WO2010010837A1 - Support de culture de cellules et procédé de culture de cellules - Google Patents

Support de culture de cellules et procédé de culture de cellules Download PDF

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WO2010010837A1
WO2010010837A1 PCT/JP2009/062873 JP2009062873W WO2010010837A1 WO 2010010837 A1 WO2010010837 A1 WO 2010010837A1 JP 2009062873 W JP2009062873 W JP 2009062873W WO 2010010837 A1 WO2010010837 A1 WO 2010010837A1
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cell culture
temperature
cells
group
blemmer
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PCT/JP2009/062873
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English (en)
Japanese (ja)
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一仁 伊原
泰光 藤野
幸司 宮崎
賢一 大久保
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コニカミノルタホールディングス株式会社
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Priority to JP2010521684A priority Critical patent/JPWO2010010837A1/ja
Publication of WO2010010837A1 publication Critical patent/WO2010010837A1/fr

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    • 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
    • C12N5/0068—General culture methods using substrates
    • 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

Definitions

  • the present invention relates to a cell culture support for in vitro cell culture used in the fields of humans, cell culture, tissue culture and the like, and a cell culture method using this cell culture support.
  • the monolayer cell culture method which is widely used as a general method for culturing animal cells, is not placed in the original culture environment of complex cells that have been in vivo, and therefore has a differentiation function that continues to survive. Although it is difficult to maintain and cells survive or proliferate, it is well known that the complex system of the living body is not accurately reproduced, resulting in termination of differentiation function and difficulty in control.
  • primary hepatocytes with highly differentiated metabolic functions in the body tend to lose their functions within a monolayer culture period.
  • the ability to metabolize ammonia which is one of the important functions of hepatocytes, is usually lost after about 10 generations from the start of culture. It has been known.
  • co-culture with blood vessel-derived cells is considered effective (in view of the structure of the liver in the living body). It cannot be stably co-cultured by mixing cells and fibroblasts or hepatocytes and endothelial cells.
  • thermoresponsive polymer patterned on the substrate is Nn-propyl methacrylamide monomer It is composed of a polymer, an N-isopropylacrylamide monomer polymer, and respective homopolymers. Since the two single polymers have a difference in temperature-responsive performance, they do not peel evenly when peeling at a low temperature after cell culture, and some cells are damaged.
  • Patent Document 2 As a method for patterning a cell adhesion layer on a cell culture substrate, for example, as in Patent Document 2, there is a method of patterning a cell adhesion layer on a substrate with a cell adhesion material by an inkjet method. It is merely applied to the film and a certain line width is drawn, and a technique for separately coating a plurality of polymers is not mentioned.
  • PEG polyethylene glycol
  • supercritical carbon dioxide is used as a means for fixing PEG to the substrate, which makes the process complicated and increases the size of the apparatus.
  • a cell culture support having a surface coated with two or more kinds of temperature-responsive polymers on a substrate, wherein at least one of the temperature-responsive polymers contains an acrylic resin.
  • R 1 and R 2 represent a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, and an aryl group.
  • R 5 , R 6 , and R 7 represent a hydrogen atom or a methyl group;
  • 3 represents a hydrogen atom, an alkyl group having 1 to 30 carbon atoms, a cycloalkyl group, or a poly (oxyethylene) group represented by — (CH 2 CH 2 O) n — (CH 2 CH (CH 3 ) O) m —R 0.
  • Represents an alkyl group having a number of 3 or more and 22 or less, and x, y, and z represent mass% of each component, 0 ⁇ x ⁇ 80, 0 ⁇ y ⁇ 80, 0 ⁇ x ⁇ 40, where x + y + z 100 .) 3.
  • a cell culture method comprising culturing two or more different cells on the cell culture support according to any one of 1 to 4 above.
  • a cell culture method comprising superposing cells cultured by the cell culture method described in 6 above.
  • the cell culture support of the present invention has made it possible to cultivate many types of cells in the same layer, and to achieve a technique for peeling only the cells from the substrate without destroying the cells.
  • Temperature responsive polymer In the present invention, it is preferable to apply a temperature-responsive polymer on a general cell culture substrate.
  • the temperature-responsive polymer is a polymer material in which hydrophilicity and hydrophobicity are reversibly changed by a temperature change.
  • a preferred temperature-responsive polymer is a polymer obtained by copolymerizing an acrylic resin monomer and an acrylamide monomer.
  • a more preferred temperature-responsive polymer is a polymer represented by the general formula (1).
  • a cell culture substrate having a temperature-responsive polymer on the surface exhibits excellent adhesion to cells under hydrophobic conditions, so that the cells can be cultured and proliferated appropriately, and under hydrophilic conditions, Since cell adhesion can be reduced, cells can be detached without the use of proteolytic enzymes or chemicals, so that cells can be easily removed without causing cell damage or substrate contamination. Recovery is possible.
  • the critical temperature The temperature at which the hydrophilicity and hydrophobicity of the temperature-responsive polymer changes is called the critical temperature, and the temperature at which it becomes hydrophobic at high temperatures and hydrophilic at low temperatures is called the lower critical temperature.
  • Many of the cells used for cell culture are derived from constant temperature animals. Therefore, the cells are often cultured around 37 degrees near the human body temperature, and it is better that the cells adhere to the cell culture support at that temperature. In other words, it is preferable that the surface of the cell culture support is hydrophobic at around 37 degrees.
  • the cell culture support is detached from the cell culture support at a low temperature so as not to cause protein denaturation due to heat. Better. That is, it is preferable that the cell culture support surface has a hydrophilic property at a low temperature.
  • the lower critical temperature is preferably in the temperature range of about 20 ° C. or more and 40 ° C. or less because cells can be cultured and detached suitably.
  • At least one of the temperature-responsive polymers contains an acrylic resin.
  • the acrylic resin in the present invention may be a general acrylic resin such as acrylic acid, methacrylic acid, methyl acrylate or methyl methacrylate, or a derivative thereof.
  • an acrylic resin which is a polycyclic hydrocarbon compound composed of an alicyclic hydrocarbon skeleton may be used.
  • the polycyclic hydrocarbon compound has an aliphatic polycyclic structure and a three-dimensional crosslinked structure.
  • tricyclodecane dimethanol dimethacrylate tricyclodecane dimethanol diacrylate, adamantyl methacrylate, adamantyl acrylate, etc.
  • isobornyl methacrylate, isobornyl acrylate, vinyl norbornene and the like may be included.
  • a polymer in which an acrylic resin monomer and an acrylamide monomer are copolymerized improves cell detachability.
  • the reason is that the amide has the same chemical structure as the peptide bond of the protein in the living body, and thus the affinity between the cell and the polymer is increased, which is considered to cause cell damage at the time of detachment.
  • a polymer obtained by copolymerizing an acrylamide monomer and an acrylic resin monomer as a support for cell culture, a material satisfying both cell detachability and temperature responsiveness was obtained.
  • An acrylamide polymer is well known as a temperature-responsive polymer, and it can be obtained by polymerizing an acrylamide monomer.
  • acrylamide monomers that give such polymers are preferably N-substituted acrylamide derivatives, N, N-disubstituted acrylamide derivatives, N-substituted methacrylamide derivatives, N, N-disubstituted methacrylamide derivatives, and the like.
  • N-isopropylacrylamide, N-isopropylmethacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxy Ethyl acrylamide, N-ethoxyethyl methacrylamide, N-tetrahydrofurfuryl acrylamide, N-tetrahydrofurfuryl methacrylamide, N-ethyl acrylamide, N-ethyl-N-methyl acrylamide, N, - diethyl acrylamide, N- methyl -N-n-propyl acrylamide, N- methyl -N- isopropylacrylamide, N- acryloyl Lupi Peri Dinh include N- acryloyl pyrrolidine.
  • acrylamide polymers examples include poly (N-isopropylacrylamide), poly (Nn-propylacrylamide), poly (N-cyclopropylmethacrylamide), poly (N-isopropylmethacrylamide), poly (Nn).
  • -Propylmethacrylamide poly (N-ethoxyethylacrylamide), poly (N-ethoxyethylmethacrylamide), poly (N-tetrahydrofurfurylacrylamide), poly (N-tetrafurfurylmethacrylamide), poly (N- Ethyl acrylamide), poly (N, N-diethylacrylamide), poly (N-acryloylpiperidine), and poly (N-acryloylpyrrolidine).
  • a copolymer obtained by polymerizing a plurality of different water-soluble acrylamide monomers selected from these is used as the polymer of the water-soluble acrylamide monomer. It is also effective.
  • a polymer comprising the water-soluble acrylamide monomer is preferred, but a copolymer of the water-soluble acrylamide monomer and another water-soluble acrylamide monomer or an organic solvent-soluble acrylamide monomer may also be a hydrophilic and hydrophobic polymer. Any material that exhibits both sexes can be used.
  • Specific examples of acrylamide monomers used for copolymerization include acrylamides such as N-alkylacrylamide, N, N-dialkylacrylamide, and acrylamide, or N-alkylmethacrylamide, N, N-dialkylmethacrylamide, and methacrylamide. And other methacrylamides. More preferably, N-alkylacrylamide or N, N-dialkylacrylamide is used.
  • alkyl group one having 1 to 4 carbon atoms is preferably selected.
  • acryloylmorpholine, N, N-dimethylaminopropylacrylamide, N-acryloylmethyl homopiperazine, N-acryloylmethylpiperazine, and the like can also be used.
  • the acrylic resin may be copolymerized with other monomers based on an acrylic resin monomer.
  • the temperature-responsive polymer is preferably a compound represented by the following general formula (1). is there.
  • R 1 and R 2 represent a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, or an aryl group.
  • R 5 , R 6 and R 7 each represent a hydrogen atom or a methyl group
  • R 3 represents a hydrogen atom, an alkyl group having 1 to 30 carbon atoms, a cycloalkyl group, or — (CH 2 CH 2 O) n — ( It represents a polyoxyalkylene group represented by CH 2 CH (CH 3 ) O) m —R 0 .
  • n represents an integer of 1 to 300
  • m represents an integer of 0 to 60.
  • R 0 represents a hydrogen atom or an alkyl group having 1 to 30 carbon atoms.
  • R 4 represents an alkyl group having 3 to 22 carbon atoms.
  • the alkyl group having 1 to 8 carbon atoms represented by R 1 and R 2 may be linear or branched.
  • it represents a group such as a methyl group, an ethyl group, an isopropyl group, an isobutyl group, or a dodecyl group, and may be substituted.
  • the substituent include groups such as a halogen atom, a hydroxy group, a carboxy group, and an acyl group, and examples thereof include a hydroxyethyl group and a hydroxypropyl group.
  • the cycloalkyl group include a cyclopropyl group, a cyclopentyl group, and a cyclohexyl group.
  • Examples of the aryl group represented by R 1 and R 2 include a phenyl group and a substituted phenyl group such as a tolyl group. Moreover, it is preferable that at least one of R 1 and R 2 is a hydrogen atom.
  • the alkyl group having 1 to 30 carbon atoms represented by R 3 may be linear or branched.
  • Examples of the branched alkyl group include a methyl group, an ethyl group, an isopropyl group, and an isobutyl group.
  • An unsubstituted alkyl group and a substituted alkyl group are included, and as an unsubstituted alkyl group, groups, such as a methyl group, an ethyl group, isopropyl group, a dodecyl group, are represented, for example.
  • examples of the substituent in the substituted alkyl group include groups such as a halogen atom, a hydroxy group, and a carboxy group, and examples thereof include a hydroxyethyl group and a hydroxypropyl group.
  • examples of the cycloalkyl group include a cyclopropyl group, a cyclopentyl group, a cyclohexyl group, and the like, but may be a substituted cycloalkyl group.
  • Examples of the alkyl group having 1 to 30 carbon atoms represented by R 0 include groups such as methyl, ethyl, propyl, butyl, octyl, decyl, dodecyl, stearyl.
  • the alkyl group having 3 to 22 carbon atoms represented by R 4 may be a linear or branched alkyl group, and is a propyl group, isopropyl group, butyl group, t-butyl group, hexyl group, dodecyl group, stearyl group. Etc.
  • the production of the copolymer of the general formula (1) can be obtained by copolymerization of each component monomer.
  • examples of the monomer containing a group represented by R 1 , R 2 , or R 5 typically include diacetone acrylamide, acrylamide, N-isopropyl acrylamide (NIPAM), N-ethyl acrylamide, N -Pyrrolidinyl acrylamide, N-cyclopropyl acrylamide, N-diethyl acrylamide, N-methyl, N-isopropyl acrylamide, N-propyl acrylamide, N-methyl, N-isopropyl acrylamide, N-piperidinyl acrylamide , N-propylacrylamide, N-cyclopropylmethacrylamide, N-ethylmethallylamide, N-isopropylmethacrylamide and the like.
  • NIPAM N-isopropyl acrylamide
  • NIPAM N-ethyl acrylamide
  • N -Pyrrolidinyl acrylamide N-cyclopropyl acrylamide
  • the monomer containing R 3 and R 6 and the monomer containing a group represented by R 4 and R 7 can be selected from the following.
  • Examples include acrylic acid, methacrylic acid, methyl methacrylate, butyl methacrylate and the like.
  • (polyoxyalkylene) acrylate and methacrylate are commercially available hydroxy poly (oxyalkylene) materials such as “Pluronic” (Pluronic (manufactured by Asahi Denka Kogyo Co., Ltd.)), Adeka polyether (Asahi Denka Kogyo ( Co., Ltd.), Carbowax [Carbowax (Glico Products)], Triton [Toriton (Rohm and Haas)] and P.I. E.
  • G made by Daiichi Kogyo Seiyaku Co., Ltd.
  • G can be manufactured by making it react with acrylic acid, methacrylic acid, acrylic chloride, methacrylic chloride, acrylic anhydride, etc. by a well-known method.
  • Blemmer 50POEP-800B Blemmer 50AOEP-800B, Blemmer PLE-200, Blemmer ALE-200, Blemmer ALE-800, Blemmer PSE-400, Blemmer PSE-1300, Blemmer ASE series, Blemmer PKEP series, Blemmer AKEP series, Blemmer AE-300 , Blemmer ANE-1300, Blemmer PNEP series, Blemmer PNPE series, Blemmer 43ANE -500, Bremer 70ANEP-550, etc., and Kyoeisha Chemical Co., Ltd.
  • light ester MC light ester 130MA, light ester 041MA, light acrylate BO-A, light acrylate EC-A, light acrylate MTG-A, light acrylate 130A, light Examples thereof include acrylate DPM-A, light acrylate P-200A, light acrylate NP-4EA, and light acrylate NP-8EA, which can be selected and used.
  • the polymers made of these copolymers have both hydrophilicity and lipophilicity, and their properties reversibly change depending on the lower critical temperature. In order to change the lower critical temperature, it can be arbitrarily determined by changing the copolymerization ratio of x, y, z in the general formula (1).
  • the lower critical temperature can be increased by increasing the number of repeating units of the oxyethylene group.
  • the lower critical temperature can be lowered by making the substituents of R 4 and R 7 hydrophobic.
  • copolymerization may be random copolymerization or block copolymerization. Random copolymerization is preferred in order to sharpen the hydrophilic and hydrophobic changes at the lower critical temperature.
  • the polymerization initiator is a polymerization of an acrylic resin monomer and an acrylamide monomer, and is therefore an initiator that generates radicals.
  • azo initiators and peroxide initiators can be used.
  • Oil-soluble peroxide-based or azo-based initiators are preferred.
  • benzoyl peroxide, lauroyl peroxide, octanoyl peroxide, benzoyl peroxide, orthomethoxybenzoyl peroxide, methyl ethyl ketone peroxide, diisopropyl Peroxide initiators such as peroxydicarbonate, cumene hydroperoxide, cyclohexanone peroxide, t-butyl hydroperoxide, diisopropylbenzene hydroperoxide, 2,2′-azobisisobutyronitrile, 2,2 '-Azobis (2,4-dimethylvaleronitrile), 2,2'-azobis (2,3-dimethylbutyronitrile), 2,2'-azobis (2-methylbutyronitrile), 2,2' -Azobis (2,3,3-trime Rubutyronitrile), 2,2'-azobis (2-isopropylbuty
  • organic peroxides such as tertiary isobutyl hydroperoxide, cumene hydroperoxide, paramentane hydroperoxide, hydrogen peroxide, and the like are preferable.
  • UV curable initiators are also preferred as polymerization initiators, such as acetophenone, acetophenone benzyl ketal, 1-hydroxycyclohexyl phenyl ketone, 2,2-dimethoxy-2-phenylacetophenone, xanthone, fluorenone, benzaldehyde, fluorene, anthraquinone.
  • Triphenylamine Triphenylamine, carbazole, 3-methylacetophenone, 4-chlorobenzophenone, 4,4'-dimethoxybenzophenone, 4,4'-diaminobenzophenone, Michler's ketone, benzoin propyl ether, benzoin ethyl ether, benzyldimethyl ketal, 1- ( 4-Isopropylphenyl) -2-hydroxy-2-methylpropan-1-one, 2-hydroxy-2-methyl-1-phenylpropan-1-one And the like of the photoradical initiator.
  • polymerization initiators are preferably used in an amount of 0.01 to 20% by mass, particularly 0.1 to 10% by mass, based on the monomer.
  • an organic solvent or water may or may not be used as a reaction site. Since it is necessary to remove the solvent in a later step, it is preferable not to use a solvent.
  • the organic solvent include alcohols such as methanol, ethanol and isopropyl alcohol, esters such as ethyl acetate and methyl acetate, ketones such as methyl ethyl ketone and acetone, ethers such as ether and isopropyl ether, and cyclic ethers such as tetrahydrofuran and dioxane.
  • alcohols such as methanol, ethanol and isopropyl alcohol
  • esters such as ethyl acetate and methyl acetate
  • ketones such as methyl ethyl ketone and acetone
  • ethers such as ether and isopropyl ether
  • cyclic ethers such as tetrahydrofuran and dioxane.
  • toluene which is
  • the boiling point of the solvent is preferably 50 ° C. or higher, more preferably 70 ° C. or higher. However, if it becomes as high as 150 ° C. or higher, man-hours are required for subsequent handling.
  • the solid content concentration is preferably 10% by mass or more and 40% by mass or less, and the viscosity of the final solution containing the copolymer is 30% by mass.
  • the degree of polymerization is preferably 10 mPa ⁇ s or more and 500 mPa ⁇ s or less in terms of%.
  • the residual monomer amount is set to 1% by mass or less, and the reaction is terminated. This measurement is performed with a gas chromatograph.
  • the reaction liquid containing the copolymer can be mixed with a poor solvent and precipitated, and further dissolved and precipitated repeatedly to be isolated as a solid content.
  • thermo-responsive polymer used in the present invention
  • No. 1 in the Examples is used.
  • examples thereof include polymers represented by E to L and the like.
  • a known and commonly used organic crosslinking agent may be used during polymerization for the purpose of improving the characteristics.
  • concentration of the organic crosslinking agent to be used is not particularly limited and can be selected according to the purpose.
  • organic crosslinking agents examples include conventionally known N, N′-methylenebisacrylamide, N, N′-propylenebisacrylamide, di (acrylamidomethyl) ether, 1,2-diacrylamide ethylene glycol, 1,3- Bifunctional compounds such as diacryloylethyleneurea, ethylene diacrylate, N, N'-diallyl tartaramide, N, N'-bisacrylylcystamine, and trifunctional compounds such as triallyl cyanurate and triallyl isocyanurate Can be exemplified.
  • Examples of the base material for the cell culture support according to the present invention include various polymer materials, glass, modified glass, wool cloth, cotton cloth, paper, metal (for example, aluminum) and the like. From the viewpoint of handling, as a substrate in the cell culture support of the present invention, a plastic material (for example, cellulose acetate, polyester, polyethylene terephthalate, polyethylene naphthalate, polyamide, polyimide, cellulose triacetate or polycarbonate, polystyrene, polymethyl methacrylate, etc. And polystyrene is particularly preferred in the present invention.
  • the thickness of the support is about 50 to 3000 ⁇ m, preferably 70 to 1800 ⁇ m.
  • the base material for cell culture is surface-treated by glow discharge, corona discharge, vacuum plasma treatment, atmospheric pressure plasma treatment or silane coupling treatment on the surface of the base material in order to make the temperature-responsive polymer easy to adhere. May be.
  • Temperature-responsive polymer coating method As a method of coating the surface of the substrate with the temperature-responsive polymer of the present invention, a method in which the substrate is coated with a monomer and polymerized, and a method in which the monomer is polymerized in advance to form a temperature-responsive polymer are applied. There is a way. Either method may be used, but from the viewpoint of patterning, it is preferable to polymerize after coating on a substrate with a monomer.
  • the method for applying the temperature-responsive polymer or its monomer on the substrate for example, bar coater method, curtain coating method, dipping method, air knife method, hopper coating method, reverse roll coating method, gravure.
  • Known methods such as a coating method, an extrusion coating method, and a vacuum deposition method (sputtering method) can be used.
  • a lithography method, an inkjet method, or a super inkjet method it is preferable to apply them by a lithography method, an inkjet method, or a super inkjet method.
  • the light source is, for example, a low pressure, medium pressure, high pressure mercury lamp, metal halide lamp or ultraviolet region having an operating pressure of 0.1 kPa to 1 MPa.
  • lamps such as xenon lamps, cold cathode fluorescent lamps, hot cathode fluorescent lamps, and LEDs having a light emission wavelength of 2 are used.
  • the application amount of the temperature-responsive polymer is preferably selected in accordance with the purpose of cell culture, but for the purpose of culturing cells and then peeling the cells from the substrate. is, 0.1 [mu] g / cm 2 or more, preferably 5.0 [mu] g / cm 2 or less, 0.5 [mu] g / cm 2 or more, 3.0 [mu] g / cm 2 or less being more preferred.
  • the material constituting the temperature-responsive polymer according to the present invention When adding the temperature-responsive polymer according to the present invention to the surface of the base material in a predetermined pattern, it is preferable to add the material constituting the temperature-responsive polymer in a liquefied state.
  • a method for adding the material constituting the liquefied temperature-responsive polymer in a predetermined pattern it is preferable to use a printing method such as a screen printing method or an ink jet method. By using such a printing method, the material constituting the temperature-responsive polymer can be more easily added in an arbitrary pattern to the surface of the substrate for cell culture.
  • a method may be employed in which a mask perforated in a predetermined pattern is prepared and placed on the substrate surface, and then a solution containing a material constituting the temperature-responsive polymer is spray applied.
  • an inkjet method with high individual correspondence is particularly preferable.
  • screen printing method and spray coating method are also possible, but when complex patterns are to be formed, mixing, overlapping, and boundary lines due to blurring between adjacent different types of temperature-responsive polymers Prone to problems such as opening gaps. From this point of view, an inkjet method having a high resolution is more preferable.
  • the material constituting the temperature-responsive polymer is heated and softened, or the material constituting the temperature-responsive polymer is changed to a predetermined solvent.
  • dissolve the method of melt
  • the line width that can be patterned is preferably 1 ⁇ m or more and 1000 ⁇ m or less, more preferably 3 ⁇ m or more and 100 ⁇ m or less.
  • the cells cultured on the cell culture support of the present invention are brought to a temperature lower than the lower critical temperature of the temperature-responsive polymer, and the cultured cells are detached by hydrophilizing the support surface.
  • the surface of the cell culture support of the present invention is processed with a temperature-responsive polymer having two or more kinds of lower critical temperatures, two or more kinds of different cells are co-located in the same layer. Can be cultured. By making at least one of these cells a cell having an ability to regenerate blood vessels such as vascular endothelial cells, the cells are less likely to be insufficient in nutrients and gas exchange even if the number of layers is increased, which is the object of the present invention. It has become possible to establish a cell culture method capable of precisely and precisely expressing the differentiation function of various types of cells and maintaining the tissue function.
  • the number of cells in the laminate can be confirmed by, for example, cutting out a permeable membrane on which the laminate is mounted, fixing the formalin, preparing a paraffin-embedded section, and observing under a microscope.
  • the cell to be subjected to the method of the present invention is not particularly limited, but an adherent animal cell is preferable.
  • the origin of the cell is not particularly limited, and those derived from any animal such as human, mouse, rat and the like can be used.
  • Adhesive animal cells can target both primary cultured cells and established cells.
  • the method of the present invention is particularly suitable for culturing primary cultured cells in which it is difficult to maintain cell functions.
  • Primary cultured cells are cartilage, bone, skin, nerve, oral cavity, digestive tract, liver, pancreas, kidney, glandular tissue, adrenal gland, heart, muscle, tendon, adipose tissue, connective tissue, genital organ, eyeball, blood vessel, bone marrow or blood It may be derived from any of these tissues. It is suitable to seed one cell for one kind of temperature-responsive polymer on the surface of the cell culture support, and a single kind of cell derived from a single tissue can be used as the cell. . When a plurality of temperature-responsive polymers are present on the surface, a plurality of different types of cells can be used.
  • the cell culture thus obtained can be used as cells for medical biomaterials, for example.
  • the regenerative medical biomaterial refers to a material used as a substitute for tissue of animals such as humans.
  • Regenerative medical biomaterials include artificial pancreas, artificial spleen, artificial kidney, artificial organs like artificial heart, artificial digestive tract, artificial blood vessel, artificial skin, artificial nerve, artificial bone, depending on the type of cultured cells Examples include artificial cartilage, cochlear implant, artificial lens, artificial cornea, etc., or a part thereof.
  • biomedical materials for regenerative medicine are also used for experimental animal substitute cells, anticancer drug sensitivity tests, drug discovery support, and the like.
  • Blemmer PME-400 methacrylate with-(EO) m -CH 3 (m ⁇ 9)
  • Blemmer PSE-400 methacrylate with-(EO) m -C 18 H 37 (m ⁇ 9) (EO; Ethyleneoxy group)
  • EO Ethyleneoxy group
  • NIPAM N-isopropylacrylamide (manufactured by Kojin)
  • DEAA N-diethylacrylamide (manufactured by Kojin)
  • DAAM Diacetone acrylamide (Kyowa Hakko)
  • BMA Butyl methacrylate (Tokyo Chemicals)
  • the isolated polymer was dissolved in pure water at 25 ° C. to prepare a polymer solution having a concentration of 10% by mass. Thereafter, the temperature of the solution was raised, and the temperature at which the polymer was precipitated was defined as the lower critical temperature.
  • the results are shown in Table 1. However, since the polymer solutions C and D were not dissolved in pure water at 25 ° C. and the polymer had already precipitated, the lower critical temperature was set to none.
  • a monomer / MEK solution of the type shown in Table 3 (M to X shown in Table 2) is filled in ink tank-1 and ink tank-2 shown in Table 3 instead of ink, and has a nozzle diameter of 25 ⁇ m, a driving frequency of 12 kHz, Using an on-demand type ink jet printer with a maximum recording density of 720 ⁇ 720 dpi using a piezo type recording head having 128 nozzles and a nozzle density of 180 dpi (dpi in the present invention represents the number of dots per 2.54 cm).
  • the temperature-responsive polymer was adsorbed on the surface of the polystyrene cell culture dish.
  • the temperature-responsive polymer was adsorbed on the surface of the polystyrene cell culture dish.
  • the cultured cells were seeded on the cell culture support prepared above, and the cells were cultured.
  • the cells to be cultured were patterned using the difference in the lower critical temperature on the surface of the patterned temperature-responsive polymer surface.
  • the culture is performed using a minimum essential eagle medium (manufactured by SIGMA) containing 10% fetal bovine serum (manufactured by ICN) (containing pyruvic acid (manufactured by ICN) and non-essential amino acids (manufactured by ICN) as additives). It was performed in a 37 ° C. incubator filled with% carbon dioxide gas.
  • the cell culture array was allowed to stand in a 20 ° C. constant temperature bath for 5 minutes and then the surface was observed with an optical microscope. As a result, the cells were patterned on the cell culture array and adhered. In addition, it was confirmed that the cells had proliferated sufficiently.
  • the extracted cells were treated with trypsin-EDTA, and each cell was separated into individual states, followed by trypan blue staining to measure the number of viable cells. It was confirmed that the number of cells, which was 8.2 ⁇ 10 2 at the start of 18 cultures, increased to 5.3 ⁇ 10 3 after the culture.
  • the following results are shown in Table 3 with the value after the cell culture as 100%. In the present invention, it was confirmed that the culture efficiency was good for comparison.
  • the membrane was covered, the medium was gently aspirated, and the cell culture support material was incubated at 20 ° C. for 30 minutes and cooled, so that the cells on any cell culture support material were detached together with the covered membrane. .
  • the covered membrane and cells were placed on the same cultured cells that were normally grown on the same cell culture support material, and the two were adhered in a 37 ° C. incubator filled with 5% carbon dioxide gas. After the two cell sheets adhered, the PVDF membrane was peeled off. By repeating the same operation, a 20-layer cell sheet was prepared.
  • the covered membrane could be easily peeled from any cell sheet.
  • the 20 stacked cell sheets retained the cells, the desmosome structure between the cells, and the basement membrane-like protein between the cells and the substrate.
  • the bleed resistance was good, the detachment of the cell from the support was remarkably improved, and the tissue functionalization of the stacked cells could be expressed. It was found that this technology can greatly contribute to the progress of cell culture methods for complex tissues in the future.

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Abstract

L'objectif de l'invention est de réaliser la culture de plusieurs types de cellules en une seule couche et de détacher du matériau de base seulement les cellules, uniformément et sans rompre les cellules. L'invention concerne un support de culture cellulaire qui se caractérise en ce qu'il comprend un matériau de base et deux polymères répondant à la température ou davantage, appliqués sur des zones différentes sur la surface du matériau de base, au moins un des polymères répondant à la température comprenant une résine acrylique. Elle concerne également un procédé de culture de cellules.
PCT/JP2009/062873 2008-07-25 2009-07-16 Support de culture de cellules et procédé de culture de cellules WO2010010837A1 (fr)

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WO2012029882A1 (fr) * 2010-08-31 2012-03-08 学校法人東京女子医科大学 Substrat réagissant à la température pour culture cellulaire et procédé de production dudit substrat
JP2013055898A (ja) * 2011-09-07 2013-03-28 Dainippon Printing Co Ltd ディスペンス液
EP2574664A1 (fr) 2011-09-30 2013-04-03 Centrum Materialów Polimerowych i Weglowych PAN Méthode pour la préparation d'un revêtement thermosensible, substrat avec revêtement thermosensible et son utilisation
JP2014097031A (ja) * 2012-11-15 2014-05-29 Dainippon Printing Co Ltd 温度応答性を有する細胞培養基材の製造方法
JP2016194041A (ja) * 2015-03-31 2016-11-17 東洋インキＳｃホールディングス株式会社 インクジェット用インク、印刷物、およびインクジェット記録方法
JP2018052967A (ja) * 2017-11-28 2018-04-05 株式会社ファンケル 脂質二重膜からの内包物の放出率調整剤

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012029882A1 (fr) * 2010-08-31 2012-03-08 学校法人東京女子医科大学 Substrat réagissant à la température pour culture cellulaire et procédé de production dudit substrat
CN103080295A (zh) * 2010-08-31 2013-05-01 学校法人东京女子医科大学 细胞培养用温度应答性基材及其制造方法
CN103080295B (zh) * 2010-08-31 2014-08-27 学校法人东京女子医科大学 细胞培养用温度应答性基材及其制造方法
JP5846584B2 (ja) * 2010-08-31 2016-01-20 学校法人東京女子医科大学 細胞培養用温度応答性基材及びその製造方法
US9279102B2 (en) 2010-08-31 2016-03-08 Tokyo Women's Medical University Temperature-responsive substrate for cell culture and production method thereof
JP2013055898A (ja) * 2011-09-07 2013-03-28 Dainippon Printing Co Ltd ディスペンス液
EP2574664A1 (fr) 2011-09-30 2013-04-03 Centrum Materialów Polimerowych i Weglowych PAN Méthode pour la préparation d'un revêtement thermosensible, substrat avec revêtement thermosensible et son utilisation
JP2014097031A (ja) * 2012-11-15 2014-05-29 Dainippon Printing Co Ltd 温度応答性を有する細胞培養基材の製造方法
JP2016194041A (ja) * 2015-03-31 2016-11-17 東洋インキＳｃホールディングス株式会社 インクジェット用インク、印刷物、およびインクジェット記録方法
JP2018052967A (ja) * 2017-11-28 2018-04-05 株式会社ファンケル 脂質二重膜からの内包物の放出率調整剤

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