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WO2019078251A1 - Adhésif et son utilisation - Google Patents

Adhésif et son utilisation Download PDF

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Publication number
WO2019078251A1
WO2019078251A1 PCT/JP2018/038656 JP2018038656W WO2019078251A1 WO 2019078251 A1 WO2019078251 A1 WO 2019078251A1 JP 2018038656 W JP2018038656 W JP 2018038656W WO 2019078251 A1 WO2019078251 A1 WO 2019078251A1
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Prior art keywords
cells
cell
hydrogel
adhesive
embedded
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English (en)
Japanese (ja)
Inventor
昌治 竹内
悠加 ▲吉▼江
晃世 横溝
ミンハオ ニエ
雄矢 森本
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University of Tokyo NUC
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University of Tokyo NUC
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09J—ADHESIVES; NON-MECHANICAL ASPECTS OF ADHESIVE PROCESSES IN GENERAL; ADHESIVE PROCESSES NOT PROVIDED FOR ELSEWHERE; USE OF MATERIALS AS ADHESIVES
    • C09J11/00—Features of adhesives not provided for in group C09J9/00, e.g. additives
    • C09J11/02—Non-macromolecular additives
    • C09J11/06—Non-macromolecular additives organic
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09J—ADHESIVES; NON-MECHANICAL ASPECTS OF ADHESIVE PROCESSES IN GENERAL; ADHESIVE PROCESSES NOT PROVIDED FOR ELSEWHERE; USE OF MATERIALS AS ADHESIVES
    • C09J201/00—Adhesives based on unspecified macromolecular compounds
    • 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 adhesives and their use. Specifically, the present invention relates to an adhesive, a cell structure containing the adhesive, and a method of producing a cell structure using the adhesive.
  • Priority is claimed on Japanese Patent Application No. 2017-202468, filed Oct. 19, 2017, the content of which is incorporated herein by reference.
  • Alginic acid hydrogel is obtained by gelling alginic acid in the presence of divalent ions such as calcium ions and barium ions, and is used for microcapsule-immobilized culture in which cells are immobilized and cultured.
  • Patent Document 1 discloses a method of producing a blood vessel tissue using cells and an alginate hydrogel.
  • the alginic acid hydrogel can be adhered to each other by modifying the alginic acid hydrogel with an external stimulus such as light or heat.
  • an external stimulus such as light or heat.
  • this method can not be applied to alginate hydrogels embedded with biomolecules or cells that are susceptible to external stimuli.
  • the alginate hydrogel is negatively charged in the aqueous solvent. Therefore, a method is also conceivable in which a polymer having a positive charge such as chitosan is adsorbed on the surface of alginic acid hydrogel to bond alginic acid hydrogels to each other. However, this method is not preferable because a polymer having a positive charge such as chitosan exhibits toxicity to cells embedded in alginate hydrogel.
  • the present invention has been made in view of the above circumstances, and provides an adhesive capable of adhering an anionic water-soluble substance easily and quickly without external stimulation.
  • the present invention also provides a cell structure three-dimensionally formed using the adhesive and a method of manufacturing the same.
  • the adhesive according to the first aspect of the present invention comprises nanoparticles, the surface of which is coated with a cationic water-soluble polymer.
  • the cationic water-soluble polymer may be polyethyleneimine or a salt thereof.
  • the adhesive according to the first aspect may be used for adhesion of alginic acid hydrogel or cell tissue.
  • At least one selected from the group consisting of extracellular matrix and cells may be contained inside the alginate hydrogel.
  • the cell structure according to the second aspect of the present invention includes an alginate hydrogel in which cells are embedded, and the adhesive according to the first aspect.
  • the method for producing a cell structure according to the third aspect of the present invention comprises a cell embedding step of embedding cells in an alginate hydrogel, and the alginic acid hydrogel in which a plurality of the cells are embedded in the first aspect. Bonding using such an adhesive.
  • the anionic water-soluble substance can be adhered easily and quickly without external irritation.
  • cells can be cultured three-dimensionally.
  • a three-dimensionally formed cell structure can be easily obtained.
  • FIG. 7 is a scanning electron microscopic image of cationic nanoparticles (CNP) in Production Example 1.
  • the scale bar is 50 nm.
  • 5 is a graph showing the particle size distribution of CNP in Production Example 1.
  • FIG. 2 is a schematic view of an apparatus used for a tensile test in Test Example 1; The scale bar is 1 mm. It is a graph which shows the result of the tension tension test in example 1 of an examination. The graph on the left is the result of a tensile test of one plate-like alginate hydrogel.
  • the graph on the right shows the results of a tensile test of two plate-like alginate hydrogels adhered using CNP. It is an image which shows the result of the cell survival test in Experiment 2.
  • the upper image is an image showing the result of HeLa cells containing CNP solution.
  • the lower image is an image showing the results of HeLa cells containing a polyethyleneimine solution.
  • the scale bars are each 200 ⁇ m.
  • 7 is a graph showing cell viability in each condition in Test Example 2. It is an image which shows the fibrous alginic acid hydrogel which embedded the fluorescent substance of the bundle shape which adhered in CNP in Experiment 3.
  • FIG. The scale bars are each 200 ⁇ m.
  • FIG. 15 is a schematic view showing a 3D printer in Test Example 5.
  • FIG. 16 is a schematic view showing the production steps of the cell structure in Test Example 5. It is an image which shows a time-dependent change of the cell structure cultured on each condition in Experiment 5.
  • the scale bar is 250 ⁇ m.
  • the adhesive according to one embodiment of the present invention comprises nanoparticles surface-coated with a cationic water soluble polymer.
  • the anionic water-soluble substance can be adhered easily and quickly without external irritation.
  • the adhesive of the present embodiment includes nanoparticles (hereinafter, may be referred to as "CNP") whose surfaces are coated with a cationic water-soluble polymer, and electrostatics are generated between the CNP and the anionic water-soluble substance. It is possible to bond by the generation of a chemical interaction.
  • CNP nanoparticles
  • FIG. 1 is a schematic configuration view showing an example of nanoparticles (CNP) whose surface is coated with a cationic water-soluble polymer, which is included in the adhesive of the present embodiment.
  • CNP (10) shown in FIG. 1 is composed of a cationic water-soluble polymer 1 that covers the surface and a core 2.
  • the average particle diameter of CNP (10) is preferably 1 nm or more and 100 nm or less, more preferably 5 nm or more and 70 nm or less, and still more preferably 20 nm or more and 50 nm or less.
  • an anionic water-soluble substance of micro order can be more effectively adhered.
  • visible light can be transmitted, and the location of the adhesive can be made transparent.
  • the charge on the surface of CNP (10) may be, for example, about 10 mV or more and 50 mV or less.
  • the cationic water-soluble polymer 1 may be a polymer having a cationic functional group.
  • the cationic functional group include, but are not limited to, primary to quaternary amino groups and guanidine groups.
  • the cationic water-soluble polymer 1 is a polymer obtained by polymerizing the monomer (cationic monomer) which has the said cationic functional group.
  • cationic monomers examples include vinylamine, allylamine, ethyleneimine, 3- (N, N-dimethylaminopropyl)-(meth) acrylamide, 3- (N, N-dimethylaminopropyl)-(meth) acrylate, amino Styrene, 2- (N, N-dimethylaminoethyl)-(meth) acrylamide, 2- (N, N-dimethylaminoethyl)-(meth) acrylate and salts thereof, and diallyldialkylammonium halides and the like It can be mentioned. These cationic monomers may be used alone or in combination of two or more.
  • the cationic water-soluble polymer 1 may copolymerize the said cationic monomer and another monomer.
  • the other monomer may be a hydrophilic monomer, or may be a hydrophobic monomer depending on the blending ratio.
  • the hydrophilic monomer may be neutral one in an aqueous solvent, and examples thereof include dimethyl acrylamide, acrylic acid and methacrylic acid having polyethylene glycol side chains, and the like. These may be used alone or in combination of two or more.
  • hydrophobic monomer examples include those shown in (i) to (v) below. These may be used alone or in combination of two or more.
  • acrylic esters such as methyl acrylate, ethyl acrylate, isopropyl acrylate, n-butyl acrylate, 2-ethylhexyl acrylate;
  • Methacrylates such as methyl methacrylate, ethyl methacrylate, isopropyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, n-hexyl methacrylate, cyclohexyl methacrylate, lauryl methacrylate, glycidyl methacrylate and the like ;
  • aromatic olefins such as styrene and ⁇ -methylstyrene;
  • vinyl esters such as vinyl acetate;
  • Vinyl nitriles such as acrylonitrile
  • the cationic water-soluble polymer 1 is preferably polyethylene imine obtained by polymerizing ethylene imine or a salt thereof.
  • Polyethyleneimine is a polymer obtained by ring-opening polymerization of ethyleneimine by a known method.
  • the salt of polyethylenimine is one in which a part or all of the amino groups in polyethylenimine are neutralized with acid.
  • the acid used for neutralization may be an inorganic acid or an organic acid.
  • an inorganic acid hydrochloric acid, a sulfuric acid, phosphoric acid, nitric acid etc. are mentioned, for example.
  • Examples of the organic acid include acetic acid, formic acid, propionic acid and the like.
  • the hydrophobic polymer As a material which comprises the core 2, it is preferable that it is a hydrophobic polymer.
  • a hydrophobic polymer By being a hydrophobic polymer, an emulsion containing spherical CNP can be easily produced by the production method described later.
  • the hydrophobic polymer is a polymer obtained by polymerizing a hydrophobic monomer.
  • the hydrophobic monomer may be one having a solubility in water at 25 ° C. of 10 g / dL or less, and specifically, the same as those exemplified for the other monomers described above can be mentioned.
  • the hydrophobic polymer may be a polymer obtained by using the above-mentioned hydrophobic monomer in combination with a crosslinkable monomer.
  • crosslinkable monomer examples include ethylene glycol di (meth) acrylate, hexanediol di (meth) acrylate, divinyl benzene, methylene bis acrylamide, trimethylolpropane tri (meth) acrylate, tetraallyl ethane and the like. These may be used alone or in combination of two or more.
  • the hydrophobic polymer is preferably polystyrene obtained by polymerizing styrene.
  • Method of producing CNP] CNP contained in the adhesive of the present embodiment can be obtained by emulsion polymerization of the cationic monomer and the hydrophobic monomer in a water solvent in the presence of a radical polymerization initiator.
  • the blending amount of the cationic monomer with respect to the mass of the hydrophobic monomer is preferably 0.5% by mass or more and 30% by mass or less, and more preferably 0.5% by mass or more and 15% by mass or less
  • the content is 0.5% by mass or more and 5% by mass or less.
  • radical polymerization initiator examples include those shown in the following (i) to (v). These may be used alone or in combination of two or more.
  • oil-soluble azo compounds such as 2,2'-azobisisobutyronitrile, 2,2'-azobis (2,4-dimethylvaleronitrile);
  • organic peroxides such as benzoyloxy peroxide, ditertiary butyl peroxide and the like;
  • inorganic peroxides such as potassium persulfate, sodium persulfate
  • the radical polymerization initiator is preferably one shown in the above (ii), and 2,2'-azobis (2-amidinopropane) hydrochloride, 2,2'-azobis [2- (2-imidazoline) -2-yl) propane] or a hydrochloride thereof, or 2,2'-azobis [2-methyl-N- (2-hydroxyethyl) propionamide] is preferable.
  • the blending amount of the radical polymerization initiator based on the mass of the hydrophobic monomer can be, for example, 0.001% by mass or more and 2% by mass or less.
  • any solvent containing water as a main component may be used, and examples thereof include distilled water, ion exchanged water, tap water, industrial water and the like.
  • soap free emulsion polymerization the method which does not use the emulsifier of low molecular weight called soap free emulsion polymerization as emulsion polymerization.
  • the polymer forms fine particles in an aqueous solvent by balancing the hydrophilicity and hydrophobicity of the cationic water-soluble polymer and the hydrophobic polymer, CNP can be easily obtained.
  • the total blending amount of the cationic water-soluble polymer and the hydrophobic polymer with respect to the mass of the whole polymerization system is usually 1% by mass to 70% by mass, and 10% by mass to 60% by mass Preferably, the content is 20% by mass or more and 60% by mass or less.
  • the polymerization temperature and time are selected depending on the polymerizability of the monomer, the decomposition temperature and the half life of the initiator, and the like.
  • the polymerization temperature can be usually 30 ° C. or more and 130 ° C. or less, and preferably 50 ° C. or more and 100 ° C. or less.
  • the polymerization time can be usually from 1 hour to 10 hours.
  • the adhesive of the present embodiment may be in powder form or in liquid form.
  • the adhesive agent of this embodiment may contain the other component in the extent which does not impair the cationic property of CNP other than CNP.
  • examples of other components include stabilizers, thickeners, preservatives and the like.
  • the adhesive of the present embodiment may contain, for example, an aqueous solvent.
  • the aqueous solvent is not particularly limited, and examples thereof include water, physiological saline, physiological saline having a buffer effect, and the like.
  • the buffered saline include phosphate buffered saline (PBS), tris buffered saline (TBS), HEPES buffered saline and the like.
  • a water-soluble organic solvent may be contained.
  • the water-soluble organic solvent include lower alcohols, acetone, dioxane, ethylene glycol and the like.
  • the lower alcohol may be a monohydric alcohol having 1 to 3 carbon atoms, and specific examples thereof include methanol, ethanol and propanol.
  • the adhesive of the present embodiment can be obtained by mixing the above-mentioned CNP and, if necessary, other components.
  • the adhesive of this embodiment is suitably used for adhesion of an anionic water-soluble substance.
  • anionic water-soluble substance examples include heparin, hyaluronic acid, dextran sulfate, polystyrene sulfonic acid, polyacrylic acid, polymethacrylic acid, polyphosphoric acid, sulfated polysaccharides, curdlan, polyalginic acid (alginic acid hydrogel), And alkali metal salts thereof and the like.
  • Other anionic water-soluble substances also include, for example, nucleic acids, cells, and peptides and proteins negatively charged in aqueous solvents.
  • polyalginic acid (alginic acid hydrogel) is preferable as the anionic water-soluble substance to be adhered to the adhesive of the present embodiment.
  • Alginic acid hydrogel is biocompatible and has been applied to microencapsulation of cells and the like.
  • the alginate hydrogel can be shaped freely, such as in the form of particles, sheets (plates), tubes (fibers), and the like. Therefore, the adhesive of the present embodiment can be used to adhere alginic acid hydrogel in which cells are embedded, to construct a three-dimensional structure having a desired shape.
  • alginic acid hydrogel means the insoluble gel obtained by forming a salt with alginic acid and bivalent metal ion (a calcium ion, barium ion, etc.).
  • the alginate hydrogel to which the adhesive of the present embodiment is to be adhered may be a single component thereof, or may contain other components such as an extracellular matrix, cells, etc. which will be described later.
  • the adhesive of this embodiment does not show cytotoxicity, as shown in the below-mentioned Example. Therefore, it can also be used to directly adhere cell tissues.
  • cell tissue refers to a collection of cells and extracellular matrix.
  • CNP contained in the adhesive of the present embodiment has a nano-order average particle diameter, for example, to adhere various parts constituting a micro robot, a micro device, an artificial muscle (soft actuator), etc. Are preferably used.
  • a cell structure according to an embodiment of the present invention includes an alginic acid hydrogel in which cells are embedded, and the above-mentioned adhesive.
  • the cell structure of the present embodiment is formed three-dimensionally, cells can be cultured three-dimensionally, and various tissue-like structures or organ-like structures can be obtained.
  • tissue refers to a unit of structure assembled in a pattern based on a certain lineage in which one type of stem cell differentiates, and has one role as a whole.
  • the cell structure of the present embodiment can reproduce, for example, epithelial tissue, connective tissue, muscle tissue, nerve tissue and the like.
  • the term "organ” refers to an organ composed of two or more types of tissues, and is responsible for one function as a whole.
  • the cell structure of the present embodiment can reproduce, for example, the stomach, intestine, liver, kidney and the like.
  • FIG. 2 is a schematic view showing an example of the cell structure of the present embodiment.
  • an alginic acid hydrogel 20 a in which a first cell is embedded and an alginic acid hydrogel 20 b in which a second cell is embedded are adhered via an adhesive 10.
  • the alginate hydrogel 20 a in which the first cell is embedded and the alginate hydrogel 20 b in which the second cell is embedded respectively include the cell 3 and the alginate hydrogel 4.
  • the first cell and the second cell may be identical or different.
  • FIG. 2 as the cell structure of the present embodiment, one in which two alginate hydrogels in which cells are embedded is adhered is illustrated, but the invention is not limited thereto. That is, for example, three or more alginic acid hydrogels in which cells are embedded may be adhered.
  • the shape of the alginate hydrogel by which the cell was embedded illustrated what is a sheet-like (plate-like), it is not limited to this. That is, for example, the shape of alginic acid hydrogel in which cells are embedded may be tube-like (fiber-like), particulate or the like. Moreover, the thing of the same shape may be adhere
  • ⁇ Cell> There is no particular limitation on the cells contained in the cell structure of the present embodiment, and for example, germ cells (sperm, egg, etc.), somatic cells constituting a living body, stem cells, progenitor cells, cancer separated from living body Cells, cells (cell lines) isolated from living organisms that acquire immortalization ability and stably maintained in vitro, artificially genetically modified cells isolated from living organisms, artificially exchanged nuclei from living organisms Cells, and the like, without being limited thereto. Alternatively, cell clusters (spheroids) of these cells may be used. Alternatively, a piece of tissue separated from normal tissue or cancer tissue of a living body may be used as it is.
  • somatic cells constituting a living body include skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, Examples include, but are not limited to, cells collected from any tissue such as bone, cartilage, blood vessel tissue, blood, heart, eye, brain, nerve tissue and the like.
  • somatic cells more specifically, for example, fibroblasts, bone marrow cells, immune cells (eg, B lymphocytes, T lymphocytes, neutrophils, macrophages, monocytes, etc.), erythrocytes, platelets, bone cells, Bone marrow cells, pericytes, dendritic cells, epidermal keratinocytes (keratinocytes), adipocytes, mesenchymal cells, epithelial cells, endothelial cells, vascular endothelial cells, lymphatic endothelial cells, hepatocytes, islet cells (for example, ⁇ Cells, ⁇ cells, ⁇ cells, ⁇ cells, PP cells etc., chondrocytes, cumulus cells, glial cells, neurons (neurons), oligodendrocytes, microglia, astrocytes, cardiomyocytes, esophageal cells, A muscle cell (for example, smooth muscle cell, skeletal muscle cell etc.), a melan
  • Stem cells are cells that combine the ability to replicate themselves with the ability to differentiate into cells of multiple other lineages.
  • Examples of stem cells include embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells Muscle stem cells, germ stem cells, enteric stem cells, cancer stem cells, hair follicle stem cells and the like, but the present invention is not limited thereto.
  • Progenitor cells are cells in the process of differentiating from the stem cells to specific somatic cells or germ cells.
  • Cancer cells are cells derived from somatic cells and having infinite proliferation ability, and are malignant neoplasms that infiltrate surrounding tissues or cause metastasis.
  • cancers from which cancer cells are derived include, for example, breast cancer (eg, invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast cancer, etc.), prostate cancer (eg, hormone-dependent prostate cancer, hormone independent) Prostate cancer, etc.), pancreatic cancer (eg, pancreatic duct cancer etc.), gastric cancer (eg, papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous cell carcinoma etc.), lung cancer (eg, non-small cell lung cancer, small cell lung cancer, malignant mesothelium) Tumors), colon cancer (eg, gastrointestinal stromal tumor), rectal cancer (eg, gastrointestinal stromal tumor), colon cancer (eg, familial colorectal cancer, hereditary non-polyposis colorectal
  • a cell line is a cell which has acquired infinite proliferation ability by artificial manipulation in vitro.
  • Examples of cell lines include HCT116, Huh7, HEK293 (human fetal kidney cells), HeLa (human cervical cancer cell line), HepG2 (human hepatoma cell line), UT7 / TPO (human leukemia cell line), CHO (Chinese hamster ovary cell line), MDCK, MDBK, BHK, C-33A, HT-29, AE-1, 3D9, Ns0 / 1, Jurkat, NIH3T3, PC12, S2, S2, Sf9, Sf21, High Five, Vero, etc. Is not limited to these.
  • the animal from which the cells are derived may be a vertebrate or invertebrate.
  • the vertebrates are not particularly limited and include, for example, mammals, amphibians, reptiles, amphibians, fish and the like.
  • the invertebrate is not particularly limited, and examples thereof include insects, crustaceans, molluscs, protozoa and the like.
  • the animal from which cells are derived is preferably a vertebrate, and more preferably a mammal.
  • mammals include, but are not limited to, humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, rats and the like.
  • a mammal is preferably a human.
  • the type of cells contained in the cell structure of the present embodiment may be one type, or two or more types.
  • the cell structure of the present embodiment may further contain other components in addition to the cell-embedded alginic acid hydrogel and the adhesive.
  • Examples of other components include media, physiologically active substances, extracellular matrices, and the like.
  • the medium may be a basic culture solution containing components (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, etc.) necessary for the survival and growth of cells, and can be appropriately selected according to the type of cells. . Specifically, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), RPMI-1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle 'as a medium. s Medium: Nutrient Mixture F-12 (DMEM / F-12), Glasgow Minimum Essential Medium (Glasgow MEM), etc., and the like, but it is not limited thereto.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimum Essential Medium
  • BME Basal Medium Eagle
  • s Medium Nutrient Mixture F-12 (DMEM / F-12), Glasgow Minimum Essential Medium (Glasgow MEM), etc., and the like, but it is not limited thereto.
  • physiologically active substances include, but are not limited to, cell growth factors, differentiation inducers, cell adhesion factors and the like.
  • the cells included in the cell structure of the present embodiment are stem cells or progenitor cells, a cell structure that reproduces a desired tissue by inducing differentiation of the stem cells or the progenitor cells by including a differentiation inducing factor. You can build the body.
  • extracellular matrix for example, it is reconstituted from collagen (type I, type II, type III, type V, type XI, etc.), mouse EHS tumor extract (including type IV collagen, laminin, heparan sulfate proteoglycan etc.) Base membrane components (trade name: Matrigel), glycosaminoglycans, hyaluronic acid, proteoglycans, gelatin and the like, without being limited thereto.
  • the method for producing a cell structure according to an embodiment of the present invention is a method comprising a cell embedding step and an adhesion step.
  • the cell embedding step is a step of embedding cells in an alginate hydrogel.
  • a solution of alginic acid or a salt thereof, a solution containing a divalent metal salt, and a suspension of cells may be mixed.
  • the concentration of alginic acid or a salt thereof in a solution of alginic acid or a salt thereof can be, for example, 0.5% by mass or more and 3% by mass or less, and can be, for example, 1% by mass or more and 2% by mass or less.
  • the divalent metal salt examples thereof include barium salt, calcium salt, magnesium salt and the like.
  • barium salt barium chloride, barium fluoride, barium bromide, barium hydroxide, barium carbonate, barium phosphate etc. are mentioned, for example.
  • calcium salt calcium chloride, calcium fluoride, calcium bromide, calcium peroxide, calcium hydroxide, calcium carbonate, calcium nitrate, calcium sulfate, calcium phosphate etc. are mentioned, for example.
  • magnesium salt magnesium chloride, magnesium fluoride, magnesium bromide, magnesium peroxide, magnesium hydroxide, magnesium hydroxide, magnesium carbonate, magnesium nitrate, magnesium sulfate, magnesium phosphate etc. are mentioned, for example, It is not limited to these.
  • the concentration of the divalent metal salt in the solution containing the divalent metal salt can be, for example, 50 mM or more and 200 mM or less, and can be, for example, 75 mM or more and 150 mM or less.
  • the cell suspension may be suspended in an aqueous solvent or medium.
  • aqueous solvent and the medium include the same as those exemplified for the adhesive and the cell structure.
  • the cell suspension may further contain an extracellular matrix, a physiologically active substance, and the like.
  • extracellular matrix and the physiologically active substance the same ones as exemplified in the above-mentioned cell structure can be mentioned.
  • the type of cells used may be used singly or in combination of two or more.
  • the shape of the alginate hydrogel in which the cells obtained in the cell embedding step are embedded is not particularly limited, and examples thereof include sheet (plate), tube (fiber), and particles.
  • the adhesion step is a step of adhering a plurality of cell-embedded alginate hydrogels using the above-mentioned adhesive.
  • the number of alginic acid hydrogels in which cells are embedded may be two or may be three or more. Moreover, the alginate hydrogel in which cells are embedded may be adhered to one another, which has the same shape, or two or more different shapes may be adhered.
  • the adhesive is applied to the surface to which the cell-embedded alginate hydrogel is to be attached. Then, the alginic acid hydrogels in which the cells are embedded are superposed and allowed to adhere simply by leaving them to stand at room temperature for about 10 minutes to 2 hours.
  • a method of applying the adhesive for example, a method of dropping the adhesive onto the surface to be adhered of the alginate hydrogel in which the cells are embedded using a micropipette or the like can be mentioned.
  • a method of immersing alginate hydrogel in which cells are embedded in a solution containing an adhesive for example, a method of immersing alginate hydrogel in which cells are embedded in a solution containing an adhesive.
  • steps may be provided in addition to the cell embedding step and the adhesion step.
  • Other steps include a washing step and a removing step.
  • the washing step may be performed after the cell embedding step and before the adhesion step or after the cell embedding step and the adhesion step.
  • the removal step may be performed after the cell embedding step and the adhesion step.
  • the washing step may be performed to wash the alginate hydrogel in which the cells obtained in the cell embedding step are embedded. Or you may carry out in order to wash the cell structure obtained in the said adhesion process.
  • the alginic acid hydrogel or cell structure in which the cells are embedded can be washed once or more (for example, twice or more and about three times or less) using an aqueous solvent or a medium.
  • aqueous solvent or medium used in the washing step include the same as those exemplified for the adhesive and the cell structure.
  • the removing step is a step for removing the alginate hydrogel contained in the cell structure. This makes it possible to obtain a cell structure in which the cells are directly adhered via the adhesive.
  • a solution containing a solubilizing agent for alginic acid hydrogel is added to the cell structure obtained in the above-mentioned adhesion step.
  • a method to add the method of dripping the solution containing the solubilizer of alginic acid hydrogel in a cell structure etc. is mentioned, for example.
  • a method of immersing the cell structure in a solution containing a solubilizing agent of alginic acid hydrogel can be mentioned.
  • solubilizing agent for alginic acid hydrogel any agent may be used as long as it removes divalent metal ions that have formed a salt with alginic acid or decomposes alginic acid itself.
  • Specific examples of the solubilizer for alginic acid hydrogel include, but are not limited to, chelating agents and enzymes.
  • a chelating agent for example, citric acid, ethylenediamine (Ethylenediamine), ethylenediaminetetraacetic acid (Ethylene Diamine Tetraacetic Acid; EDTA), Nitrilo Triacetic Acid (NTA), Diethylenetriamine Pentaacetic Acid (DTPA), N- (2-hydroxyethyl) ethylenediamine-N, N ', N'-triacetic acid (Hydroxyethyl Ethyleamine Diamine Triacetic Acid; HEDTA), glycol ether diamine tetraacetic acid (Glycol Ether Diamine Tetraacetic Acid; EDTA, EGTA), triethylenetetramine-N, N, N ', N' ', N' ', N' ', N' '-'- hexaacetic acid (TTHA), N- (2-hydroxyethyl) imino Diacetic acid (Hydroxyethyl Imino Diagnostic Acid; H
  • Examples of the enzyme include alginate lyase and the like.
  • the citric acid may also be in the form of a citrate such as sodium citrate or potassium citrate.
  • the concentration of citric acid in the solution containing the solubilizing agent of alginic acid hydrogel can be, for example, 0.1 mM or more and 100 mM or less.
  • the concentration of EDTA in the solution containing the solubilizing agent of alginic acid hydrogel can be, for example, 0.5 mM or more and 100 mM or less.
  • the concentration of alginate lyase in the solution containing the alginate hydrogel solubilizer may be, for example, 0.04 mg / mL or more and 400 mg / mL or less it can.
  • the solution containing the solubilizer of alginic acid hydrogel may contain, for example, an oligosaccharide, a polysaccharide thickener, etc., in order to increase the specific gravity.
  • oligosaccharides include disaccharides such as sucrose, lactose and maltose; fructooligosaccharides, galactooligosaccharides, mannan oligosaccharides and the like.
  • polysaccharide thickeners examples include pectin, guar gum, xanthan gum, carrageenan and the like.
  • the invention is an adhesive
  • the adhesive comprises cationic nanoparticles
  • Examples of the cationic nanoparticles include adhesives whose core is polystyrene and whose surface is coated with polyethyleneimine or a salt thereof, and the like.
  • the invention is an adhesive
  • the adhesive comprises cationic nanoparticles
  • the cationic nanoparticle has a central portion made of polystyrene, and the surface is coated with polyethyleneimine or a salt thereof,
  • the adhesive etc. whose average particle diameter of the said cationic nanoparticle is 1 nm-100 nm (preferably, 20 nm-50 nm) are mentioned.
  • the invention is the use of an alginate hydrogel or an adhesive for adhering cellular tissue
  • the adhesive comprises cationic nanoparticles,
  • the cationic nanoparticles have a central portion made of polystyrene and a surface coated with polyethyleneimine or a salt thereof.
  • Alginic acid hydrogel or the use of the above adhesive for adhering cellular tissue, etc. may be mentioned.
  • the invention is the use of an alginate hydrogel or an adhesive for adhering cellular tissue
  • the adhesive comprises cationic nanoparticles
  • the cationic nanoparticle has a central portion made of polystyrene, and the surface is coated with polyethyleneimine or a salt thereof,
  • the average particle diameter of the cationic nanoparticles is 1 nm or more and 100 nm or less (preferably, 20 nm or more and 50 nm or less).
  • Alginic acid hydrogel or the use of the above adhesive for adhering cellular tissue, etc. may be mentioned.
  • the invention is a cell structure
  • the cell structure is Two or more alginic acid hydrogels in which cells (for example, human skin fibroblasts etc.) are embedded;
  • An adhesive comprising cationic nanoparticles, Including
  • the cationic nanoparticle has a central portion made of polystyrene, and the surface is coated with polyethyleneimine or a salt thereof,
  • attach via the said adhesive agent are mentioned.
  • the invention is a cell structure
  • the cell structure is Two or more alginic acid hydrogels in which cells (for example, human skin fibroblasts etc.) are embedded;
  • An adhesive comprising cationic nanoparticles, Including
  • the cationic nanoparticle has a central portion made of polystyrene, and the surface is coated with polyethyleneimine or a salt thereof,
  • the average particle diameter of the cationic nanoparticles is 1 nm or more and 100 nm or less (preferably, 20 nm or more and 50 nm or less),
  • attach via the said adhesive agent are mentioned.
  • the present invention relates to a method of producing a cell structure, A solution containing cells of 1.0 ⁇ 10 5 cells / mL or more and 1.0 ⁇ 10 10 cells / mL or less (eg, human skin fibroblasts etc.) and extracellular matrix, and 0.5% by mass or more and 3% by mass A mixed solution obtained by mixing a sodium alginate solution of 5% or less and a solution containing 50 mM or more and 200 mM or less of a divalent metal salt (preferably calcium chloride) using a 3D printer to obtain a desired mixed solution
  • the present invention relates to a method of producing a cell structure, A solution containing cells of 1.0 ⁇ 10 5 cells / mL or more and 1.0 ⁇ 10 10 cells / mL or less (eg, human skin fibroblasts etc.) and extracellular matrix, and 0.5% by mass or more and 3% by mass A mixed solution obtained by mixing a sodium alginate solution of 5% or less and a solution containing 50 mM or more and 200 mM or less of a divalent metal salt (preferably calcium chloride) using a 3D printer to obtain a desired mixed solution
  • Production Example 1 Production of Adhesive 160 g of water, polyethylene imine (manufactured by MERCK, molecular weight: 600,000 to 1,000,000, 50 v / w% aqueous solution) 300 ⁇ L in a nitrogen atmosphere in a reactor capable of heating, cooling and stirring And 8.5 g of styrene were charged and stirred. Next, 25 ⁇ g of ammonium peroxodisulfate (manufactured by Kanto Chemical Co., Ltd.) was added as a polymerization initiator, and a polymerization reaction was carried out while stirring at 70 ° C. for 2 hours in a nitrogen atmosphere. With the polymerization, the polymerization system gradually became cloudy and became an emulsified state.
  • ammonium peroxodisulfate manufactured by Kanto Chemical Co., Ltd.
  • CNP cationic nanoparticles
  • the surface potential of CNP was measured using a zeta potential meter (manufactured by Malvern, ZetasizernanoZSP). As a result, the surface potential of CNP was 35.7 mV.
  • FIG. 3B the result of having measured the particle diameter of CNP using the particle diameter distribution measuring apparatus (Malvern company make, ZetasizernanoZSP) is shown to FIG. 3B. From FIG. 3B, the average particle size of CNP was about 20 nm.
  • Test Example 1 Tensile Test Preparation of plate-like alginic acid hydrogel A 3.0 mass% aqueous solution of sodium alginate, a 0.2 mass% aqueous solution of calcium carbonate and a 2-fold molar amount of gluconolactone with respect to the calcium carbonate are mixed to obtain a plate. Two alginate gel hydrogels (20 mm long ⁇ 5 mm wide ⁇ 1 mm thick) were prepared. One of them was cut, and the CNP obtained in Production Example 1 was applied and adhered.
  • FIG. 4A Tension Test As shown in FIG. 4A, one sheet of alginic acid hydrogel (single plate) obtained in “1.” and CNP bonded alginic acid hydrogel (connected plate) are sandwiched by anchors and clamps at the top and the bottom, respectively. A tensile test was performed using a tester (Instron TE200N). The respective pulling behavior is shown in FIG. 4B. In FIG. 4B, the left graph shows the tensile behavior of an alginate hydrogel (single plate) and the right graph shows the tensile behavior of a CNP-bonded alginate hydrogel (connected plate).
  • the medium was replaced with a medium containing 10 mg / mL of CNP, or a medium containing 10 mg / mL of polyethyleneimine (PEI) as a control, and cultured at 37 ° C. for 1 minute.
  • PHI polyethyleneimine
  • cells were stained using Calcein-AM (staining live cells) and Ethmdium homodimer (staining dead cells) to calculate cell viability.
  • the stained image of the cells is shown in FIG. 5A (upper: CNP-containing medium, lower: PEI-containing medium).
  • the scale bar is 200 ⁇ m.
  • each cell survival rate is shown to FIG. 5B.
  • “None” indicates cells cultured in a simple medium without adding CNP and PEI.
  • Test Example 3 Bundling and Slicing of Fibrous Alginate Hydrogel 1. Preparation of filamentous alginate hydrogel embedded with fluorescent substance Reference 1 (H. Onoe et al., "Metre-long cell-laden microfibrils exitbit tissue morphology and functions", Nature Materials, Vol. 12, p584-590, A fibrous alginate hydrogel in which a fluorescent material was embedded was prepared using the method described in 2013.). In addition, as a fluorescent substance, the following were each embedded.
  • Red FluoSpheres, carboxylate-modified 0.2 ⁇ m, red (excitation wavelength: 580 nm, fluorescence wavelength: 605 nm)
  • Green FluoSpheres, carboxylate-modified 0.2 ⁇ m, yellow-green (excitation wavelength: 505 nm, fluorescence wavelength: 515 nm)
  • Blue FluoSpheres, carboxylate-modified 0.2 ⁇ m, blue Fluorescent (excitation wavelength: 365 nm, fluorescence wavelength: 415 nm)
  • FIG. 6A An image taken with a fluorescent microscope (IX71 manufactured by Olympus Corporation) of a fibrous alginate hydrogel in which the bundled fluorescent material is embedded is shown in FIG. 6A.
  • the upper left is a bundle of fibrous alginic acid hydrogel in which fluorescent materials of respective colors are embedded.
  • the lower left is a bundle of fibrous alginic acid hydrogel embedded with a blue fluorescent substance.
  • the upper right is a bundle of fibrous alginic acid hydrogel embedded with a green fluorescent substance.
  • the lower right is a bundle of a fibrous alginic acid hydrogel embedded with a red fluorescent substance.
  • the scale bar is 200 ⁇ m.
  • “with shell” refers to a fibrous alginate hydrogel in which a fluorescent substance is embedded, which is further covered with an alginate hydrogel in which the fluorescent substance is not embedded, and which has a layer separation structure.
  • the right image is an enlarged image of the left image.
  • the scale bar of the image on the left is 200 ⁇ m.
  • the scale bars in the right image are each 100 ⁇ m.
  • Test Example 4 Bundling and Slicing of Filamentous Alginate Hydrogel Embedding Cells Preparation of fibrous alginate hydrogel in which cells are embedded Using the method described in the above-mentioned reference 1, a fibrous alginate hydrogel in which cells are embedded was prepared. In addition, as a cell, the suspension of the human skin fibroblast of 6.0 * 10 ⁇ 7 > cellscells / mL was used. In addition, cells were previously stained using Calcein-AM (staining live cells), Ethmdium homodimer (staining dead cells) and Hoechst (staining nuclei).
  • a three-dimensional structure of CNP-adhered cell-embedded fibrous alginic acid hydrogel obtained in "2.” is placed in a 10 cm dish containing a medium, and a shaking stirrer (NISSIN Co., Ltd.) , NA-301), and incubated for 5 days at 37 ° C. in a 5% CO 2 environment.
  • a three-dimensional structure of CNP-attached cell-embedded fibrous alginic acid hydrogel was placed in a 10 cm dish containing a culture medium and statically cultured at 37 ° C. in a 5% CO 2 environment for 5 days .
  • the time course of each cell structure is shown in FIG. 8C.
  • the anionic water-soluble substance can be adhered easily and quickly without external irritation.
  • cells can be cultured three-dimensionally.
  • a three-dimensionally formed cell structure can be easily obtained.
  • SYMBOLS 1 cationic water-soluble polymer, 2 ... core, 3 ... cell, 4 ... alginic acid hydrogel, 10 ... cationic nanoparticle, 20a ... alginic acid hydrogel in which the 1st cell was embedded, 20b ... 2nd cell Embedded alginate hydrogel, 100 ... cell structure

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Abstract

L'adhésif selon la présente invention comprend des nanoparticules dont les surfaces sont revêtues d'un polymère cationique soluble dans l'eau. Une structure cellulaire de la présente invention comprend un hydrogel d'acide alginique dans lequel une cellule est incorporée, et l'adhésif susmentionné. Un procédé de fabrication d'une structure cellulaire selon la présente invention comprend une étape d'incorporation de cellule pour incorporer une cellule dans un hydrogel d'acide alginique, et une étape de liaison pour lier l'hydrogel d'acide alginique dans lequel une pluralité de cellules sont incorporées, à l'aide de l'adhésif.
PCT/JP2018/038656 2017-10-19 2018-10-17 Adhésif et son utilisation Ceased WO2019078251A1 (fr)

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WO2022270549A1 (fr) 2021-06-23 2022-12-29 持田製薬株式会社 Nouvelle fibre de gel d'alginate réticulée revêtue de polymère

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