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WO2014208778A1 - Cell-carrying patterned nano-thin film - Google Patents

Cell-carrying patterned nano-thin film Download PDF

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Publication number
WO2014208778A1
WO2014208778A1 PCT/JP2014/067852 JP2014067852W WO2014208778A1 WO 2014208778 A1 WO2014208778 A1 WO 2014208778A1 JP 2014067852 W JP2014067852 W JP 2014067852W WO 2014208778 A1 WO2014208778 A1 WO 2014208778A1
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Prior art keywords
thin film
cell
nano thin
patterned
cells
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French (fr)
Japanese (ja)
Inventor
弘和 梶
俊宣 藤枝
森　好弘
松彦 西澤
阿部　俊明
展裕 永井
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Tohoku University NUC
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Tohoku University NUC
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Priority to JP2015524158A priority Critical patent/JPWO2014208778A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/06—Plates; Walls; Drawers; Multilayer plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2535/00—Supports or coatings for cell culture characterised by topography
    • C12N2535/10—Patterned coating

Definitions

  • the present invention relates to a cell delivery method that enables cell transplantation (delivery) in a narrow space in a living body simply, efficiently, and minimally invasively. More specifically, the present invention relates to a cell-supported patterned nanothin film characterized in that a nanothin film having a micropattern is used as a base material and cells are supported on the nanothin film.
  • An object of the present invention is to provide a new means capable of performing cell transplantation (delivery) in a narrow space in a living body such as an eyeball in a simple, efficient and minimally invasive manner.
  • the present inventors have drawn and released a cell-supported patterned nano-thin film obtained by culturing cells on a nano-thin film having a micro pattern, by means of a thin tube such as an injection needle.
  • the cell-supported patterned nano-thin film can maintain its original shape even if it is sucked and released as such, and the cells adhered to the membrane surface can be detached while remaining alive. It was found that it can be held stably without doing.
  • the present inventors have found that the cell-supported patterned nano thin film can be introduced into a living body with minimal invasiveness using a capillary such as an injection needle and transplanted (delivered).
  • the present invention is based on these findings and has the following characteristics.
  • a patterned nano thin film having a thickness of less than 500 nm and a diameter or the longest diagonal line of 10 ⁇ m to 20 mm is used as a base material, and cells are supported on the patterned nano thin film.
  • Cell-patterned patterned nano thin film is used as a base material, and cells are supported on the patterned nano thin film.
  • Cell-patterned patterned nano thin film is used as a base material, and cells are supported on the patterned nano thin film.
  • Cell-patterned patterned nano thin film is used as a base material, and cells are supported on the patterned nano thin film.
  • Cell-patterned patterned nano thin film is used as a base material, and cells are supported on the patterned nano thin film.
  • Cell-patterned patterned nano thin film is
  • [3] The cell-supported patterned nano thin film according to [1] or [2], wherein the patterned nano thin film is coated with an extracellular matrix.
  • [4] The cell-supported patterned nano thin film according to any one of [1] to [3], wherein the patterned nano thin film includes nanoparticles made of a metal, a semiconductor, a ceramic, or a magnetic material.
  • [5] The cell-supported patterned nano thin film according to any one of [1] to [4], wherein the patterned nano thin film contains a functional substance.
  • [6] The cell-supported patterned nano thin film according to any one of [1] to [5], wherein the cell is a retinal pigment epithelial cell.
  • FIG. 1 shows a simplified diagram of a method for producing a patterned nanofilm.
  • FIG. 2 shows a photograph of the patterned nanofilm.
  • the nano thin film (a) which consists of the polylactic acid glycolic acid copolymer (PLGA) and magnetic nanoparticle (MNPs) transcribe
  • the diameter is 1000 ⁇ m.
  • FIG. 4 shows the surface profile of the patterned nanofilm.
  • A A patterned nano-thin film composed only of PLGA without mixing MNPs.
  • B Patterned nano thin film produced using PLGA mixed with MNPs. The inset is an optical image of the nanothin film, and the broken line indicates the scan portion.
  • FIG. 5-1 shows the morphology evaluation of RPE cells cultured on patterned nanofilms.
  • A Phase contrast micrograph of RPE cells after 1 day of culture.
  • B Fluorescence micrograph of RPE cells after 1 day of culture (green: live cells, red: dead cells).
  • FIG. 5-2 shows the morphology evaluation of RPE cells cultured on patterned nanofilms.
  • FIG. 6 shows an evaluation of cell viability after mechanical stress loading due to syringe operation on the cell-supported patterned nano thin film.
  • A RPE cell-supported patterned nano thin film (400 ⁇ m diameter) before and after being sucked and released with a 25 G (inner diameter 320 ⁇ m) injection needle, phase contrast micrographs and fluorescence micrographs showing green cells on the patterned nano thin film (green: Live cells, red: dead cells).
  • B Cell viability before and after mechanical stress loading of cell-supported patterned nanothin films with different diameters (injection needle: SN, intravenous indwelling catheter: IC).
  • C Phase contrast micrographs and fluorescence micrographs (green) of RPE cells on each thin film before and after mechanical stress loading in nano-thin films (thickness 170 nm) and micrometer-thick thin films (5.5 ⁇ m).
  • FIG. 7 shows a simplified diagram of (a) a method for introducing a cell-supported patterned nano thin film into the retina suffering from macular degeneration.
  • B shows the result of introduction of RPE cell-supported patterned nanothin into the subretinal macular region of the extracted porcine eyeball. Photomicrograph of patterned nanofilm (stained with rhodamine B) introduced under the macula retina.
  • FIG. 8 shows the result of introduction of the RPE cell-supported patterned nanothin film under the rat eyeball retina.
  • A-1 It is a photograph figure which shows the fundus optical coherence tomogram (OCT) of a rat one week after the RPE cell carrying patterned nano thin film was introduced under the eyeball retina. The arrow indicates the introduced RPE cell-supported patterned nanofilm.
  • A-2) It is a photograph figure which shows the fundus OCT of the control rat in which the RPE cell carrying patterned nano thin film is not introduced.
  • B It is a photograph figure which shows the posterior eye part of the rat eyeball by which the extracted RPE cell carrying
  • FIG. 1 A photographic diagram showing the result of hematoxylin-eosin (HB) staining of a retinal tissue section prepared from an eyeball extracted from a rat one week after the RPE cell-supported patterned nanothin film was introduced under the eyeball retina. is there. Arrows indicate cells introduced by the RPE cell-carrying nanofilm.
  • HB hematoxylin-eosin
  • the “cell-supported patterned nano thin film” in the present invention is characterized in that cells are supported on a patterned nano thin film as a substrate.
  • the “nano thin film” means a sheet made of a biocompatible polymer having a thickness of less than 500 nm, preferably about 400 nm or less, more preferably about 300 nm or less, and further preferably about 200 nm or less.
  • the lower limit of the thickness of the “nano thin film” is not particularly limited, but can be about 20 nm or more, preferably about 40 nm or more, more preferably about 60 nm or more, more preferably about 80 nm or more, and still more preferably about 100 nm or more.
  • the “nanothin film” can be a sheet made of a biocompatible polymer having a thickness of about 20 nm to 300 nm, preferably about 100 nm to 200 nm.
  • biocompatible polymer usable in the present invention include polylactic acid, polyglycolic acid, polyhydroxybutyric acid, polycaprolactone, polybutylene succinate, polydioxanone, polydimethylsiloxane, polymethyl methacrylate, polystyrene, polyacetic acid.
  • the biocompatible polymer is a biodegradable polymer, and particularly preferably a polylactic acid glycolic acid copolymer (hereinafter referred to as “PLGA”).
  • PLGA polylactic acid glycolic acid copolymer
  • the “patterned nano thin film” means a nano thin film having a fine shape and size (micro pattern).
  • the shape of the nano thin film is not particularly limited, and may be a circle, an ellipse, or a polygon (for example, a quadrangle, a star, etc.), but the diameter or the length of the longest diagonal is about 10 ⁇ m to 20 mm, preferably about
  • the thickness is about 50 ⁇ m to 15 mm, more preferably about 100 ⁇ m to 10 mm, more preferably about 150 ⁇ m to 5 mm, still more preferably about 200 ⁇ m to 3 mm, and particularly preferably about 300 ⁇ m to 1 mm.
  • the thickness and diameter of the patterned nano thin film or the length of the longest diagonal line is not limited to the above range, and the patterned nano thin film supporting cells can be sucked and released by a capillary tube, and the suction is performed. Even if a mechanical stress due to release is loaded, many of the loaded cells (for example, 50% or more, 60% or more, 70% or more, 80% or more of the number of loaded cells before the mechanical stress is loaded) , 90% or more, 95% or more cells) can be appropriately changed as long as the cells adhere and survive on the patterned nano thin film.
  • the cell-supported patterned nano thin film of the present invention can be obtained by a technique including the following steps [1] to [5].
  • the patterned nano thin film in the present invention can be produced based on a known technique (Japanese Patent No. 5028422), and the outline of a production method combining a micro stamp method and a spin coating method is shown below and in FIG.
  • the method for producing the patterned nano thin film is not limited to this method.
  • a micropattern substrate that is, “stamp” in which a specific micropattern is engraved in a convex shape on a substrate (for example, polydimethylsiloxane (PDMS), metal, silicon, glass, etc.) is manufactured.
  • the micro-pattern substrate can be manufactured using a photolithography technique according to a conventional method.
  • a positive photoresist is applied thereon.
  • the resist is exposed through a photomask (electron irradiation, ultraviolet irradiation, X-ray irradiation, etc.).
  • the resist on the substrate is developed, and the resist in the exposed area is removed.
  • micropattern substrate can be obtained (shown as (a) in FIG. 1).
  • a biocompatible polymer layer is formed on the surface of the obtained micropattern substrate (stamp surface on which the micropattern is engraved).
  • a biocompatible polymer or a component of a biocompatible polymer (for example, a monomer or the like that is a component of a biodegradable polymer) (hereinafter referred to as “biocompatible polymer or the like”) is appropriate.
  • a solvent eg, dichloromethane, chloroform, acetone, ethyl acetate, etc.
  • Dissolve in a solvent eg, dichloromethane, chloroform, acetone, ethyl acetate, etc.
  • spin a solution of this biocompatible polymer or the like It is applied to the surface of the micropattern substrate by a coating method.
  • the thickness of the biocompatible polymer applied on the surface of the micropattern substrate can be adjusted, and the thickness of the patterned nano thin film that is the final product can be adjusted. Can be adjusted.
  • a layer made of the biocompatible polymer can be formed on the surface of the micropattern substrate by polymerizing and / or crosslinking the applied biocompatible polymer or the like (in FIG. 1, as (b)). Show).
  • polymerization includes polycondensation, polyaddition, addition condensation, ring-opening polymerization, addition polymerization (radical polymerization, anionic polymerization, cationic polymerization), solid phase polymerization by heat, photopolymerization, radiation polymerization, plasma polymerization, etc.
  • crosslinking can be performed using a known crosslinking agent (for example, alkyl dimidates, acyldiazides, diisocyanates, bismaleimides, triazinyl, diazo compounds, glutaraldehyde, etc.).
  • the solution of biocompatible polymer or the like can contain nanoparticles made of metal, semiconductor, ceramic, magnetic material, etc., preferably magnetic nanoparticles.
  • the nanoparticles have a particle diameter of about 1 nm to 500 nm, preferably about 1 nm to 50 nm.
  • the patterned nano thin film can be moved / assembled by using magnetic force, and the operability can be improved.
  • the nanoparticles can be included in a solution of biocompatible polymer or the like at a concentration of 0.1 mg / mL to 10 mg / mL, preferably 1 mg / mL to 5 mg / mL.
  • a support substrate having a water-soluble sacrificial layer is prepared.
  • One surface of a supporting substrate eg, silicon, glass, etc.
  • polysaccharide Coating is performed using a water-soluble polymer such as a polymer electrolyte or a salt thereof. Coating can be performed by applying a water-soluble polymer on a support substrate by a casting method, a spin coating method, or the like, and drying. Thereby, a support substrate provided with a water-soluble sacrificial layer soluble in an aqueous solvent can be obtained (shown as (d) in FIG. 1).
  • PVA polyvinyl alcohol
  • a derivative thereof polyisopropylacrylamide or a derivative thereof, polyether or a derivative thereof
  • polysaccharide Coating is performed using a water-soluble polymer such as a polymer electrolyte or a salt thereof. Coating can be performed by applying a water-soluble polymer on a support substrate by a casting method, a spin coating method, or the like, and drying. Thereby, a support substrate provided with a water-soluble sacrificial layer soluble in an aqueous solvent can
  • the biocompatible polymer layer on the micropattern substrate is stamp-baked on the water-soluble sacrificial layer of the support substrate (shown as (e) in FIG. 1). Baking the biocompatible polymer onto the water-soluble sacrificial layer can be performed by heat treatment. As a result, the biocompatible polymer is transferred onto the water-soluble sacrificial layer while maintaining the micropattern, and a support substrate (biocompatible polymer-supported support substrate) supporting the biocompatible polymer can be obtained ( (Indicated as (f) in FIG. 1). Next, the surface provided with the biocompatible polymer of the obtained biocompatible polymer-supported support substrate is coated with an extracellular matrix that promotes cell adhesion and proliferation.
  • extracellular matrix examples include type I collagen, type IV collagen, fibronectin, poly-D-lysine (PDL), laminin, poly-L-ornithine / laminin (PLO / LM), and the like. Is mentioned.
  • the extracellular matrix coating is performed by applying an extracellular matrix solution dissolved in an appropriate solvent (for example, at 0.01 ⁇ g / mL to 5 ⁇ g / mL) onto a support substrate by a spin coating method or the like and then drying it. It can be carried out. Thereby, a biocompatible polymer-supported support substrate coated with an extracellular matrix can be obtained.
  • a cell suspension is dropped onto a support substrate carrying a biocompatible polymer coated with an extracellular matrix, and the cells are incubated for about 1 to 5 hours to deposit the cells on the support substrate.
  • the support substrate is placed in a medium and further cultured for 1 to 3 days.
  • the water-soluble sacrificial layer on the support substrate dissolves, so that the patterned nano thin film having a predetermined micropattern is released from the support substrate (shown as (g) in FIG. 1).
  • the extracellular matrix layer directly coated thereon also dissolves, so that the cells grow only on the patterned nanofilm.
  • the target cell-supported patterned nano thin film By culturing until the cells are confluent on the patterned nano thin film, the target cell-supported patterned nano thin film can be obtained.
  • the cell culture conditions and the medium composition general ones suitable for the cells to be used can be used.
  • a functional substance can be supported in or on the patterned nano thin film.
  • “Functional substance” means a substance (for example, protein, polypeptide, compound, etc.) having a function of controlling cell proliferation, differentiation, physiological activity, etc., or a substance that enables visualization of a nano thin film.
  • Such functional substances include growth / growth factors (eg, fibroblast growth factor (FGF), epidermal growth factor (EGF), bone morphogenetic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic Factor (BDNF), etc.), intraocular pressure-lowering agent, neuroprotective agent, antibiotic, anticancer agent, visualization probe (contrast agent, nanoparticle, fluorescent dye, etc.).
  • FGF fibroblast growth factor
  • EGF epidermal growth factor
  • BMP bone morphogenetic protein
  • NEF nerve growth factor
  • BDNF brain-derived neurotrophic Factor
  • intraocular pressure-lowering agent eg, intraocular pressure-lowering agent, neuroprotective agent, antibiotic, anticancer agent, visualization probe (contrast agent, nanoparticle, fluorescent dye, etc.
  • the functional material can be bound to the patterned nanofilm using chemical or physical techniques.
  • the chemical method includes chemical bonding via a functional group.
  • a biocompatible polymer is modified in advance with an appropriate functional group (for example
  • a functional substance can be bound to the obtained patterned nano thin film through the introduced functional group.
  • physical methods include electrostatic interaction, hydrophobic interaction, hydrogen bonding, intermolecular force, and the like between the patterned nano thin film and the functional material.
  • the binding of the functional substance to the patterned nano thin film can be performed before the coating with the extracellular matrix, after the coating, or simultaneously with the coating in the manufacturing process of the cell-supported patterned nano thin film.
  • Examples of cells that can be supported on the patterned nanothin film include cells that can be transplanted to a patient by cell transplantation therapy (except cells that float in body fluids).
  • RPE retinal pigment epithelium
  • photoreceptor cells hepatocytes
  • cardiomyocytes skeletal muscle cells
  • smooth muscle cells vascular endothelial cells
  • kidney cells islet cells
  • nerve cells and the like examples include retinal pigment epithelium (RPE) cells, photoreceptor cells, hepatocytes, cardiomyocytes, skeletal muscle cells, smooth muscle cells, vascular endothelial cells, kidney cells, islet cells, nerve cells and the like.
  • RPE retinal pigment epithelium
  • the cell-supported patterned nano thin film of the present invention can stably hold cells, and even when mechanical stress such as suction and release by a capillary tube is applied, many cells (for example, the mechanical stress is applied). 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more or more of the number of supported cells before the treatment) can be retained on the patterned nano thin film.
  • the “capillary tube” include an injection needle and a catheter. The size (gauge) and length of the “capillary tube” can be appropriately selected according to factors such as the size of the cell-supported patterned nano thin film and the site into which the cell-supported patterned nano thin film is introduced.
  • the cell-supported patterned nano thin film of the present invention is introduced into the living body by drawing the cell-supported patterned nano thin film together with physiological saline from the tip of the injection needle or catheter and holding it in the injection syringe or catheter.
  • the tip can be inserted into or near the diseased site, and the cell-supported patterned nano thin film can be released from the injection needle or the tip of the catheter and placed.
  • the cells supported at or near the diseased site can be delivered.
  • One or more cell-supported patterned nano thin films can be introduced into the living body.
  • FIG. 7A shows an outline of cell delivery using the cell-supported patterned nano thin film of the present invention to the retina suffering from macular degeneration.
  • a 18-25 gauge fine injection needle or catheter is inserted into the eyeball from the sclera, and a small amount of physiological saline is injected from the retinal side of the diseased site or its vicinity into the subretinal region, thereby patterning the cell. Secure space for introducing nano thin films.
  • the RPE cell-carrying patterned nano thin film is released from the injection needle or catheter into the space, and the injected physiological saline is sucked and removed, whereby the cell-carrying patterned nano thin film can be placed under the retina.
  • the introduced RPE cells are proliferated or replaced with cells in the diseased part at the diseased part (for example, macular degeneration site), and the disease can be completely cured or its symptoms can be reduced.
  • the introduced patterned nano thin film can be decomposed and absorbed in vivo.
  • Patterned nano thin films were fabricated by combining spin coating and micro stamping methods.
  • MNPs magnetic nanoparticles
  • PDMS polydimethylsiloxane
  • a polylactic acid glycolic acid copolymer (PLGA) solution (5 mg / mL) was spin-coated (4000 rpm, 40 s).
  • PLGA polylactic acid glycolic acid copolymer
  • FIG. 2A shows a patterned nano thin film composed of PLGA and MNPs transferred on a support substrate
  • FIG. 2B is an enlarged view thereof.
  • the patterned nano thin film released from the support substrate has excellent flexibility and self-supporting ability, and was able to be sucked by a catheter having an inner diameter smaller than the diameter of the nano thin film itself (24G (inner diameter 470 ⁇ m) catheter) ( The patterned nanothin film in the photograph is colored with rhodamine) (FIG. 2 (c)).
  • the average film thickness of the patterned nano thin film was about 170 nm regardless of the presence or absence of mixing of MNPs. (FIGS. 4A to 4B).
  • Example 1 Culture of Retinal Pigment Epithelium (RPE) Cells on Patterned Nano Thin Film
  • a suspension of RPE cells (on a support substrate on which a PLGA layer was transferred from a micropattern substrate and spin coated with a type I collagen solution ( 1.5 ⁇ 10 6 cells / mL) was added dropwise at a time, and incubated at 37 ° C. for about 1 hour to deposit the cells on a support substrate.
  • the support substrate on which the cells were deposited was placed in a medium (DMEM, 4% (v / v) FBS, 1% penicillin streptomycin) and cultured according to a conventional method.
  • DMEM fetal bovine serum
  • the living cell and the dead cell were dye
  • the results are shown in FIGS. 5-1 (a)-(b). Since PVA, which is a water-soluble sacrificial layer, is dissolved in the medium, it was confirmed that RPE cells adhered and proliferated and grew only on the patterned nanofilm. No dead cells were detected on the patterned nanofilm.
  • the RPE cell was dye
  • MNPs (+) patterned nano thin film prepared using PLGA mixed with MNPs and a patterned nano thin film (hereinafter referred to as “MNPs (+) patterned nano thin film”) composed only of PLGA without mixing MNPs.
  • MNPs (+) patterned nano thin film composed only of PLGA without mixing MNPs.
  • AFM atomic force microscope
  • the MNPs (+) patterned nano thin film (left) root mean square roughness (RMS): 2.86 nm
  • RMS root mean square roughness
  • ⁇ patterned nano thin film
  • RMS 0.489 nm
  • the results are shown in FIG. It was confirmed that the cells on the MNPs (+) patterned nanofilm had higher proliferation activity after 1 day of culture than the cells on the MNPs ( ⁇ ) patterned nanofilm. Furthermore, immunostaining of cells cultured on MNPs (+) patterned nanofilm or MNPs (-) patterned nanofilm for 2 days, respectively, using anti-ZO-1 antibody and anti-F-actin antibody as primary antibodies Went. The results are shown in Fig. 5-2 (f)-(g). The cells on the MNPs (+) patterned nanofilm (FIG. 5-2 (f)) are more ZO-1 than the cells on the MNPs ( ⁇ ) patterned nanofilm (FIG. 5-2 (g)). (Tight junction) and F-actin formation were promoted, and it was confirmed that the cell morphology showed a hexagonal morphology characteristic of the retinal pigment epithelium.
  • RPE cell-supported patterned nano thin film (having a diameter of 300 ⁇ m, 400 ⁇ m, 500 ⁇ m, and 1000 ⁇ m) is sucked and released once by a 25 G (inner diameter 320 ⁇ m) injection needle, and the pattern before and after the mechanical stress loading Regarding the RPE cells on the activated nanofilm, changes in tissue morphology were confirmed with a phase-contrast microscope, and cell survival was confirmed using a life / death determination kit (Cellstein-Double Staining Kit, Dojindo Laboratories). The results are shown in FIG. As is apparent from the photograph of FIG.
  • FIG. 6 (b) shows the effect of the size of the patterned nano thin film on cell viability before and after the mechanical stress loading. As is clear from the results, there was no effect of the diameter of the patterned nano thin film or the type of syringe needle (injection needle: SN, intravenous indwelling catheter: IC), and in any case mechanical stress A cell viability of 80% or more was obtained after loading.
  • FIG. 6 (c) shows the observation results of each thin film before and after the mechanical stress loading
  • FIG. 6 (d) shows the change in the cell adhesion area with respect to each thin film.
  • the value of a cell adhesion area is shown by the relative value which makes the cell adhesion area before mechanical stress load 100%.
  • the patterned nanothin film carrying RPE cells produced using nanothin films more than 80% of the cells remain attached to the surface of the thin film, and most of the cells survive, and the monolayer formed on the patterned nanothin film It was confirmed that the layer structure was maintained stably.
  • the RPE cell-supported patterned micro thick thin film produced using the micro thick thin film most cells were detached from the film due to the load of mechanical stress. It is considered that the patterned microthin film is less flexible than the patterned nanothin film, and therefore the load on the cells due to mechanical stress is increased, and most of the cells are detached from the film.
  • FIG. 7B shows a micrograph of the RPE cell-carrying nanothin film introduced below the retina of the porcine eyeball macular region. It was confirmed that the injected RPE cell-carrying nanothin film was stretched under the retina and maintained the original circular shape.
  • the RPE cell-supported patterned nano thin film prepared according to the method described in Example 2 above was sucked into the syringe using a 30G injection needle, and formed under the retina from the injection needle inserted in the same manner as described above. Released into the space. Finally, the RPE cell-supported patterned nano thin film was placed under the retina by aspirating and removing the injected physiological saline. The control rats were similarly injected with saline alone and aspirated / removed. The treated rats were bred according to the usual method.
  • the nano thin film which consists of a biocompatible polymer which carries the cell which can be attracted
  • a cell-supporting nano thin film having such characteristics can be introduced into a small space in a living body using a small tube such as an injection needle in a minimally invasive manner and transplanted (delivered), thereby reducing the burden on patients and tissues.
  • All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

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Abstract

The objective of the present invention is to provide a new means capable of easy, efficient and minimally invasive cell transplantation (delivery) to a narrow space in an organism such as in an eyeball. This cell-carrying patterned nano-thin film is characterised by having, as a substrate, a nano-thin film that has a micropattern and by carrying cells atop the nano-thin film.

Description

細胞担持パターン化ナノ薄膜Cell-supported patterned nano thin film

　本発明は、生体内の狭小な空間に、簡便に、かつ効率的に、また低侵襲に細胞移植（送達）することを可能とする細胞送達法に関する。
　より詳細には、本発明は、マイクロパターンを有するナノ薄膜を基材とし、当該ナノ薄膜上に細胞を担持することを特徴とする、細胞担持パターン化ナノ薄膜に関する。 The present invention relates to a cell delivery method that enables cell transplantation (delivery) in a narrow space in a living body simply, efficiently, and minimally invasively.
More specifically, the present invention relates to a cell-supported patterned nanothin film characterized in that a nanothin film having a micropattern is used as a base material and cells are supported on the nanothin film.

　近年、再生医療の分野で難治性疾患に対する細胞移植療法の開発が広く進められている。例えば、眼科領域における難治性疾患の１つに加齢黄斑変性があるが、これは網膜組織中心の黄斑部が障害されることにより発症する失明リスクの極めて高い疾患である。今日までに、治療法として網膜色素上皮（以下、「ＲＰＥ」と記載する。）細胞の移植が試みられている。例えば、ＲＰＥ細胞の懸濁液を網膜下に直接注入することによって細胞移植を行う方法が検討されているが（非特許文献１）、注入された細胞の生着率が低いことが問題となっている。また、ＲＰＥ細胞のみから成るシートを移植する方法も検討されているが、ＲＰＥ細胞のシートは脆弱であるため扱いが非常に難しい。近年では、ｉＰＳ細胞より誘導したＲＰＥ細胞をシート化し患部に移植する試みも検討されているが、網膜下へのＲＰＥ細胞の効率的な送達法は依然として確立されておらず、当該分野においては新たな細胞送達法が切望されている。
　一方、生体分解性ポリマーからなるナノメートルオーダーの膜厚を有する薄膜（いわゆる「ナノ薄膜」）が開発され、当該ナノ薄膜に機能性物質を担持させ皮膚や生体組織に貼付して使用する複合体が報告されている（特許文献１−２）。 In recent years, cell transplantation therapy for intractable diseases has been widely developed in the field of regenerative medicine. For example, age-related macular degeneration is one of the intractable diseases in the ophthalmological field, and this is a disease with an extremely high risk of blindness that develops when the macular portion at the center of the retinal tissue is damaged. To date, transplantation of retinal pigment epithelium (hereinafter referred to as “RPE”) cells has been attempted as a therapeutic method. For example, a method of transplanting cells by directly injecting a suspension of RPE cells under the retina has been studied (Non-Patent Document 1), but the problem is that the survival rate of the injected cells is low. ing. In addition, a method of transplanting a sheet composed only of RPE cells has been studied, but the sheet of RPE cells is very difficult to handle because it is fragile. In recent years, attempts have been made to form a sheet of RPE cells derived from iPS cells and transplant them to the affected area. However, an efficient delivery method of RPE cells under the retina has not been established, New cell delivery methods are eagerly desired.
On the other hand, a thin film (so-called “nanothin film”) made of biodegradable polymer and having a film thickness on the order of nanometers has been developed, and a functional substance is carried on the nanothin film and used on a skin or living tissue. Has been reported (Patent Documents 1-2).

特許第５０２８４２２号公報Japanese Patent No. 5028422 特開２０１０−６３４１１号公報JP 2010-63411 A

Ｃｅｌｌ　Ｔｒａｎｓｐｌａｎｔ，１４，７９９−８０８（２００５）Cell Transplant, 14, 799-808 (2005)

　本発明は、眼球内のような生体内の狭小な空間に、簡便に、かつ効率的に、また低侵襲に細胞移植（送達）することができる新たな手段を提供することを目的とする。
　本発明者らは上記課題を解決すべく鋭意検討した結果、マイクロパターンを有するナノ薄膜上で細胞を培養して得られる細胞担持パターン化ナノ薄膜が、注射針等の細管で吸引し、放出することが可能であり、かつそのように吸引・放出しても、当該細胞担持パターン化ナノ薄膜は元の形状を維持することが可能であり、また膜表面に接着した細胞を生存したまま、剥離することなく安定的に保持できることを見出した。そしてかかる特徴により、当該細胞担持パターン化ナノ薄膜は注射針等の細管を用いて低侵襲に生体内に導入し細胞移植（送達）できることを見出した。
　本発明は、これらの知見に基づくものであり、以下の特徴を有する。
［１］　厚さ５００ｎｍ未満であり、かつ直径又は最も長い対角線の長さが１０μｍ~２０ｍｍであるパターン化ナノ薄膜を基材とし、該パターン化ナノ薄膜上に細胞を担持することを特徴とする、細胞担持パターン化ナノ薄膜。
［２］　パターン化ナノ薄膜が、生体適合性高分子からなる、［１］の細胞担持パターン化ナノ薄膜。
［３］　パターン化ナノ薄膜が細胞外基質でコーティングされている、［１］又は［２］の細胞担持パターン化ナノ薄膜。
［４］　パターン化ナノ薄膜が金属、半導体、セラミック、又は磁性体からなるナノ粒子を含む、［１］~［３］のいずれかの細胞担持パターン化ナノ薄膜。
［５］　パターン化ナノ薄膜が機能性物質を含む、［１］~［４］のいずれかの細胞担持パターン化ナノ薄膜。
［６］　細胞が網膜色素上皮細胞である、［１］~［５］のいずれかの細胞担持パターン化ナノ薄膜。
［７］　細管より組織中に放出され、該組織に細胞を送達するための、［１］~［６］のいずれかの細胞担持パターン化ナノ薄膜。
［８］　［１］~［７］のいずれかの細胞担持パターン化ナノ薄膜を細管より生体組織中に放出することを含む、生体組織への細胞移植方法。
　本明細書は本願の優先権の基礎である日本国特許出願２０１３−１３７２５３号の明細書および／または図面に記載される内容を包含する。 An object of the present invention is to provide a new means capable of performing cell transplantation (delivery) in a narrow space in a living body such as an eyeball in a simple, efficient and minimally invasive manner.
As a result of intensive studies to solve the above-mentioned problems, the present inventors have drawn and released a cell-supported patterned nano-thin film obtained by culturing cells on a nano-thin film having a micro pattern, by means of a thin tube such as an injection needle. The cell-supported patterned nano-thin film can maintain its original shape even if it is sucked and released as such, and the cells adhered to the membrane surface can be detached while remaining alive. It was found that it can be held stably without doing. Based on these characteristics, the present inventors have found that the cell-supported patterned nano thin film can be introduced into a living body with minimal invasiveness using a capillary such as an injection needle and transplanted (delivered).
The present invention is based on these findings and has the following characteristics.
[1] A patterned nano thin film having a thickness of less than 500 nm and a diameter or the longest diagonal line of 10 μm to 20 mm is used as a base material, and cells are supported on the patterned nano thin film. Cell-patterned patterned nano thin film.
[2] The cell-supported patterned nano thin film according to [1], wherein the patterned nano thin film is made of a biocompatible polymer.
[3] The cell-supported patterned nano thin film according to [1] or [2], wherein the patterned nano thin film is coated with an extracellular matrix.
[4] The cell-supported patterned nano thin film according to any one of [1] to [3], wherein the patterned nano thin film includes nanoparticles made of a metal, a semiconductor, a ceramic, or a magnetic material.
[5] The cell-supported patterned nano thin film according to any one of [1] to [4], wherein the patterned nano thin film contains a functional substance.
[6] The cell-supported patterned nano thin film according to any one of [1] to [5], wherein the cell is a retinal pigment epithelial cell.
[7] The cell-supported patterned nano thin film according to any one of [1] to [6], which is released into a tissue from a capillary tube and delivers cells to the tissue.
[8] A method for transplanting cells into a living tissue, comprising releasing the cell-supported patterned nano thin film according to any one of [1] to [7] into a living tissue from a thin tube.
This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2013-137253, which is the basis of the priority of the present application.

　図１は、パターン化ナノ薄膜の作製方法の簡略図を示す。
　図２は、パターン化ナノ薄膜の写真図を示す。支持基板上に転写されたポリ乳酸グリコール酸共重合体（ＰＬＧＡ）と磁性ナノ粒子（ＭＮＰｓ）から成るナノ薄膜（ａ）及びその顕微鏡写真（ｂ）。２４Ｇ（内径４７０μｍ）カテーテル内に吸引されたパターン化ナノ薄膜（直径１ｍｍ）（ｃ）。
　図３（ａ）−（ｄ）は、異なるサイズのパターン化ナノ薄膜の顕微鏡写真図を示す。（ａ）直径１０００μｍ。（ｂ）直径５００μｍ。（ｃ）直径４００μｍ。（ｄ）直径３００μｍ。
　図４は、パターン化ナノ薄膜の表面プロファイルを示す。（ａ）ＭＮＰｓを混合させずＰＬＧＡのみから成るパターン化ナノ薄膜。（ｂ）ＭＮＰｓを混合したＰＬＧＡを用いて作製したパターン化ナノ薄膜。挿入図はナノ薄膜の光学イメージであり、破線はスキャン部を示す。
　図５−１は、パターン化ナノ薄膜上で培養したＲＰＥ細胞の形態評価を示す。（ａ）培養１日後のＲＰＥ細胞の位相差顕微鏡写真。（ｂ）培養１日後のＲＰＥ細胞の蛍光顕微鏡写真（緑：生細胞、赤：死細胞）。（ｃ）培養１日後のＲＰＥ細胞をカルセインＡＭ（緑）にて、パターン化ナノ薄膜をローダミンＢ（赤）にて染色した結果を示す共焦点顕微鏡写真。（ｄ）パターン化ナノ薄膜表面の原子間力顕微鏡（ＡＦＭ）写真。（ｅ）ＭＮＰｓ混合の有無による、パターン化ナノ薄膜上の細胞増殖活性への影響を示す特性図。
　図５−２は、パターン化ナノ薄膜上で培養したＲＰＥ細胞の形態評価を示す。（ｆ）ＭＮＰｓを含むＰＬＧＡナノ薄膜上および（ｇ）ＭＮＰｓを含まないＰＬＧＡナノ薄膜上で２日間培養したＲＰＥ細胞の免疫染色写真（左上：位相差顕微鏡写真。右上：（赤）ＺＯ−１タイトジャンクション、（青）核。左下：（緑）Ｆ−アクチン、（青）核。右下：重ね合わせ）。
　図６は、細胞担持パターン化ナノ薄膜に対するシリンジ操作に伴う力学的ストレス負荷後の細胞生存率評価示す。（ａ）ＲＰＥ細胞担持パターン化ナノ薄膜（４００μｍ径）を２５Ｇ（内径３２０μｍ）注射針で吸引・放出した前後の、パターン化ナノ薄膜上の細胞を示す位相差顕微鏡写真と蛍光顕微鏡写真（緑：生細胞、赤：死細胞）。（ｂ）異なる直径を有する細胞担持パターン化ナノ薄膜の力学的ストレス負荷前後の細胞生存率（注射針：Ｓ．Ｎ．，静脈留置カテーテル：Ｉ．Ｃ．）。（ｃ）細胞を担持したナノ薄膜（厚さ１７０ｎｍ）およびマイクロメートル厚の薄膜（５．５μｍ）における力学的ストレス負荷前後の、各薄膜上のＲＰＥ細胞の位相差顕微鏡写真と蛍光顕微鏡写真（緑：生細胞、赤：死細胞）。（ｄ）シリンジによる吸引・放出前後の各薄膜上の細胞接着面積。
　図７は、（ａ）黄班変性症を患う網膜への、細胞担持パターン化ナノ薄膜の導入方法の簡略図を示す。（ｂ）摘出したブタ眼球の黄斑部網膜下へのＲＰＥ細胞担持パターン化ナノ薄の導入結果を示す。黄斑部網膜下に導入されたパターン化ナノ薄膜（ローダミンＢで染色）の顕微鏡写真。
　図８は、ラットの眼球網膜下へのＲＰＥ細胞担持パターン化ナノ薄膜の導入結果を示す。（ａ−１）眼球網膜下にＲＰＥ細胞担持パターン化ナノ薄膜が導入されて一週間後のラットの眼底光干渉断層像（ＯＣＴ）を示す写真図である。矢印は導入したＲＰＥ細胞担持パターン化ナノ薄膜を示す。（ａ−２）ＲＰＥ細胞担持パターン化ナノ薄膜が導入されていないコントロールラットの眼底ＯＣＴを示す写真図である。（ｂ）摘出された、ＲＰＥ細胞担持パターン化ナノ薄膜が導入されたラット眼球の後眼部を示す写真図である。矢印は導入したＲＰＥ細胞担持パターン化ナノ薄膜を示す。（ｃ）眼球網膜下にＲＰＥ細胞担持パターン化ナノ薄膜が導入されて一週間後のラットより摘出した眼球より作製された網膜組織切片を、ヘマトキシリン・エオジン（ＨＢ）染色した結果を示す写真図である。矢印はＲＰＥ細胞担持ナノ薄膜によって導入された細胞を示す。 FIG. 1 shows a simplified diagram of a method for producing a patterned nanofilm.
FIG. 2 shows a photograph of the patterned nanofilm. The nano thin film (a) which consists of the polylactic acid glycolic acid copolymer (PLGA) and magnetic nanoparticle (MNPs) transcribe | transferred on the support substrate, and its micrograph (b). Patterned nanofilm (1 mm diameter) aspirated into a 24G (inner diameter 470 μm) catheter (c).
3 (a)-(d) show micrographs of patterned nanofilms of different sizes. (A) The diameter is 1000 μm. (B) 500 μm in diameter. (C) Diameter 400 μm. (D) Diameter 300 μm.
FIG. 4 shows the surface profile of the patterned nanofilm. (A) A patterned nano-thin film composed only of PLGA without mixing MNPs. (B) Patterned nano thin film produced using PLGA mixed with MNPs. The inset is an optical image of the nanothin film, and the broken line indicates the scan portion.
FIG. 5-1 shows the morphology evaluation of RPE cells cultured on patterned nanofilms. (A) Phase contrast micrograph of RPE cells after 1 day of culture. (B) Fluorescence micrograph of RPE cells after 1 day of culture (green: live cells, red: dead cells). (C) Confocal micrograph showing the result of staining RPE cells one day after culture with calcein AM (green) and the patterned nanofilm with rhodamine B (red). (D) Atomic force microscope (AFM) photograph of the patterned nano thin film surface. (E) The characteristic figure which shows the influence on the cell growth activity on a patterned nano thin film by the presence or absence of MNPs mixing.
FIG. 5-2 shows the morphology evaluation of RPE cells cultured on patterned nanofilms. (F) Immunostaining photograph of RPE cells cultured on PLGA nanofilm containing MNPs and (g) PLGA nanofilm containing no MNPs for 2 days (upper left: phase contrast micrograph; upper right: (red) ZO-1 tight Junction, (blue) nucleus, lower left: (green) F-actin, (blue) nucleus, lower right: overlay).
FIG. 6 shows an evaluation of cell viability after mechanical stress loading due to syringe operation on the cell-supported patterned nano thin film. (A) RPE cell-supported patterned nano thin film (400 μm diameter) before and after being sucked and released with a 25 G (inner diameter 320 μm) injection needle, phase contrast micrographs and fluorescence micrographs showing green cells on the patterned nano thin film (green: Live cells, red: dead cells). (B) Cell viability before and after mechanical stress loading of cell-supported patterned nanothin films with different diameters (injection needle: SN, intravenous indwelling catheter: IC). (C) Phase contrast micrographs and fluorescence micrographs (green) of RPE cells on each thin film before and after mechanical stress loading in nano-thin films (thickness 170 nm) and micrometer-thick thin films (5.5 μm). : Live cells, red: dead cells). (D) Cell adhesion area on each thin film before and after suction / release by syringe.
FIG. 7 shows a simplified diagram of (a) a method for introducing a cell-supported patterned nano thin film into the retina suffering from macular degeneration. (B) shows the result of introduction of RPE cell-supported patterned nanothin into the subretinal macular region of the extracted porcine eyeball. Photomicrograph of patterned nanofilm (stained with rhodamine B) introduced under the macula retina.
FIG. 8 shows the result of introduction of the RPE cell-supported patterned nanothin film under the rat eyeball retina. (A-1) It is a photograph figure which shows the fundus optical coherence tomogram (OCT) of a rat one week after the RPE cell carrying patterned nano thin film was introduced under the eyeball retina. The arrow indicates the introduced RPE cell-supported patterned nanofilm. (A-2) It is a photograph figure which shows the fundus OCT of the control rat in which the RPE cell carrying patterned nano thin film is not introduced. (B) It is a photograph figure which shows the posterior eye part of the rat eyeball by which the extracted RPE cell carrying | support patterned nano thin film was introduce | transduced. The arrow indicates the introduced RPE cell-supported patterned nanofilm. (C) A photographic diagram showing the result of hematoxylin-eosin (HB) staining of a retinal tissue section prepared from an eyeball extracted from a rat one week after the RPE cell-supported patterned nanothin film was introduced under the eyeball retina. is there. Arrows indicate cells introduced by the RPE cell-carrying nanofilm.

１．細胞担持パターン化ナノ薄膜
　本発明における「細胞担持パターン化ナノ薄膜」は、基材であるパターン化ナノ薄膜上に細胞を担持してなることを特徴とする。
　本発明において「ナノ薄膜」とは、５００ｎｍ未満、好ましくはおよそ４００ｎｍ以下、より好ましくはおよそ３００ｎｍ以下、さらに好ましくはおよそ２００ｎｍ以下の厚みを有する生体適合性高分子からなるシートを意味する。「ナノ薄膜」の厚みの下限は特に限定されないが、およそ２０ｎｍ以上、好ましくはおよそ４０ｎｍ以上、より好ましくはおよそ６０ｎｍ以上、さらに好ましくはおよそ８０ｎｍ以上、よりさらに好ましくはおよそ１００ｎｍ以上とすることができる。例えば、本発明において「ナノ薄膜」は、およそ２０ｎｍ~３００ｎｍ、好ましくはおよそ１００ｎｍ~２００ｎｍ程度の厚みを有する生体適合性高分子からなるシートとすることができる。
　本発明において利用可能な「生体適合性高分子」としては、例えばポリ乳酸、ポリグリコール酸、ポリヒドロキシ酪酸、ポリカプロラクトン、ポリブチレンサクシネート、ポリジオキサノン、ポリジメチルシロキサン、ポリメチルメタクリレート、ポリスチレン、ポリ酢酸ビニル、ポリ（３，４−エチレンジオキシチオフェン）、タンパク質（コラーゲン、ゼラチン、ラミニン、ファイブロネクチン、エラスチン）、多糖類（キトサン、アルギン酸、ヒアルロン酸、コンドロイチン硫酸、セルロース）、核酸（ＤＮＡ，ＲＮＡ）及びそれらの共重合体等が挙げられる。好ましくは、生体適合性高分子は生体分解性ポリマーであり、特に好ましくはポリ乳酸グリコール酸共重合体（以下、「ＰＬＧＡ」と記載する。）である。
　本発明において「パターン化ナノ薄膜」とは、微細な形状及び大きさ（マイクロパターン）を有するナノ薄膜を意味する。ナノ薄膜の形状は特に限定されず、円形、楕円形、多角形（例えば四角形、星形等）とすることができるが、その直径又は最も長い対角線の長さがおよそ１０μｍ~２０ｍｍ、好ましくはおよそ５０μｍ~１５ｍｍ、より好ましくはおよそ１００μｍ~１０ｍｍ、さらに好ましくはおよそ１５０μｍ~５ｍｍ、よりさらに好ましくはおよそ２００μｍ~３ｍｍ、とりわけ好ましくはおよそ３００μｍ~１ｍｍ程度である。ただし、パターン化ナノ薄膜の厚み並びに直径又は最も長い対角線の長さは上記範囲に限定されず、細胞を担持させたパターン化ナノ薄膜を細管により吸引・放出することが可能であり、かつ当該吸引・放出による力学的ストレスが負荷されたとしても担持された細胞の多く（例えば、当該力学的ストレスが負荷される前の担持細胞数の５０％以上、６０％以上、７０％以上、８０％以上、９０％以上、９５％以上またはそれ以上の細胞）がパターン化ナノ薄膜上に接着・生存している限り、適宜変更することができる。
　本発明の細胞担持パターン化ナノ薄膜は以下の［１］~［５］の工程を含む手法により得ることができる。本発明におけるパターン化ナノ薄膜は公知の手法（特許第５０２８４２２号公報）に基づいて作製することができ、以下及び図１に、マイクロスタンプ法とスピンコーティング法を組み合わせた作製方法の概略を示すが、パターン化ナノ薄膜の作製方法は当該手法に限定されない。
［１］　基板（例えば、ポリジメチルシロキサン（ＰＤＭＳ）、金属、シリコン、ガラス等）に特定のマイクロパターンが凸状に刻みこまれたマイクロパターン基板（すなわち“スタンプ”）を作製する。マイクロパターン基板は常法に従い、フォトリソグラフィー技術を利用して作製することができる。例えば、基板表面をオクタデシルトリメトキシシラン（ＯＤＭＳ）、オクタデシルジメチルクロロシラン、トリアルコキシヘキサデシルシラン等の長鎖状疎水性分子でコーティングした後、その上にポジ型フォトレジストを塗布する。次に、前記レジストにフォトマスクを透過させて露光（電子照射、紫外線照射、Ｘ線照射等）する。続いて、基体上のレジストを現像し、感光した領域のレジストを除去する。そして、Ｏ２プラズマ処理、ＣＯプラズマ処理又はハロゲンガスを用いた反応性イオンエッチング処理により、レジストで保護されていない領域の長鎖状疎水性分子を除去する。最後にアセトン、テトラヒドロフラン（ＴＨＦ）、ジクロロメタン等を用いてレジストを除去することで、マイクロパターン基板を得ることができる（図１中、（ａ）として示す）。
［２］　得られたマイクロパターン基板の表面（マイクロパターンが刻みこまれたスタンプ面）に生体適合性高分子層を形成する。生体適合性高分子又は生体適合性高分子の構成要素（例えば、生体分解性ポリマーの構成要素であるところのモノマー等）（以下、「生体適合性高分子等」と記載する。）を適当な溶媒（例えば、ジクロロメタン、クロロホルム、アセトン、酢酸エチル等）中に１ｍｇ／ｍＬ~１００ｍｇ／ｍＬ、好ましくは５ｍｇ／ｍＬ~４０ｍｇ／ｍＬの濃度で溶解し、この生体適合性高分子等の溶液をスピンコーティング法にてマイクロパターン基板表面に塗布する。スピンコーターの回転速度、回転時間を調節することにより、マイクロパターン基板表面上に塗布される生体適合性高分子等の厚みを調節することができ、最終生成物であるパターン化ナノ薄膜の厚みを調節することができる。次いで、塗布された生体適合性高分子等を重合及び／又は架橋することによって、マイクロパターン基板表面上に生体適合性高分子からなる層を形成することができる（図１中、（ｂ）として示す）。ここで「重合」としては、重縮合、重付加、付加縮合、開環重合、付加重合（ラジカル重合、アニオン重合、カチオン重合）、熱による固相重合、光重合、放射線重合、プラズマ重合等を挙げることができる。また「架橋」は、公知の架橋剤（例えば、アルキルジミデート類、アシルジアジド類、ジイソシアネート類、ビスマレイミド類、トリアジニル類、ジアゾ化合物、グルタルアルデヒド等）を用いて行うことができる。
　生体適合性高分子等の溶液には、金属、半導体、セラミック、磁性体等からなるナノ粒子、好ましくは磁性体ナノ粒子を含めることができる。当該ナノ粒子は１ｎｍ~５００ｎｍ、好ましくは１ｎｍ~５０ｎｍ程度の粒子径を有する。当該ナノ粒子を生体適合性高分子等の溶液中に含め、生体適合性高分子等と共にマイクロパターン基板表面上に塗布することによって、最終生成物であるパターン化ナノ薄膜の表面に当該ナノ粒子に起因する凹凸を形成することができる。パターン化ナノ薄膜の表面に凹凸を形成することによって、細胞が接着できる表面積を増すことができると共に、細胞の増殖活性を増大させることができる。また、パターン化ナノ薄膜に磁性体ナノ粒子含めた場合には、磁力を用いてパターン化ナノ薄膜を移動／集合させることができ操作性を高めることができる。ナノ粒子は生体適合性高分子等の溶液中に０．１ｍｇ／ｍＬ~１０ｍｇ／ｍＬ、好ましくは１ｍｇ／ｍＬ~５ｍｇ／ｍＬの濃度で含めることができる。
［３］　水溶性犠牲層を備える支持基板を作製する。支持基板（例えば、シリコン、ガラス等）（図１中、（ｃ）として示す）の一表面を、ポリビニルアルコール（ＰＶＡ）若しくはその誘導体、ポリイソプロピルアクリルアミド若しくはその誘導体、ポリエーテル若しくはその誘導体、多糖類、高分子電解質又はその塩等の水溶性高分子を用いてコーティングする。コーティングはキャスト法、スピンコーティング法等により水溶性高分子を支持基板上に塗布し、乾燥させることにより行うことができる。これにより、水性溶媒に可溶な水溶性犠牲層を備える支持基板を得ることができる（図１中、（ｄ）として示す）。
［４］　マイクロパターン基板上の生体適合性高分子層を、支持基板の水溶性犠牲層上にスタンプ・ベイクする（図１中、（ｅ）として示す）。生体適合性高分子の水溶性犠牲層上へのベイクは熱処理により行うことができる。これにより、生体適合性高分子がマイクロパターンを維持したまま水溶性犠牲層上に転写され、生体適合性高分子を担持する支持基板（生体適合性高分子担持支持基板）を得ることができる（図１中、（ｆ）として示す）。
　次いで、得られた生体適合性高分子担持支持基板の生体適合性高分子を備える面を、細胞の接着及び増殖を促進する細胞外基質によりコーティングする。本発明において利用可能な「細胞外基質」としては、例えば、Ｉ型コラーゲン、ＩＶ型コラーゲン、フィブロネクチン、ポリ−Ｄ−リジン（ＰＤＬ）、ラミニン、ポリ−Ｌ−オルニチン／ラミニン（ＰＬＯ／ＬＭ）等が挙げられる。細胞外基質のコーティングは、適当な溶媒中に溶解した（例えば０．０１μｇ／ｍＬ~５μｇ／ｍＬにて）細胞外基質溶液をスピンコーティング法等により支持基板上に塗布し、その後乾燥させることにより行うことができる。これにより、細胞外基質によりコーティングされた生体適合性高分子担持支持基板を得ることができる。
［５］　細胞外基質によりコーティングされた生体適合性高分子担持支持基板に細胞懸濁液を滴下して、およそ１時間~５時間ほどインキュベートして細胞を支持基板上に沈着させる。次いで、当該支持基板を培地中に入れさらに１日~３日間ほど培養する。当該培養の間に、支持基板上の水溶性犠牲層は溶解するため、所定のマイクロパターンを有するパターン化ナノ薄膜が支持基板より遊離する（図１中、（ｇ）として示す）。水溶性犠牲層の溶解に伴い、その上に直接コーティングされた細胞外基質層も溶解するため、細胞はパターン化ナノ薄膜上でのみで増殖する。パターン化ナノ薄膜上で細胞がコンフルエントになるまで培養することによって、目的の細胞担持パターン化ナノ薄膜を得ることができる。細胞の培養条件及び培地の組成は、用いる細胞に適した一般的なものを使用することができる。
　本発明の細胞担持パターン化ナノ薄膜において、パターン化ナノ薄膜中又はその表面には、機能性物質を担持させることができる。「機能性物質」とは、細胞の増殖、分化、生理活性等を制御する機能を有する物質（例えば、タンパク質、ポリペプチド、化合物等）やナノ薄膜の可視化を可能にする物質を意味する。このような機能性物質としては、成長／増殖因子（例えば、線維芽細胞増殖因子（ＦＧＦ）、上皮成長因子（ＥＧＦ）、骨形成タンパク質（ＢＭＰ）、神経成長因子（ＮＧＦ）、脳由来神経栄養因子（ＢＤＮＦ）等）、眼圧降下剤、神経保護剤、抗生物質、抗がん剤、可視化プローブ（造影剤、ナノ粒子、蛍光色素等）が挙げられる。機能性物質はパターン化ナノ薄膜に対して化学的又は物理的手法を用いて結合させることができる。化学的手法としては、官能基を介した化学的結合が挙げられ、例えば生体適合性高分子等を予め適当な官能基（例えばアミノ基、カルボキシル基、ヒドロキシル基、アルデヒド基等）で修飾しておき、得られたパターン化ナノ薄膜に対して当該導入した官能基を介して機能性物質を結合させることができる。物理的手法としては、パターン化ナノ薄膜と機能性物質との間の静電的相互作用、疎水性相互作用、水素結合、分子間力などによる結合が挙げられる。機能性物質のパターン化ナノ薄膜への結合は、上記細胞担持パターン化ナノ薄膜の製造工程において、細胞外基質によるコーティングの前、又は当該コーティングの後、又は当該コーティングと同時に行うことができる。
２．細胞
　本発明の細胞担持パターン化ナノ薄膜において、パターン化ナノ薄膜に担持させ得る細胞としては、細胞移植療法にて患者に移植され得る細胞（ただし、体液中に浮遊する細胞は除く）が挙げられ、例えば網膜色素上皮（ＲＰＥ）細胞、視細胞、肝細胞、心筋細胞、骨格筋細胞、平滑筋細胞、血管内皮細胞、腎細胞、膵島細胞、神経細胞等が挙げられる。これらの細胞は、本発明の細胞担持パターン化ナノ薄膜が導入される患者より単離されたものであっても良いし、あるいは、ＥＳ細胞、幹細胞又はｉＰＳ細胞から誘導されたものであっても良い。
３．細胞担持パターン化ナノ薄膜を用いた細胞送達
　本発明の細胞担持パターン化ナノ薄膜は優れた柔軟性と自己支持性を有し、その直径又は最も長い対角線の長さよりも小さな内径を有する細管で吸引することができ、また当該細管より放出することができる。また、本発明の細胞担持パターン化ナノ薄膜は細胞を安定に保持することができ、細管による吸引及び放出といった力学的ストレスが負荷されても、細胞の多く（例えば、当該力学的ストレスが負荷される前の担持細胞数の５０％以上、６０％以上、７０％以上、８０％以上、９０％以上、９５％以上またはそれ以上の細胞）をパターン化ナノ薄膜上に保持することができる。
　「細管」としては例えば、注射針やカテーテル等が挙げられる。「細管」のサイズ（ゲージ）や長さは、細胞担持パターン化ナノ薄膜の大きさや、細胞担持パターン化ナノ薄膜を導入する部位等の要因に応じて適宜選択することができる。
　本発明の細胞担持パターン化ナノ薄膜の生体内への導入は、生理食塩水と共に細胞担持パターン化ナノ薄膜を注射針又はカテーテル先端より吸引し、注射シリンジ又はカテーテル内に保持し、注射針又はカテーテル先端を疾患部位又はその近傍に挿入し、細胞担持パターン化ナノ薄膜を注射針又はカテーテル先端より放出して留置することにより行うことができる。細胞担持パターン化ナノ薄膜の生体内への導入により、前記疾患部位又はその近傍に担持する細胞を送達することができる。生体内には一又はそれ以上の細胞担持パターン化ナノ薄膜を導入することができる。例えば、本発明によれば、黄班変性症の治療を目的とする、黄班部網膜への網膜色素上皮（ＲＰＥ）細胞移植において、患者及び眼組織に大きな負担をかけることなく細胞移植（送達）を行うことができる。図７（ａ）に黄班変性症を患う網膜への、本発明の細胞担持パターン化ナノ薄膜を用いた細胞送達の概略を示す。例えば、１８~２５ゲージの微細な注射針又はカテーテルを強膜より眼球内部に挿入し、疾患部位又はその近傍の網膜側より網膜下まで刺しいれ少量の生理食塩水を注入し、細胞担持パターン化ナノ薄膜を導入するスペースを確保する。次いで、当該スペースに注射針又はカテーテルよりＲＰＥ細胞担持パターン化ナノ薄膜を放出し、注入した生理食塩水を吸引・除去することによって、細胞担持パターン化ナノ薄膜を網膜下に留置することができる。導入されたＲＰＥ細胞は疾患部（例えば、黄班変性部位）にて増殖又は疾患部における細胞と置換され、疾患を完治又はその症状を軽減することができる。また、導入されたパターン化ナノ薄膜は生体内で分解吸収され得る。
　次に実施例を挙げ、本発明をさらに詳しく説明するが、本発明はこれらに限定されるものではない。 1. Cell-supported patterned nano thin film The “cell-supported patterned nano thin film” in the present invention is characterized in that cells are supported on a patterned nano thin film as a substrate.
In the present invention, the “nano thin film” means a sheet made of a biocompatible polymer having a thickness of less than 500 nm, preferably about 400 nm or less, more preferably about 300 nm or less, and further preferably about 200 nm or less. The lower limit of the thickness of the “nano thin film” is not particularly limited, but can be about 20 nm or more, preferably about 40 nm or more, more preferably about 60 nm or more, more preferably about 80 nm or more, and still more preferably about 100 nm or more. . For example, in the present invention, the “nanothin film” can be a sheet made of a biocompatible polymer having a thickness of about 20 nm to 300 nm, preferably about 100 nm to 200 nm.
Examples of the “biocompatible polymer” usable in the present invention include polylactic acid, polyglycolic acid, polyhydroxybutyric acid, polycaprolactone, polybutylene succinate, polydioxanone, polydimethylsiloxane, polymethyl methacrylate, polystyrene, polyacetic acid. Vinyl, poly (3,4-ethylenedioxythiophene), protein (collagen, gelatin, laminin, fibronectin, elastin), polysaccharide (chitosan, alginic acid, hyaluronic acid, chondroitin sulfate, cellulose), nucleic acid (DNA, RNA) And copolymers thereof. Preferably, the biocompatible polymer is a biodegradable polymer, and particularly preferably a polylactic acid glycolic acid copolymer (hereinafter referred to as “PLGA”).
In the present invention, the “patterned nano thin film” means a nano thin film having a fine shape and size (micro pattern). The shape of the nano thin film is not particularly limited, and may be a circle, an ellipse, or a polygon (for example, a quadrangle, a star, etc.), but the diameter or the length of the longest diagonal is about 10 μm to 20 mm, preferably about The thickness is about 50 μm to 15 mm, more preferably about 100 μm to 10 mm, more preferably about 150 μm to 5 mm, still more preferably about 200 μm to 3 mm, and particularly preferably about 300 μm to 1 mm. However, the thickness and diameter of the patterned nano thin film or the length of the longest diagonal line is not limited to the above range, and the patterned nano thin film supporting cells can be sucked and released by a capillary tube, and the suction is performed. Even if a mechanical stress due to release is loaded, many of the loaded cells (for example, 50% or more, 60% or more, 70% or more, 80% or more of the number of loaded cells before the mechanical stress is loaded) , 90% or more, 95% or more cells) can be appropriately changed as long as the cells adhere and survive on the patterned nano thin film.
The cell-supported patterned nano thin film of the present invention can be obtained by a technique including the following steps [1] to [5]. The patterned nano thin film in the present invention can be produced based on a known technique (Japanese Patent No. 5028422), and the outline of a production method combining a micro stamp method and a spin coating method is shown below and in FIG. The method for producing the patterned nano thin film is not limited to this method.
[1] A micropattern substrate (that is, “stamp”) in which a specific micropattern is engraved in a convex shape on a substrate (for example, polydimethylsiloxane (PDMS), metal, silicon, glass, etc.) is manufactured. The micro-pattern substrate can be manufactured using a photolithography technique according to a conventional method. For example, after coating the substrate surface with long-chain hydrophobic molecules such as octadecyltrimethoxysilane (ODMS), octadecyldimethylchlorosilane, trialkoxyhexadecylsilane, a positive photoresist is applied thereon. Next, the resist is exposed through a photomask (electron irradiation, ultraviolet irradiation, X-ray irradiation, etc.). Subsequently, the resist on the substrate is developed, and the resist in the exposed area is removed. Then, long chain hydrophobic molecules in the region not protected by the resist are removed by O 2 plasma treatment, CO plasma treatment, or reactive ion etching treatment using a halogen gas. Finally, by removing the resist using acetone, tetrahydrofuran (THF), dichloromethane or the like, a micropattern substrate can be obtained (shown as (a) in FIG. 1).
[2] A biocompatible polymer layer is formed on the surface of the obtained micropattern substrate (stamp surface on which the micropattern is engraved). A biocompatible polymer or a component of a biocompatible polymer (for example, a monomer or the like that is a component of a biodegradable polymer) (hereinafter referred to as “biocompatible polymer or the like”) is appropriate. Dissolve in a solvent (eg, dichloromethane, chloroform, acetone, ethyl acetate, etc.) at a concentration of 1 mg / mL to 100 mg / mL, preferably 5 mg / mL to 40 mg / mL, and spin a solution of this biocompatible polymer or the like. It is applied to the surface of the micropattern substrate by a coating method. By adjusting the rotation speed and rotation time of the spin coater, the thickness of the biocompatible polymer applied on the surface of the micropattern substrate can be adjusted, and the thickness of the patterned nano thin film that is the final product can be adjusted. Can be adjusted. Next, a layer made of the biocompatible polymer can be formed on the surface of the micropattern substrate by polymerizing and / or crosslinking the applied biocompatible polymer or the like (in FIG. 1, as (b)). Show). Here, “polymerization” includes polycondensation, polyaddition, addition condensation, ring-opening polymerization, addition polymerization (radical polymerization, anionic polymerization, cationic polymerization), solid phase polymerization by heat, photopolymerization, radiation polymerization, plasma polymerization, etc. Can be mentioned. The “crosslinking” can be performed using a known crosslinking agent (for example, alkyl dimidates, acyldiazides, diisocyanates, bismaleimides, triazinyl, diazo compounds, glutaraldehyde, etc.).
The solution of biocompatible polymer or the like can contain nanoparticles made of metal, semiconductor, ceramic, magnetic material, etc., preferably magnetic nanoparticles. The nanoparticles have a particle diameter of about 1 nm to 500 nm, preferably about 1 nm to 50 nm. By including the nanoparticles in a solution of biocompatible polymer and applying it onto the surface of the micropattern substrate together with the biocompatible polymer and the like, the nanoparticle is applied to the surface of the patterned nano thin film that is the final product. The resulting irregularities can be formed. By forming irregularities on the surface of the patterned nano thin film, the surface area to which cells can adhere can be increased, and the proliferation activity of the cells can be increased. In addition, when magnetic nano particles are included in the patterned nano thin film, the patterned nano thin film can be moved / assembled by using magnetic force, and the operability can be improved. The nanoparticles can be included in a solution of biocompatible polymer or the like at a concentration of 0.1 mg / mL to 10 mg / mL, preferably 1 mg / mL to 5 mg / mL.
[3] A support substrate having a water-soluble sacrificial layer is prepared. One surface of a supporting substrate (eg, silicon, glass, etc.) (shown as (c) in FIG. 1) is coated with polyvinyl alcohol (PVA) or a derivative thereof, polyisopropylacrylamide or a derivative thereof, polyether or a derivative thereof, polysaccharide Coating is performed using a water-soluble polymer such as a polymer electrolyte or a salt thereof. Coating can be performed by applying a water-soluble polymer on a support substrate by a casting method, a spin coating method, or the like, and drying. Thereby, a support substrate provided with a water-soluble sacrificial layer soluble in an aqueous solvent can be obtained (shown as (d) in FIG. 1).
[4] The biocompatible polymer layer on the micropattern substrate is stamp-baked on the water-soluble sacrificial layer of the support substrate (shown as (e) in FIG. 1). Baking the biocompatible polymer onto the water-soluble sacrificial layer can be performed by heat treatment. As a result, the biocompatible polymer is transferred onto the water-soluble sacrificial layer while maintaining the micropattern, and a support substrate (biocompatible polymer-supported support substrate) supporting the biocompatible polymer can be obtained ( (Indicated as (f) in FIG. 1).
Next, the surface provided with the biocompatible polymer of the obtained biocompatible polymer-supported support substrate is coated with an extracellular matrix that promotes cell adhesion and proliferation. Examples of the “extracellular matrix” that can be used in the present invention include type I collagen, type IV collagen, fibronectin, poly-D-lysine (PDL), laminin, poly-L-ornithine / laminin (PLO / LM), and the like. Is mentioned. The extracellular matrix coating is performed by applying an extracellular matrix solution dissolved in an appropriate solvent (for example, at 0.01 μg / mL to 5 μg / mL) onto a support substrate by a spin coating method or the like and then drying it. It can be carried out. Thereby, a biocompatible polymer-supported support substrate coated with an extracellular matrix can be obtained.
[5] A cell suspension is dropped onto a support substrate carrying a biocompatible polymer coated with an extracellular matrix, and the cells are incubated for about 1 to 5 hours to deposit the cells on the support substrate. Next, the support substrate is placed in a medium and further cultured for 1 to 3 days. During the culture, the water-soluble sacrificial layer on the support substrate dissolves, so that the patterned nano thin film having a predetermined micropattern is released from the support substrate (shown as (g) in FIG. 1). As the water-soluble sacrificial layer dissolves, the extracellular matrix layer directly coated thereon also dissolves, so that the cells grow only on the patterned nanofilm. By culturing until the cells are confluent on the patterned nano thin film, the target cell-supported patterned nano thin film can be obtained. As the cell culture conditions and the medium composition, general ones suitable for the cells to be used can be used.
In the cell-supported patterned nano thin film of the present invention, a functional substance can be supported in or on the patterned nano thin film. “Functional substance” means a substance (for example, protein, polypeptide, compound, etc.) having a function of controlling cell proliferation, differentiation, physiological activity, etc., or a substance that enables visualization of a nano thin film. Such functional substances include growth / growth factors (eg, fibroblast growth factor (FGF), epidermal growth factor (EGF), bone morphogenetic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic Factor (BDNF), etc.), intraocular pressure-lowering agent, neuroprotective agent, antibiotic, anticancer agent, visualization probe (contrast agent, nanoparticle, fluorescent dye, etc.). The functional material can be bound to the patterned nanofilm using chemical or physical techniques. The chemical method includes chemical bonding via a functional group. For example, a biocompatible polymer is modified in advance with an appropriate functional group (for example, amino group, carboxyl group, hydroxyl group, aldehyde group, etc.). In addition, a functional substance can be bound to the obtained patterned nano thin film through the introduced functional group. Examples of physical methods include electrostatic interaction, hydrophobic interaction, hydrogen bonding, intermolecular force, and the like between the patterned nano thin film and the functional material. The binding of the functional substance to the patterned nano thin film can be performed before the coating with the extracellular matrix, after the coating, or simultaneously with the coating in the manufacturing process of the cell-supported patterned nano thin film.
2. Cells In the cell-supported patterned nanothin film of the present invention, examples of cells that can be supported on the patterned nanothin film include cells that can be transplanted to a patient by cell transplantation therapy (except cells that float in body fluids). Examples include retinal pigment epithelium (RPE) cells, photoreceptor cells, hepatocytes, cardiomyocytes, skeletal muscle cells, smooth muscle cells, vascular endothelial cells, kidney cells, islet cells, nerve cells and the like. These cells may be isolated from a patient into which the cell-supported patterned nanofilm of the present invention is introduced, or may be derived from ES cells, stem cells, or iPS cells. good.
3. Cell delivery using cell-supported patterned nanofilm The cell-supported patterned nanofilm of the present invention has excellent flexibility and self-supporting ability, and is aspirated with a capillary having an inner diameter smaller than its diameter or the length of the longest diagonal. And can be released from the tubule. In addition, the cell-supported patterned nano thin film of the present invention can stably hold cells, and even when mechanical stress such as suction and release by a capillary tube is applied, many cells (for example, the mechanical stress is applied). 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more or more of the number of supported cells before the treatment) can be retained on the patterned nano thin film.
Examples of the “capillary tube” include an injection needle and a catheter. The size (gauge) and length of the “capillary tube” can be appropriately selected according to factors such as the size of the cell-supported patterned nano thin film and the site into which the cell-supported patterned nano thin film is introduced.
The cell-supported patterned nano thin film of the present invention is introduced into the living body by drawing the cell-supported patterned nano thin film together with physiological saline from the tip of the injection needle or catheter and holding it in the injection syringe or catheter. The tip can be inserted into or near the diseased site, and the cell-supported patterned nano thin film can be released from the injection needle or the tip of the catheter and placed. By introducing the cell-supported patterned nano thin film into the living body, the cells supported at or near the diseased site can be delivered. One or more cell-supported patterned nano thin films can be introduced into the living body. For example, according to the present invention, in transplantation of retinal pigment epithelium (RPE) cells to the macula retina for the purpose of treating macular degeneration, cell transplantation (delivery) is performed without imposing a heavy burden on the patient and the eye tissue. )It can be performed. FIG. 7A shows an outline of cell delivery using the cell-supported patterned nano thin film of the present invention to the retina suffering from macular degeneration. For example, a 18-25 gauge fine injection needle or catheter is inserted into the eyeball from the sclera, and a small amount of physiological saline is injected from the retinal side of the diseased site or its vicinity into the subretinal region, thereby patterning the cell. Secure space for introducing nano thin films. Subsequently, the RPE cell-carrying patterned nano thin film is released from the injection needle or catheter into the space, and the injected physiological saline is sucked and removed, whereby the cell-carrying patterned nano thin film can be placed under the retina. The introduced RPE cells are proliferated or replaced with cells in the diseased part at the diseased part (for example, macular degeneration site), and the disease can be completely cured or its symptoms can be reduced. Moreover, the introduced patterned nano thin film can be decomposed and absorbed in vivo.
EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention is not limited to these.

パターン化ナノ薄膜の作製
　パターン化ナノ薄膜の作製方法の概略を図１に示す。パターン化ナノ薄膜はスピンコーティングとマイクロスタンプ法を組み合わせて作製した。まず、ポリジメチルシロキサン（ＰＤＭＳ）製のマイクロパターン基板（図１（ａ））の表面に、磁性ナノ粒子（ＭＮＰｓ：粒径１０ｎｍ）（２．５ｍｇ／ｍＬ）を混合させた、又は混合させない、ポリ乳酸グリコール酸共重合体（ＰＬＧＡ）溶液（５ｍｇ／ｍＬ）をスピンコートした（４０００ｒｐｍ，４０ｓ）。次いで、このマイクロパターン基板（図１（ｂ））を、水溶性犠牲層であるポリビニルアルコール（ＰＶＡ）をコートしておいたガラス製の支持基板（２２ｍｍｘ２２ｍｍ）（図１（ｄ））に押し付けベイクし（１２０℃にて５分間）、ＰＬＧＡ層をマイクロパターン基板から支持基板側に転写した（図１（ｆ））。細胞培養に利用するパターン化ナノ薄膜の製造においては、支持基板にさらにＩ型コラーゲン溶液（０．５ｍｇ／ｍＬ）をスピンコートした。その後、水溶性犠牲層であるＰＶＡを水性溶媒中で溶解させることで、支持基板より遊離した、自己支持性を有するパターン化ナノ薄膜を得た（図１（ｇ））。
　得られたパターン化ナノ薄膜を図２に示す。図２（ａ）は支持基板上に転写されたＰＬＧＡとＭＮＰｓから成るパターン化ナノ薄膜を示し、図２（ｂ）はその拡大図である。また、支持基板より遊離したパターン化ナノ薄膜は優れた柔軟性と自己支持性を有し、ナノ薄膜自体の直径より小さい内径のカテーテル（２４Ｇ（内径４７０μｍ）カテーテル）で吸引することができた（写真図のパターン化ナノ薄膜はローダミンにより着色されている）（図２（ｃ））。
　パターン化ナノ薄膜は、作製に用いるマイクロパターン基板のパターン形状を変えることで、様々な形状／サイズのパターン化ナノ薄膜を作製することができた（図３（ａ）−（ｄ））。
　得られたパターン化ナノ薄膜の表面形状を触針式表面形状測定器（ＤｅｋＴａｋ）にて測定したところ、ＭＮＰｓの混合の有無にかかわらず、パターン化ナノ薄膜の平均膜厚はおよそ１７０ｎｍであった（図４（ａ）−（ｂ））。 Production of Patterned Nano Thin Film An outline of a method for producing a patterned nano thin film is shown in FIG. Patterned nano thin films were fabricated by combining spin coating and micro stamping methods. First, magnetic nanoparticles (MNPs: particle size 10 nm) (2.5 mg / mL) were mixed or not mixed on the surface of a micropattern substrate (FIG. 1 (a)) made of polydimethylsiloxane (PDMS). A polylactic acid glycolic acid copolymer (PLGA) solution (5 mg / mL) was spin-coated (4000 rpm, 40 s). Next, this micropattern substrate (FIG. 1B) is pressed against a glass support substrate (22 mm × 22 mm) (FIG. 1D) coated with polyvinyl alcohol (PVA), which is a water-soluble sacrificial layer, and baked. Then, the PLGA layer was transferred from the micropattern substrate to the support substrate side (FIG. 1 (f)). In the production of the patterned nano thin film used for cell culture, a type I collagen solution (0.5 mg / mL) was further spin-coated on a support substrate. Thereafter, PVA, which is a water-soluble sacrificial layer, was dissolved in an aqueous solvent to obtain a patterned nano thin film having self-supporting properties that was released from the support substrate (FIG. 1 (g)).
The resulting patterned nanofilm is shown in FIG. FIG. 2A shows a patterned nano thin film composed of PLGA and MNPs transferred on a support substrate, and FIG. 2B is an enlarged view thereof. Further, the patterned nano thin film released from the support substrate has excellent flexibility and self-supporting ability, and was able to be sucked by a catheter having an inner diameter smaller than the diameter of the nano thin film itself (24G (inner diameter 470 μm) catheter) ( The patterned nanothin film in the photograph is colored with rhodamine) (FIG. 2 (c)).
By changing the pattern shape of the micropatterned substrate used for the production of the patterned nano thin film, it was possible to produce patterned nano thin films having various shapes / sizes (FIGS. 3A to 3D).
When the surface shape of the obtained patterned nano thin film was measured with a stylus type surface shape measuring device (DekTak), the average film thickness of the patterned nano thin film was about 170 nm regardless of the presence or absence of mixing of MNPs. (FIGS. 4A to 4B).

パターン化ナノ薄膜上での網膜色素上皮（ＲＰＥ）細胞の培養
　上記実施例１において、ＰＬＧＡ層がマイクロパターン基板から転写されＩ型コラーゲン溶液をスピンコートした支持基板に、ＲＰＥ細胞の懸濁液（１．５×１０６細胞／ｍＬ）を０．４ｍＬずつ滴下し、３７℃にて１時間程度インキュベートし細胞を支持基板上に沈着させた。次いで、細胞が沈着した支持基板を培地（ＤＭＥＭ，４％（ｖ／ｖ）ＦＢＳ，１％ペニシリンストレプトマイシン）中に入れ、常法に従い培養した。
　得られた培養物について、生死判定キット（Ｃｅｌｌｓｔａｉｎ−Ｄｏｕｂｌｅ　Ｓｔａｉｎｉｎｇ　Ｋｉｔ，同仁化学研究所）を用いて、生細胞及び死細胞を染色した。
　結果を図５−１（ａ）−（ｂ）に示す。培地中で水溶性犠牲層であるＰＶＡが溶解されるため、ＲＰＥ細胞はパターン化ナノ薄膜上にのみ接着して増殖・成長していることが確認された。パターン化ナノ薄膜上にて死細胞は検出されなかった。
　また、得られた培養物について、ＲＰＥ細胞をカルセインＡＭで、パターン化ナノ薄膜をローダミンＢで染色した。
　結果を図５−１（ｃ）に示す。写真図より明らかなとおり、ＲＰＥ細胞がパターン化ナノ薄膜上でモノレイヤー組織を形成し存在していることが確認できた。
　また、ＭＮＰｓを混合したＰＬＧＡを用いて作製したパターン化ナノ薄膜（以下、「ＭＮＰｓ（＋）パターン化ナノ薄膜」と記載する。）及びＭＮＰｓを混合させずＰＬＧＡのみから成るパターン化ナノ薄膜（以下、「ＭＮＰｓ（−）パターン化ナノ薄膜」と記載する。）について、その表面構造を原子間力顕微鏡（ＡＦＭ）にて観察した。
　結果を図５−１（ｄ）に示す。ＭＮＰｓ（＋）パターン化ナノ薄膜（左）（自乗平均面粗さ（ＲＭＳ）：２．８６ｎｍ）の方が、ＭＮＰｓ（−）パターン化ナノ薄膜（右）（ＲＭＳ：０．４８９ｎｍ）と比べて表面が粗い構造を有することが確認できた。
　次いで、ＭＮＰｓ混合の有無による、パターン化ナノ薄膜上の細胞増殖活性への影響を調べた。ＭＮＰｓ（＋）パターン化ナノ薄膜、又はＭＮＰｓ（−）パターン化ナノ薄膜上にて培養したＲＰＥ細胞について、セルカウンティングキット（Ｃｅｌｌ　Ｃｏｕｎｔｉｎｇ　Ｋｉｔ−８（ＣＣＫ−８）：同仁化学研究所）を用いて、経時的にパターン化ナノ薄膜上の細胞数を計測し、各パターン化ナノ薄膜の細胞増殖活性への影響を評価した。
　結果を図５−１（ｅ）に示す。ＭＮＰｓ（＋）パターン化ナノ薄膜上の細胞の方が、ＭＮＰｓ（−）パターン化ナノ薄膜上の細胞に比べ培養１日後の増殖活性が高いことが確認できた。
　さらに、ＭＮＰｓ（＋）パターン化ナノ薄膜上又はＭＮＰｓ（−）パターン化ナノ薄膜上にてそれぞれ２日間培養した細胞について、抗ＺＯ−１抗体及び抗Ｆ−Ａｃｔｉｎ抗体を一次抗体として用いて免疫染色を行った。
　結果を図５−２（ｆ）−（ｇ）に示す。ＭＮＰｓ（＋）パターン化ナノ薄膜上の細胞（図５−２（ｆ））の方が、ＭＮＰｓ（−）パターン化ナノ薄膜上の細胞（図５−２（ｇ））に比べ、ＺＯ−１（タイトジャンクション）及びＦ−Ａｃｔｉｎの形成が促進されており、また、細胞の形態は網膜色素上皮に特徴的なヘキサゴナルな形態を示しているのが確認できた。 Culture of Retinal Pigment Epithelium (RPE) Cells on Patterned Nano Thin Film In Example 1 above, a suspension of RPE cells (on a support substrate on which a PLGA layer was transferred from a micropattern substrate and spin coated with a type I collagen solution ( 1.5 × 10 6 cells / mL) was added dropwise at a time, and incubated at 37 ° C. for about 1 hour to deposit the cells on a support substrate. Next, the support substrate on which the cells were deposited was placed in a medium (DMEM, 4% (v / v) FBS, 1% penicillin streptomycin) and cultured according to a conventional method.
About the obtained culture, the living cell and the dead cell were dye | stained using the life-and-death determination kit (Cellstein-Double Staining Kit, Dojindo Laboratories).
The results are shown in FIGS. 5-1 (a)-(b). Since PVA, which is a water-soluble sacrificial layer, is dissolved in the medium, it was confirmed that RPE cells adhered and proliferated and grew only on the patterned nanofilm. No dead cells were detected on the patterned nanofilm.
Moreover, about the obtained culture, the RPE cell was dye | stained with calcein AM and the patterned nano thin film with rhodamine B.
The results are shown in FIG. As is clear from the photograph, it was confirmed that RPE cells were present by forming a monolayer structure on the patterned nanofilm.
In addition, a patterned nano thin film (hereinafter referred to as “MNPs (+) patterned nano thin film”) prepared using PLGA mixed with MNPs and a patterned nano thin film (hereinafter referred to as “MNPs (+) patterned nano thin film”) composed only of PLGA without mixing MNPs. The surface structure of “MNPs (−) patterned nano thin film” was observed with an atomic force microscope (AFM).
The results are shown in FIG. The MNPs (+) patterned nano thin film (left) (root mean square roughness (RMS): 2.86 nm) is compared to the MNPs (−) patterned nano thin film (right) (RMS: 0.489 nm). It was confirmed that the surface had a rough structure.
Next, the influence of the presence or absence of MNPs on cell proliferation activity on the patterned nanofilm was examined. About MPEs (+) patterned nano thin film or RPE cells cultured on MNPs (−) patterned nano thin film, using a cell counting kit (Cell Counting Kit-8 (CCK-8): Dojindo Laboratories) The number of cells on the patterned nanofilm was measured over time, and the influence of each patterned nanofilm on the cell proliferation activity was evaluated.
The results are shown in FIG. It was confirmed that the cells on the MNPs (+) patterned nanofilm had higher proliferation activity after 1 day of culture than the cells on the MNPs (−) patterned nanofilm.
Furthermore, immunostaining of cells cultured on MNPs (+) patterned nanofilm or MNPs (-) patterned nanofilm for 2 days, respectively, using anti-ZO-1 antibody and anti-F-actin antibody as primary antibodies Went.
The results are shown in Fig. 5-2 (f)-(g). The cells on the MNPs (+) patterned nanofilm (FIG. 5-2 (f)) are more ZO-1 than the cells on the MNPs (−) patterned nanofilm (FIG. 5-2 (g)). (Tight junction) and F-actin formation were promoted, and it was confirmed that the cell morphology showed a hexagonal morphology characteristic of the retinal pigment epithelium.

ＲＰＥ細胞担持ナノ薄膜の注射針による吸引・放出試験
　上記実施例２に記載の手法にしたがって、ＭＮＰｓ（＋）パターン化ナノ薄膜上でＲＰＥ細胞を培養して得られた培養物（以下、「ＲＰＥ細胞担持パターン化ナノ薄膜」と呼ぶ。）について、２５Ｇ（内径３２０μｍ）注射針での吸引・放出に伴う力学的ストレスによる、組織形態の変化及び細胞の生存について評価した。具体的には、ＲＰＥ細胞担持パターン化ナノ薄膜（３００μｍ、４００μｍ、５００μｍ、１０００μｍの直径を有する）を２５Ｇ（内径３２０μｍ）注射針で１回ずつ吸引・放出し、当該力学的ストレス負荷前後のパターン化ナノ薄膜上のＲＰＥ細胞について、組織形態の変化を位相差顕微鏡にて、また細胞の生存を生死判定キット（Ｃｅｌｌｓｔａｉｎ−Ｄｏｕｂｌｅ　Ｓｔａｉｎｉｎｇ　Ｋｉｔ，同仁化学研究所）を用いて確認した。
　結果を図６に示す。図６（ａ）の写真図より明らかなとおり、ＲＰＥ細胞担持パターン化ナノ薄膜に対して力学的ストレスを負荷したとしても、パターン化ナノ薄膜上のＲＰＥ細胞において、組織形態及び細胞の生存に大きな変化は生じないことが確認できた。
　図６（ｂ）は、パターン化ナノ薄膜のサイズによる、上記力学的ストレス負荷前後の細胞生存率への影響を示す。結果より明らかなとおり、パターン化ナノ薄膜の直径やシリンジ針の種類（注射針：Ｓ．Ｎ．，静脈留置カテーテル：Ｉ．Ｃ．）による影響はみられず、いずれの場合においても力学的ストレス負荷後に８０％以上の細胞生存率が得られた。
　また、ナノ薄膜（厚さ１７０ｎｍ）を用いて作製されたＲＰＥ細胞担持パターン化ナノ薄膜、及び厚さのみ異なるマイクロ厚薄膜（厚さ５．５μｍ）を用いて作製されたＲＰＥ細胞担持パターン化マイクロ厚薄膜に対して、上記力学的ストレスを負荷し、各薄膜上の組織形態の変化及び細胞の生存について上記と同様に調べた。
　力学的ストレス負荷前後における、各薄膜の各顕微鏡における観察結果を図６（ｃ）に、各薄膜に対する細胞接着面積の変化を図６（ｄ）に示す。なお、図６（ｄ）において細胞接着面積の値は力学的ストレス負荷前における細胞接着面積を１００％とする相対値にて示す。
　ナノ薄膜を用いて作製されたＲＰＥ細胞担持パターン化ナノ薄膜では、８割以上の細胞が薄膜表面に接着維持され、かつほとんどの細胞が生存しており、パターン化ナノ薄膜上に形成されたモノレイヤー構造を安定に維持していることが確認できた。一方、マイクロ厚薄膜を用いて作製されたＲＰＥ細胞担持パターン化マイクロ厚薄膜では、力学的ストレスの負荷により大部分の細胞が膜から剥離してしまった。パターン化マイクロ厚薄膜は、パターン化ナノ薄膜に比べて柔軟性に乏しく、そのため力学的ストレスによる細胞への負荷が大きくなり大部分の細胞が膜から剥離してしまったと考えられる。 Inhalation / release test of RPE cell-supported nano thin film with injection needle According to the method described in Example 2 above, a culture obtained by culturing RPE cells on MNPs (+) patterned nano thin film (hereinafter referred to as “RPE”). Changes in tissue morphology and cell survival due to mechanical stress associated with suction / release with a 25 G (inner diameter 320 μm) injection needle were evaluated. Specifically, RPE cell-supported patterned nano thin film (having a diameter of 300 μm, 400 μm, 500 μm, and 1000 μm) is sucked and released once by a 25 G (inner diameter 320 μm) injection needle, and the pattern before and after the mechanical stress loading Regarding the RPE cells on the activated nanofilm, changes in tissue morphology were confirmed with a phase-contrast microscope, and cell survival was confirmed using a life / death determination kit (Cellstein-Double Staining Kit, Dojindo Laboratories).
The results are shown in FIG. As is apparent from the photograph of FIG. 6 (a), even when mechanical stress is applied to the patterned nanofilm bearing RPE cells, the RPE cells on the patterned nanofilm have a large effect on tissue morphology and cell survival. It was confirmed that no change occurred.
FIG. 6 (b) shows the effect of the size of the patterned nano thin film on cell viability before and after the mechanical stress loading. As is clear from the results, there was no effect of the diameter of the patterned nano thin film or the type of syringe needle (injection needle: SN, intravenous indwelling catheter: IC), and in any case mechanical stress A cell viability of 80% or more was obtained after loading.
In addition, RPE cell-supported patterned nano thin films prepared using nano thin films (thickness 170 nm) and RPE cell-supported patterned micro films prepared using micro thick thin films (thickness 5.5 μm) that differ only in thickness. The above-described mechanical stress was applied to the thick film, and changes in the tissue morphology and cell survival on each thin film were examined in the same manner as described above.
FIG. 6 (c) shows the observation results of each thin film before and after the mechanical stress loading, and FIG. 6 (d) shows the change in the cell adhesion area with respect to each thin film. In addition, in FIG.6 (d), the value of a cell adhesion area is shown by the relative value which makes the cell adhesion area before mechanical stress load 100%.
In the patterned nanothin film carrying RPE cells produced using nanothin films, more than 80% of the cells remain attached to the surface of the thin film, and most of the cells survive, and the monolayer formed on the patterned nanothin film It was confirmed that the layer structure was maintained stably. On the other hand, in the RPE cell-supported patterned micro thick thin film produced using the micro thick thin film, most cells were detached from the film due to the load of mechanical stress. It is considered that the patterned microthin film is less flexible than the patterned nanothin film, and therefore the load on the cells due to mechanical stress is increased, and most of the cells are detached from the film.

ブタ眼球網膜下への細胞担持ナノ薄膜の導入
　ブタ眼球網膜下へのＲＰＥ細胞担持パターン化ナノ薄膜の導入試験を行った。２５Ｇ（内径３２０μｍ）注射針を、摘出したブタ眼球の強膜より眼球内に挿入し、黄斑部の網膜側より網膜下まで刺しいれ少量の生理食塩水を注入し、ＲＰＥ細胞担持パターン化ナノ薄膜を導入するための空間を形成した。次いで、上記実施例２に記載の手法にしたがって作製したＲＰＥ細胞担持パターン化ナノ薄膜を２５Ｇ注射針を用いてシリンジ内に吸引し、それを前記と同様に挿入した注射針より網膜下に形成した前記空間内に放出した。最後に、注入した生理食塩水を吸引・除去することによってＲＰＥ細胞担持パターン化ナノ薄膜を網膜下に留置した。
　図７（ｂ）に、ブタ眼球黄斑部網膜下に導入したＲＰＥ細胞担持ナノ薄膜の顕微鏡写真を示す。注入されたＲＰＥ細胞担持ナノ薄膜は網膜下で伸展し、元の円形状を維持していることが確認できた。 Introduction of cell-carrying nano thin film under pig eyeball retina Introduction test of RPE cell carrying patterned nano thin film under pig eyeball retina was conducted. A 25G (inner diameter 320 μm) injection needle is inserted into the eyeball from the excised sclera of the pig eyeball, and a small amount of physiological saline is injected from the retinal side of the macula to the subretinal region. Formed a space for introducing. Subsequently, the RPE cell-supported patterned nano thin film prepared according to the method described in Example 2 above was sucked into a syringe using a 25G injection needle, and formed under the retina from the injection needle inserted in the same manner as described above. Released into the space. Finally, the RPE cell-supported patterned nano thin film was placed under the retina by aspirating and removing the injected physiological saline.
FIG. 7B shows a micrograph of the RPE cell-carrying nanothin film introduced below the retina of the porcine eyeball macular region. It was confirmed that the injected RPE cell-carrying nanothin film was stretched under the retina and maintained the original circular shape.

ラット眼球網膜下への細胞担持ナノ薄膜の導入
　Ｓｐｒａｇｕｅ−Ｄａｗｌｅｙ（ＳＤ）ラットの眼球網膜下へのＲＰＥ細胞担持パターン化ナノ薄膜の導入試験を行った。３０Ｇ（内径１４０μｍ）注射針を、麻酔したラットの眼球の強膜より眼球内に挿入し、黄斑部の網膜側より網膜下まで刺しいれ少量の生理食塩水を注入し、ＲＰＥ細胞担持パターン化ナノ薄膜を導入するための空間を形成した。次いで、上記実施例２に記載の手法にしたがって作製したＲＰＥ細胞担持パターン化ナノ薄膜を３０Ｇ注射針を用いてシリンジ内に吸引し、それを前記と同様に挿入した注射針より網膜下に形成した前記空間内に放出した。最後に、注入した生理食塩水を吸引・除去することによってＲＰＥ細胞担持パターン化ナノ薄膜を網膜下に留置した。コントロールのラットには、生理食塩水のみを注入、吸引・除去する操作を同様に行った。
　処置後のラットは通常の方法に従い、飼育した。
　処置後、１週間後の各ラットの眼底光干渉断層像（ＯＣＴ）を調べたところ、注入されたＲＰＥ細胞担持ナノ薄膜が、網膜下で伸展し、シート状の構造で保持されていることが確認できた（図８（ａ−１））。次いで、ラットの眼球を摘出し、後眼部を観察したところ、導入したナノ薄膜が確認できた（図８（ｂ））。さらに、摘出し眼球の網膜組織の切片を作製し、ヘマトキシリン・エオジン（ＨＢ）染色したところ、ＲＰＥ細胞担持ナノ薄膜によって導入された細胞が確認できた。
　これらの結果より、細胞担持パターン化ナノ薄膜を用いて生体の組織中に、低侵襲に細胞移植（送達）できることが明らかとなった。 Introduction of cell-supporting nanothin film under rat eyeball retina An introduction test of RPE cell-supported patterned nanothin film under the eyeball retina of Sprague-Dawley (SD) rat was conducted. A 30G (inner diameter 140 μm) injection needle is inserted into the eyeball from the sclera of an anesthetized rat eyeball, and a small amount of physiological saline is injected from the retinal side of the macula to the subretinal region. A space for introducing a thin film was formed. Subsequently, the RPE cell-supported patterned nano thin film prepared according to the method described in Example 2 above was sucked into the syringe using a 30G injection needle, and formed under the retina from the injection needle inserted in the same manner as described above. Released into the space. Finally, the RPE cell-supported patterned nano thin film was placed under the retina by aspirating and removing the injected physiological saline. The control rats were similarly injected with saline alone and aspirated / removed.
The treated rats were bred according to the usual method.
Examination of the fundus optical coherence tomography (OCT) of each rat one week after the treatment revealed that the injected RPE cell-carrying nanothin film was stretched under the retina and held in a sheet-like structure. It was confirmed (FIG. 8 (a-1)). Subsequently, when the rat's eyeball was extracted and the posterior eye part was observed, the introduced nano thin film could be confirmed (FIG. 8 (b)). Furthermore, when a section of the retinal tissue of the extracted eyeball was prepared and stained with hematoxylin and eosin (HB), cells introduced by the RPE cell-carrying nanothin film could be confirmed.
From these results, it became clear that cell transplantation (delivery) can be performed in a living tissue with minimal invasiveness using a cell-supported patterned nano thin film.

　本発明によれば、注射針等の細管を用いて吸引・放出することが可能な、細胞を担持する生体適合性高分子からなるナノ薄膜を提供することができる。かかる特徴を有する細胞担持ナノ薄膜は、生体内の狭小な空間にも注射針等の細管を用いて低侵襲に導入し細胞移植（送達）することができ、患者や組織の負担を低減することができる。
　本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。 ADVANTAGE OF THE INVENTION According to this invention, the nano thin film which consists of a biocompatible polymer which carries the cell which can be attracted | sucked and discharge | released using thin tubes, such as an injection needle, can be provided. A cell-supporting nano thin film having such characteristics can be introduced into a small space in a living body using a small tube such as an injection needle in a minimally invasive manner and transplanted (delivered), thereby reducing the burden on patients and tissues. Can do.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (8)

　厚さ５００ｎｍ未満であり、かつ直径又は最も長い対角線の長さが１０μｍ~２０ｍｍであるパターン化ナノ薄膜を基材とし、該パターン化ナノ薄膜上に細胞を担持することを特徴とする、細胞担持パターン化ナノ薄膜。 Cell carrying, characterized in that the substrate is a patterned nano thin film having a thickness of less than 500 nm and having a diameter or the longest diagonal line length of 10 μm to 20 mm, and carrying cells on the patterned nano thin film Patterned nano thin film. 　パターン化ナノ薄膜が、生体適合性高分子からなる、請求項１に記載の細胞担持パターン化ナノ薄膜。 The cell-supported patterned nano thin film according to claim 1, wherein the patterned nano thin film is made of a biocompatible polymer. 　パターン化ナノ薄膜が細胞外基質でコーティングされている、請求項１又は２に記載の細胞担持パターン化ナノ薄膜。 The cell-supported patterned nano thin film according to claim 1 or 2, wherein the patterned nano thin film is coated with an extracellular matrix. 　パターン化ナノ薄膜が金属、半導体、セラミック、又は磁性体からなるナノ粒子を含む、請求項１~３のいずれか１項に記載の細胞担持パターン化ナノ薄膜。 The cell-supported patterned nanothin film according to any one of claims 1 to 3, wherein the patterned nanothin film includes nanoparticles made of a metal, a semiconductor, a ceramic, or a magnetic material. 　パターン化ナノ薄膜が機能性物質を含む、請求項１~４のいずれか１項に記載の細胞担持パターン化ナノ薄膜。 The cell-supported patterned nano thin film according to any one of claims 1 to 4, wherein the patterned nano thin film contains a functional substance. 　細胞が網膜色素上皮細胞である、請求項１~５のいずれか１項に記載の細胞担持パターン化ナノ薄膜。 The cell-supported patterned nano thin film according to any one of claims 1 to 5, wherein the cell is a retinal pigment epithelial cell. 　細管より組織中に放出され、該組織に細胞を送達するための、請求項１~６のいずれか１項に記載の細胞担持パターン化ナノ薄膜。 The cell-supported patterned nano thin film according to any one of claims 1 to 6, which is released into a tissue from a capillary tube and delivers cells to the tissue. 請求項１~７のいずれか１項に記載の細胞担持パターン化ナノ薄膜を細管より生体組織中に放出することを含む、生体組織への細胞移植方法。 A method for transplanting cells into a living tissue, comprising releasing the cell-supported patterned nano thin film according to any one of claims 1 to 7 from a capillary tube into the living tissue.
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