V8379, 08-126, SRP3186). mentioned.
  • the cell adhesive substance is preferably a glycoprotein. Specifically, it is preferably selected from vitronectin, integrin, cadherin, fibronectin, laminin, tenascin, osthiopontin and bone sialoprotein. Also, it is preferably a protein having an RGD sequence as an amino acid sequence.
  • peptides examples include ECM peptide (MAPTrix (registered trademark) from Kollodis Bio Sciences) and RGD peptide (manufactured by Fujifilm Wako Pure Chemical Industries: 180-01531).
  • ECM peptide MAPTrix (registered trademark) from Kollodis Bio Sciences
  • RGD peptide manufactured by Fujifilm Wako Pure Chemical Industries: 180-01531.
  • Examples of synthetic compounds include polylysine (eg Merck products: P4707, P4832, P7280, P9155, P6407, P6282, P7405, P5899) and polyornithine (eg Merck product number P4975).
  • Examples of synthetic compounds low molecular weight include adhesamine (eg AD-00000-0201 manufactured by Nagase & Co., Ltd.) and synthetic cyclic RGD peptide (eg LS-3920.0010 manufactured by IRIS BIOTECH).
  • the ratio (mass basis) of the polymer to the cell adhesive substance in the base film-forming composition of the present embodiment is not limited as long as the base film-forming composition 48 capable of cell culture can be formed.
  • the ratio (by mass) of the polymer to the cell adhesive substance is preferably 100:0.1 to 100:100.
  • the cell adhesion substance is 0.1 or more, the cell adhesiveness is sufficiently exhibited, and when the cell adhesion substance is 100 or less, aggregation of the cells 56 after cell adhesion (formation of the cell structure 1) is suppressed. Easy to do.
  • the base film-forming composition of the present embodiment contains a solvent.
  • the solvent is not limited as long as it can dissolve the polymer, but it is preferably a water-containing solution.
  • the aqueous solution includes water, a salt-containing aqueous solution such as physiological saline or a phosphate buffer solution, or a mixed solvent in which water or a salt-containing aqueous solution and alcohol are combined.
  • Alcohols include alcohols having 2 to 6 carbon atoms, such as ethanol, propanol, isopropanol, 1-butanol, 2-butanol, isobutanol, t-butanol, 1-pentanol, 2-pentanol, 3-pentanol.
  • cell-adhesive substance in addition to the polymer, cell-adhesive substance, and solvent, other substances can be added to the base film-forming composition 48 as necessary within a range that does not impair the performance of the obtained base film 90 .
  • Other substances include pH adjusters, cross-linking agents, preservatives, surfactants, primers to improve adhesion to the container or substrate 80, anti-mold agents, sugars, and the like.
  • the coating film forming composition 38 and the base film forming composition 48 that can be used in the cell structure manufacturing apparatus 10 of the present embodiment will be specifically described, but the present invention is limited to these. not to be
  • the coating film forming composition 38 obtained above was spin-coated on a silicon wafer treated with HMDS (1,1,1,3,3,3-hexamethyldisilazane) at 1500 rpm/60 seconds, and dried for 70 minutes. C. for 24 hours to obtain a coating film 84 on the HMDS-treated silicon wafer.
  • a spectroscopic ellipsometer was used to measure the film thickness of the coating film 84 on the HMDS-treated silicon wafer. After that, it is thoroughly washed with PBS (phosphate buffered saline) and dried in an oven at 50° C. for 1 hour. bottom. The remaining film ratio was calculated from the film thickness after washing with PBS with respect to the film thickness after coating.
  • PBS phosphate buffered saline
  • a cell culture coating plate was produced by the following method (i) or (ii).
  • Mouse embryo fibroblasts (manufactured by DS Pharma Biomedical) were used as cells.
  • the medium used for cell culture was BME medium (Thermo Fisher Scientific) containing 10% FBS (Sigma-Aldrich) and L-glutamine-penicillin-streptomycin stabilizing solution (Thermo Fisher Scientific). ) was used.
  • the cells were statically cultured in a 10 cm diameter petri dish (10 mL medium) for 2 days or longer in a 37° C./CO 2 incubator while maintaining a 5% carbon dioxide concentration.
  • the cells were washed with 5 mL of PBS, and then 1 mL of 0.25 w/v% trypsin-1 mmol/L EDTA solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to peel off the cells. suspended respectively. After centrifuging this suspension (manufactured by Tomy Seiko Co., Ltd., model number LC-200, 1000 rpm/3 min, room temperature), the supernatant was removed and the above medium was added to prepare a cell suspension.
  • 0.25 w/v% trypsin-1 mmol/L EDTA solution manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
  • Cell adhesion experiment For each well of the plate prepared above, 150 ⁇ L of each cell suspension was added so that 1 ⁇ 10 4 cells/well for a 96-well cell culture plate and 5 ⁇ 10 4 cells/well for a 24-well cell culture plate. Each 0.5 mL was added so as to be cells/well. After that, it was allowed to stand in a CO 2 incubator at 37° C. for 3 days while maintaining a carbon dioxide concentration of 5%. After 3 days of culture, adhesion of cells to each well of the plate prepared above was compared based on observation (magnification: 4x) with an inverted microscope (CKX31, manufactured by Olympus). The observation results were evaluated according to the following evaluation criteria, and the cell adhesion inhibitory effect was confirmed.
  • FIG. 4 shows a photograph of the microscopic observation results of the cell adhesion test of Experimental Example A1.
  • the adhesion and spreading of the cells 56 were not observed by making the substrate surface hydrophilic. ) was observed.
  • the coating film-forming composition 38 obtained above was added to separate wells of a 96-well cell culture plate (manufactured by Corning, #9017, volume 0.36 mL, made of polystyrene) in 5 wells at a concentration of 150 ⁇ L/well. After immersion at room temperature for 1 hour, the solution was drained and dried at 50° C. for 24 hours using an oven. After that, each coated well was washed with 200 ⁇ L of pure water three times, dried in an oven at 70° C. for 1 hour, and then used for the test. As a negative control, wells of an uncoated 96-well cell culture plate (Corning #9017, volume 0.36 mL, polystyrene) were used.
  • IgG-HRP diluted solution A goat anti-mouse IgG antibody-HRP conjugate (manufactured by Southern Biotechnology Associates) was diluted with PBS to a concentration of 1 mg/g to prepare an IgG-HRP dilution.
  • the absorbance at 450 nm minus the absorbance at 650 nm was calculated to give the average absorbance of 5 wells. Taking the average absorbance in the negative control wells as the protein adsorption rate of 100%, the protein adsorption rate of the wells coated with the coating film-forming composition 38 obtained above was calculated.
  • a coating film-forming composition 38 was prepared in the same manner as in Experimental Example A1, except that the degree of polymerization and saponification of polyvinyl acetate and the composition of each solvent were changed as shown in Table 1.
  • the resulting coating film-forming composition 38 was transparent and uniform.
  • the resulting coating film-forming composition 38 was operated in the same manner as in Experimental Example A1 to form a coating film 84 on an HMDS-treated silicon wafer, and to form a cell culture coating plate and a protein adhesion suppression test coating plate. was made.
  • the remaining film ratio was determined in the same manner as in Experimental Example A1.
  • a cell adhesion experiment was performed in the same manner as in Experimental Example A1.
  • a protein adhesion experiment was performed in the same manner as in Experimental Example A1.
  • a coating film-forming composition 38 was prepared in the same manner as in Experimental Example A1, except that the degree of polymerization and saponification of polyvinyl acetate and the composition of each solvent were changed as shown in Table 1.
  • the resulting coating film-forming composition 38 was transparent and uniform.
  • the resulting coating film-forming composition 38 was operated in the same manner as in Experimental Example A1 to form a coating film 84 on an HMDS-treated silicon wafer.
  • a coated plate for cell culture was produced by the method (ii) above.
  • a coated plate for protein adhesion suppression test was produced in the same manner as in Experimental Example A1. The remaining film ratio was determined in the same manner as in Experimental Example A1.
  • FIG. 5 shows a photograph of the results of microscopic observation of the cell adhesion test of Comparative Experimental Example A3.
  • the substrate surface was not sufficiently hydrophilized, and the cells adhered and spread on the bottom surface of the plate.
  • Example A21> (Preparation of coating film-forming composition) A composition for forming a coating film was prepared in the same manner as in Experimental Example A20, except that the solution was dissolved at a concentration of 3 mg/g. The resulting coating film-forming composition was transparent and uniform.
  • ADSC Human adipose tissue-derived mesenchymal stem cells
  • the medium used for cell culture was low serum medium Mesenchymal Stem Cell Growth Medium 2 (PromoCell) containing L-glutamine-penicillin-streptomycin stabilizing solution (Thermo Fisher Scientific).
  • the cells were statically cultured in a 10 cm diameter petri dish (10 mL medium) for 2 days or longer in a 37° C./CO 2 incubator while maintaining a 5% carbon dioxide concentration.
  • TrypLE Select Enzyme manufactured by Thermo Fisher Scientific was added to detach the cells and suspended in 10 mL of the above medium. This suspension was centrifuged (manufactured by Tomy Seiko Co., Ltd., model number LC-200, 1000 rpm/3 minutes, room temperature), the supernatant was removed, and the above medium was added to prepare a cell suspension.
  • Cell adhesion experiment A cell adhesion experiment was conducted in the same manner as in Experimental Example 1, except that the cell suspension prepared by the above method was used.
  • the coating film 84 obtained from the coating film forming composition 38 of Experimental Example A21 the adhesion and spreading of the cells 56 were not observed by making the base material surface hydrophilic. ) was observed.
  • a photosensitive polyvinyl alcohol was obtained according to Synthesis Example 5 and Experimental Example A3 of JP-A-2003-292477.
  • the resulting photosensitive polyvinyl alcohol was dissolved in ethanol to a concentration of 5 mg/g to prepare coating film-forming composition 38 .
  • As polyvinyl alcohol Gosenol EG-30P (manufactured by Mitsubishi Chemical Corporation, degree of saponification: 86.3-69.0) was used.
  • the resulting coating film-forming composition 38 was operated in the same manner as in Experimental Example A1 to form a coating film 84 on an HMDS-treated silicon wafer and to produce a cell culture coating plate. The remaining film ratio was determined in the same manner as in Experimental Example A1.
  • a cell adhesion experiment was performed in the same manner as in Experimental Example A1.
  • Comparative Experimental Example A6 In Comparative Experimental Example A5, when the coating film 84 was formed on the HMDS-treated silicon wafer and the coating plate for cell culture was produced, after the drying step, an ultra-high pressure mercury lamp (ultraviolet illuminance 20 mW/cm 2 : UT- 150 (illuminance meter manufactured by USHIO)), a coating film 84 was formed on an HMDS-treated silicon wafer, and a cell culture coating plate was produced in the same manner as in Comparative Experimental Example A5, except that exposure was performed for 5 seconds. . The remaining film ratio was determined in the same manner as in Experimental Example A1. A cell adhesion experiment was performed in the same manner as in Experimental Example A1.
  • EtOH represents ethanol and PGME represents propylene glycol monomethyl ether.
  • the degree of saponification of polyvinyl acetate in Experimental Examples A1-20 and Comparative Experimental Examples A1-4 corresponds to the molar ratio (A:B) of the repeating unit (A) and the repeating unit (B).
  • the molar ratio (A:B) of polyvinyl acetate with a degree of saponification of 35.8 mol% is 64.2:35.8.
  • the molar ratio (A:B) of polyvinyl acetate with a degree of saponification of 65.4 mol% is 34.6:65.4.
  • the coating films 84 obtained from the coating film-forming compositions 38 of Experimental Examples A1 to A20 were compared with the coating films 84 obtained from the coating film-forming compositions 38 of Comparative Experimental Examples A1, 2, and 5 in terms of PBS.
  • the residual film rate was high even after washing with In the coating films 84 obtained from the coating film-forming compositions 38 of Experimental Examples A1 to A20, no cell attachment or spreading was observed, and formation of cell aggregates (spheroids) was observed in the wells.
  • the coating films 84 obtained from the coating film-forming compositions 38 of Comparative Experimental Examples A3, 4, and 5 the cells adhered and spread on the bottom surface of the plate.
  • the coating films 84 obtained from the coating film-forming compositions 38 of Experimental Examples A1 to A20 had lower protein adhesion rates than the substrates 80 without the coating film 84 and Comparative Experimental Example A3.
  • the coating films 84 obtained from the coating film-forming compositions 38 of Experimental Examples A1 to A20 have the ability to suppress cell 56 adhesion. Therefore, the coating film forming composition 38 can be preferably used as the coating film forming composition 38 for forming the coating film 84 in the coating film forming section 30 of the cell structure manufacturing apparatus 10 of the present embodiment. can.
  • the composition for forming an undercoat film will be described in more detail with reference to Experimental Example B and Comparative Experimental Example B, but the composition for forming an undercoat film is not limited to Experimental Example B below.
  • the substrate 80 on which the base film 90 is formed using the base film-forming composition 48 is referred to as the cell aggregate-producing substrate.
  • the base film forming composition 48 used in Experimental Example B and Comparative Experimental Example B is referred to as a base film forming agent.
  • GFC Gel Filtration Chromatography
  • a polymer was synthesized by dropwise polymerization in 166.62 g of 2-propanol.
  • the reaction product was reprecipitated with hexane, which is a poor solvent, and the precipitate was collected by filtration and dried under reduced pressure.
  • the weight average molecular weight of this polymer by GFC was 228,000 (hereinafter referred to as "Synthetic Example Polymer 1").
  • a polymer was synthesized by dropwise polymerization with respect to 64.65 g of 2-propanol.
  • the reaction product was reprecipitated with hexane, which is a poor solvent, and the precipitate was collected by filtration and dried under reduced pressure.
  • the weight average molecular weight of this polymer by GFC was 438,000 (hereinafter referred to as "Synthetic Example Polymer 2").
  • Example B9 A substrate for forming cell aggregates was produced in the same manner as in Experimental Example B8, except that the spot diameter of the base film-forming agent was changed to 250 ⁇ m and the center-to-center interval of the spots was changed to 350 ⁇ m.
  • Example B10> A substrate for forming cell aggregates was produced in the same manner as in Experimental Example B8, except that the spot diameter of the base film-forming agent was changed to 400 ⁇ m and the center-to-center interval of the spots was changed to 500 ⁇ m.
  • Example B11> A substrate for forming cell aggregates was produced in the same manner as in Experimental Example B8, except that the spot diameter of the base film-forming agent was changed to 700 ⁇ m and the center-to-center interval of the spots was changed to 800 ⁇ m.
  • Example B12 A substrate for forming cell aggregates was produced in the same manner as in Experimental Example B8 except that the spot diameter of the base film-forming agent was changed to 900 ⁇ m and the center-to-center interval of the spots was changed to 1000 ⁇ m.
  • Underlying film forming agent 11 was prepared by adding 4.8 g of pure water and 0.2 mL of 0.5 mg/mL vitronectin VTN-N (Gibco) and sufficiently stirring.
  • the base film-forming agent was applied using an inkjet device and dried to prepare a substrate for producing cell aggregates.
  • the base film forming agent 12 was applied using an inkjet device and dried to prepare a substrate for producing cell aggregates.
  • ⁇ Test Example B1 Cell Adhesion Confirmation Test with Mouse Fibroblasts in FBS-Free Medium of Experimental Examples B1-3 and Comparative Experimental Examples B1-2> (Preparation of cells 56)
  • Mouse fetal fibroblasts C3H10T1/T2 cells: manufactured by DS Pharma Biomedical Co., Ltd.
  • 10% FBS manufactured by Sigma-Aldrich
  • 1% glutamine/penicillin/streptmycin manufactured by Gibco
  • BME medium manufactured by Gibco
  • the cells were statically cultured in a 10 cm diameter petri dish (10 mL medium) for 2 days or longer in a 37° C./CO 2 incubator while maintaining a 5% carbon dioxide concentration. Subsequently, the cells were washed with 3 mL of PBS solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 3 mL of trypsin-EDTA solution (manufactured by PromoCell) was added, and the cells were detached by standing at room temperature for 3 minutes. Cells 56 were collected by adding 7 mL of BME medium without FBS (bovine serum) and glutamine/penicillin/streptomycin.
  • PBS solution manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
  • trypsin-EDTA solution manufactured by PromoCell
  • Cell adhesion confirmation test 2.0 mL of the cell suspension was added to the cell aggregate-producing substrates prepared in Experimental Examples B1 to B3 and Comparative Experimental Examples B1 and B2.
  • the cell density was 1.5 ⁇ 10 5 cells/cm 2 for Experimental Example B1, Experimental Example B2 and Comparative Experimental Example B1, and 3.0 ⁇ 10 5 cells/cm 2 for Experimental Example B3 and Comparative Experimental Example B2. sown as After that, it was allowed to stand in a 37° C./CO 2 incubator for 2 hours while maintaining a carbon dioxide concentration of 5%. After standing, the non-adherent cells and medium were removed, and the wells were washed with PBS to leave only the adherent cells on the wells.
  • ⁇ Test Example B2 Confirmation Test for Cell Adhesion and Cell Aggregate Formation with Mouse Fibroblasts in FBS-Free Medium of Experimental Examples B4 and 6> (Preparation of cells 56)
  • Cells 56 were prepared in the same manner as in Test Example B1.
  • Cell adhesion confirmation test 2.0 mL of the cell suspension was added to the cell aggregate-producing substrates prepared in Experimental Examples B4 and B6 so as to obtain 3.0 ⁇ 10 5 cells/cm 2 . After that, it was allowed to stand in a 37° C./CO 2 incubator for 2 hours while maintaining a carbon dioxide concentration of 5%. After standing, the non-adherent cells and medium were removed, and the wells were washed with PBS to leave only the adherent cells on the wells.
  • ⁇ Test Example B3 Cell Adhesion Confirmation Test in Serum-Free Medium Using Human Adipose Tissue-Derived Mesenchymal Stem Cells of Experimental Example B5 and Comparative Experimental Example B3> (Preparation of cells 56)
  • human adipose tissue-derived mesenchymal stem cells ADSC: manufactured by Cellsource Co., Ltd.
  • a low-serum medium Mesenchymal Stem Cell Growth Medium 2 (Takara Bio Inc., serum concentration 2%) was used for cell culture.
  • the cells were statically cultured in a 10 cm diameter petri dish (10 mL medium) for 2 days or longer in a 37° C./CO 2 incubator while maintaining a 5% carbon dioxide concentration.
  • the cells were washed with 3 mL of PBS solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 3 mL of trypsin-EDTA solution (manufactured by PromoCell) was added, and the cells were detached by standing at room temperature for 3 minutes.
  • Cells 56 were collected by adding 7 mL of serum-free Mesenchymal Stem Cell Growth Medium DXF medium. After centrifuging this suspension (manufactured by Tomy Seiko Co., Ltd., model number LC-230, 200 x g/3 minutes, room temperature), the supernatant was removed, and the above medium was added to prepare a cell suspension. .
  • Cell adhesion confirmation test 2.0 mL of the cell suspension was added to the cell aggregate-producing substrates prepared in Experimental Example B5 and Comparative Experimental Example B3 so as to obtain 3.0 ⁇ 10 5 cells/cm 2 . After that, it was allowed to stand in a 37° C./CO 2 incubator for 2 hours while maintaining a carbon dioxide concentration of 5%. After standing, the non-adherent cells and medium were removed, and the wells were washed with PBS to leave only the adherent cells on the wells. After washing, 2.0 mL of fresh medium was added, and the adherent cells 56 were observed and photographed using a stereoscopic microscope SZX16 (manufactured by Olympus Corporation). As a result, as shown in FIG.
  • ⁇ Test Example B4 Cell Adhesion Confirmation Test in Low Serum Medium Using Human Adipose Tissue-derived Mesenchymal Stem Cells of Experimental Example B7> (Preparation of cells 56) Cells 56 were prepared in the same manner as in Test Example B3, except that the culture medium after cell detachment was changed to low-serum Mesenchymal Stem Cell Growth Medium 2 medium. (Cell adhesion confirmation test) A cell adhesion confirmation test was performed on the cell aggregate-producing substrate prepared in Experimental Example B7 in the same manner as in Experimental Example B3. As a result, as shown in FIG. 9, selective adhesion of cells 56 to base film 90 on fabricated substrate 80 was confirmed. In addition, uniform adhesion occurred without gaps in cell adhesion.
  • ⁇ Test Example B5 Spheroid diameter confirmation test in serum-free medium using human adipose tissue-derived mesenchymal stem cells in Experimental Examples B8 to B12> (Preparation of cells 56)
  • a cell suspension was prepared by the method described in Test Example B3.
  • Cell adhesion, spheroid formation confirmation test 1.0 mL of the cell suspension was added to the cell aggregate-producing substrates prepared in Experimental Examples B8 to B12 so that the concentration was 2.9 ⁇ 10 5 to 6.0 ⁇ 10 5 cells/well. After that, it was allowed to stand in a 37° C./CO 2 incubator for 2 hours while maintaining a carbon dioxide concentration of 5%.
  • FIG. 13 shows the relationship between the application area of the base film for forming cell aggregates and the spheroid diameter.
  • FIG. 14 shows the result of calculating the relationship between the coating area of the base film and the volume of the spheroids and making a graph.
  • a correlation coefficient R 2 of 0.9989 indicated a strong correlation. From the above, it was shown that the spheroid diameter and spheroid volume can be controlled by controlling the coating area of the underlayer.
  • ⁇ Test Example B6 Confirmation Test for Cell Adhesion and Cell Aggregate Formation with Mouse Fibroblasts in FBS-Free Medium of Comparative Experiment Example B4 (no polymer, additive only)> (Preparation of cells 56) Cells 56 were prepared in the same manner as in Test Example B1. (Cell adhesion confirmation test) A confirmation test of cell adhesion and formation of cell aggregates was performed on the cell aggregate-producing substrate prepared in Comparative Experimental Example B4 in the same manner as in Experimental Example B2. As a result, as shown in FIG. 15, cell adhesion to the base film 90 portion on the fabricated substrate 80 was not confirmed.
  • the base film forming composition 48 can be preferably used as the base film forming composition 48 for forming the base film 90 in the base film forming section 40 of the cell structure manufacturing apparatus 10 of the present embodiment. can.

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05292957A (ja) * 1992-04-21 1993-11-09 Kurabo Ind Ltd 接着性細胞培養用被覆組成物
JP2019022527A (ja) * 2012-06-08 2019-02-14 国立研究開発法人理化学研究所 単一細胞凝集塊形成用培養容器
WO2020040247A1 (ja) * 2018-08-24 2020-02-27 日産化学株式会社 細胞培養の下地膜として使用するポリマーの製造方法及び細胞培養容器

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05292957A (ja) * 1992-04-21 1993-11-09 Kurabo Ind Ltd 接着性細胞培養用被覆組成物
JP2019022527A (ja) * 2012-06-08 2019-02-14 国立研究開発法人理化学研究所 単一細胞凝集塊形成用培養容器
WO2020040247A1 (ja) * 2018-08-24 2020-02-27 日産化学株式会社 細胞培養の下地膜として使用するポリマーの製造方法及び細胞培養容器

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