WO2010010837A1

WO2010010837A1 - Cell culture support and cell culture method

Info

Publication number: WO2010010837A1
Application number: PCT/JP2009/062873
Authority: WO
Inventors: 一仁伊原; 泰光藤野; 幸司宮崎; 賢一大久保
Original assignee: コニカミノルタホールディングス株式会社
Priority date: 2008-07-25
Filing date: 2009-07-16
Publication date: 2010-01-28
Also published as: JPWO2010010837A1

Abstract

The object aims to achieve the culture of multiple types of cells in a single layer and detach only cells from a base material evenly without disrupting the cells. Disclosed is a cell culture support which is characterized by comprising a base material and two or more temperature-responsive polymers applied on different areas on the surface of the base material, wherein at least one of the temperature-responsive polymers comprises an acrylic resin. Also disclosed is a cell culture method.

Description

Cell culture support and cell culture method

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.

Conventionally, 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.

For example, among primary cultured cells, primary hepatocytes with highly differentiated metabolic functions in the body tend to lose their functions within a monolayer culture period. For example, even when mouse primary cultured hepatocytes are cultured in a monolayer in a petri dish, 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.

For long-term culture of hepatocytes, 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.

An example of co-culture of liver parenchymal cells and rat vascular endothelial cells is known (for example, Patent Document 1), but the temperature-responsive 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.

In addition, 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.

Also, since polyethylene glycol (PEG) is used as a cell adhesion material, cells cannot be detached. In addition, 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.

International Publication No. 01/068799 Pamphlet JP 2006-325791 A

It is to enable culturing of many types of cells in the same layer and to peel only the cells evenly from the substrate without destroying the cells.

The above object of the present invention can be achieved by the following configuration.

1. 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. A cell culture support.

2. 2. The cell culture support according to 1, wherein at least one of the temperature-responsive polymers containing the acrylic resin is represented by the following general formula (1).

(In the general formula (1), 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 oxyalkylene group, wherein n represents 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, and R ₄ represents a carbon atom. 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. 3. The cell culture support according to 1 or 2 above, wherein at least one of the temperature-responsive polymers is a homopolymer of N-isopropylacrylamide or a copolymer with another monomer component.

4. 4. The cell culture support according to any one of 1 to 3, wherein the temperature-responsive polymer is patterned on a substrate by an inkjet method.

5. 5. 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.

6. 6. The cell culture method according to 5, wherein two or more kinds of different cells are detached at a temperature lower than the response temperature of the temperature-responsive polymer.

7. 7. 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.

Hereinafter, the best mode for carrying out the present invention will be described in detail.

(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.

Furthermore, since the change from hydrophobic to hydrophilic or from hydrophilic to hydrophobic is rapid, there is little influence on the cells when changing the external environment including temperature, which is preferable. 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. When the cells are detached, it is preferable that 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.

Furthermore, 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.

In the present invention, 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. Further, 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. For example, tricyclodecane dimethanol dimethacrylate, tricyclodecane dimethanol diacrylate, adamantyl methacrylate, adamantyl acrylate, etc., isobornyl methacrylate, isobornyl acrylate, vinyl norbornene and the like may be included.

Using 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. By using 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.

Examples of 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. Specifically, 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.

Examples of such acrylamide polymers 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).

In addition to the polymer from the single water-soluble acrylamide monomer as described above, 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. As the alkyl group, one having 1 to 4 carbon atoms is preferably selected. In addition, 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. In the present invention, the temperature-responsive polymer is preferably a compound represented by the following general formula (1). is there.

Here, 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, and 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 . Here, n represents an integer of 1 to 300, and 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. Moreover, x, y, z represents the mass% of each component, 0 <= x <= 80, 0 <= y <= 80, 0 <= x <= 40, and it is here x + y + z = 100.

The alkyl group having 1 to 8 carbon atoms represented by R ₁ and R ₂ may be linear or branched. For example, 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. Examples of 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. Examples of 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. Represents a group such as a dodecyl 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. In addition, 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. Also, for example, an oxolanyl group, an oxanyl group, a pyrazinyl group in which the skeleton carbon of the cycloalkyl group is substituted with a heteroatom. It may be a saturated hydrocarbon group containing a hetero atom such as a group.

The polyoxyalkylene group represented by — (CH ₂ CH ₂ O) _n — (CH ₂ CH (CH ₃ ) O) _m —R _0, which may be contained in R ₃ , includes an oxyethylene group repeating unit ( n) is preferably 1 to 300, more preferably 6 to 100, and still more preferably 8 to 50. Further, the repeating unit (m) of the oxypropylene group which may be contained in R ₃ is preferably 0 to 60, more preferably 0 to 30, and still more preferably 0 to 15. is there. These oxyethylene groups and oxypropylene groups may be mixed. 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 respective component monomers are given below, but are not limited to these as long as the condition of the general formula (1) is satisfied.

In the general formula (1), 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.

In the general formula (1), 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.

Further, for example, (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. What is marketed as G (made by Daiichi Kogyo Seiyaku Co., Ltd.) can be manufactured by making it react with acrylic acid, methacrylic acid, acrylic chloride, methacrylic chloride, acrylic anhydride, etc. by a well-known method.

As commercially available products, Blenmer PE-90, Blemmer PE-200, Blemmer PE-350, Blemmer AE-90, Blemmer AE-200, Blemmer as poly (alkylene glycol mono (meth) acrylates manufactured by NOF Corporation. AE-400, Blemmer PP-1000, Blemmer PP-500, Blemmer PP-800, Blemmer AP-150, Blemmer AP-400, Blemmer AP-550, Blemmer AP-800, Blemmer 50 PEP-300, Blemmer 70PEP-350B, Blemmer AEP series, Blemmer 55PET-400, Blemmer 30PET-800, Blemmer 55PET-800, Blemmer AET series, Blemmer 30PPT-800, Blemmer 50 PT-800, Blemmer 70PPT-800, Blemmer APT series, Brenmer 10PPB-500B, such as Brenmer 10APB-500B and the like. Similarly, Blemmer PME-100, Blemmer PME-200, Blemmer PME-400, Blemmer PME-1000, Blemmer PME-4000, Blemmer AME-400, Blemmer as alkyl-terminated polyalkylene glycol mono (meth) acrylates manufactured by NOF Corporation. 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).

For example, 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.

Mixing the monomers at a mixing ratio according to the target lower critical temperature, for example, dissolving each monomer as a solvent, or a solvent having good solubility for the copolymer, such as methyl ethyl ketone, adding a polymerization initiator, Alternatively, solution polymerization may be performed by heating or UV light.

The form of 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.

In these temperature-responsive polymers containing an acrylic resin according to the present invention, the polymerization initiator is a polymerization of an acrylic resin monomer and an acrylamide monomer, and is therefore an initiator that generates radicals. Are preferable, and azo initiators and peroxide initiators can be used.

Oil-soluble peroxide-based or azo-based initiators are preferred. For example, 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-isopropylbutyronitrile), 1,1'-azobis (cyclohexane-1-carbonitrile), 2,2'-azobis (4-methoxy-2,4-dimethylvalero) Nitrile), 2- (carbamoylazo) isobutyronitrile, 4,4′-azobis (4-cyanovaleric acid), dimethyl-2,2′-azobisisobutyrate and the like.

In particular, organic peroxides such as tertiary isobutyl hydroperoxide, cumene hydroperoxide, paramentane hydroperoxide, hydrogen peroxide, and the like are preferable.

Similarly, 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, 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.

These 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.

In the production of the copolymer according to the present invention, 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. Examples of 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. Although there is no particular limitation such as toluene, which is an aromatic hydrocarbon, or the like, it is preferable to select and use a highly soluble solvent for the monomer as a raw material and for the copolymer to be formed.

In order to prevent the polymerization temperature from becoming too low, 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.

In the copolymerization reaction, after polymerization, 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%.

In the copolymerization reaction according to the present invention, 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.

As specific examples of the temperature-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.

When applying the above-mentioned temperature-responsive polymer to the cell culture substrate of the present invention, a known and commonly used organic crosslinking agent may be used during polymerization for the purpose of improving the characteristics. The concentration of the organic crosslinking agent to be used is not particularly limited and can be selected according to the purpose. Examples of organic crosslinking agents that can be used 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.

(About cell culture supports)
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 μm, preferably 70 to 1800 μm.

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.

There is no particular limitation on 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. Moreover, when patterning two types of temperature-responsive polymers on the support of the cell culture substrate, it is preferable to apply them by a lithography method, an inkjet method, or a super inkjet method.

As the technique for immobilizing the temperature-responsive polymer on the substrate, either heat curing or UV irradiation curing may be employed.

Curing by UV irradiation is preferable from the viewpoint of quick reaction. When UV light is used, 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. Conventionally known lamps such as xenon lamps, cold cathode fluorescent lamps, hot cathode fluorescent lamps, and LEDs having a light emission wavelength of 2 are used.

In the present invention, 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.

(Patterning of temperature-responsive polymer to cell culture substrate)
In the present invention, it is sufficient that two or more types of cells can be cultured in different regions on two or more types of temperature-responsive polymers in which different regions are coated in the same layer. The same pattern is preferable in terms of systematic function expression. For this purpose, a cell culture support in which a temperature-responsive polymer is patterned on a substrate is preferred.

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. As 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. Alternatively, 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.

In addition, when forming a complicated and individual difference pattern such as a blood vessel or a nervous system using the cell culture support of the present invention, an inkjet method with high individual correspondence is particularly preferable. As other methods, 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.

In addition, as a method for making the material constituting the temperature-responsive polymer into a liquid state, 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. Although the method etc. which melt | dissolve are mentioned, the method of melt | dissolving in a solvent from the point which does not need to perform temperature adjustment is suitable.

The line width that can be patterned is preferably 1 μm or more and 1000 μm or less, more preferably 3 μm or more and 100 μm or less.

(About cells cultured on cell culture supports and their lamination)
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. In stacking the peeled cells, it is desirable to adjust the number of cells so that a laminate in which the cells are approximately 2 to 200 layers, particularly 5 to 100 layers, is obtained. If there are too many cells in the laminate, the cells in the central layer of the laminate will be undernutrition and gas exchange will be insufficient. The body is difficult to form. If it is the said range, such a problem will not arise.

Since 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.

Specifically, for example, chondrocytes, osteoblasts, epidermal keratinocytes, melanocytes, neurons, neural stem cells, glial cells, hepatocytes, intestinal epithelial cells, pancreatic β cells, pancreatic exocrine cells, renal glomerular endothelium Cells, tubular epithelial cells, breast cells, thyroid cells, salivary gland cells, adrenal cortex cells, adrenal medullary cells, cardiomyocytes, skeletal muscle cells, smooth muscle cells, adipocytes, adipose precursor cells, lens cells, corneal cells, vascular endothelium Cells, bone marrow stromal cells or lymphocytes can be used. Cells can be used singly or in combination of two or more.

The cell culture thus obtained can be used as cells for medical biomaterials, for example. In the present invention, 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.

Also, as a substitute for tissue of animals such as humans, biomedical materials for regenerative medicine are also used for experimental animal substitute cells, anticancer drug sensitivity tests, drug discovery support, and the like.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto. Unless otherwise specified, “%” in the examples represents “mass%”.

(1) Preparation of sample (1.1) Synthesis of temperature-responsive polymers A to L (comparative example, the present invention)
A 0.5 liter four-necked separable flask is equipped with a dropping device, a thermometer, a nitrogen gas inlet tube, a stirring device and a reflux condenser, and 50 g of methyl ethyl ketone (MEK) and other than NIPAM at the composition ratio shown in Table 1 The monomer (unit g) was charged and heated to 80 ° C. Further, a solution obtained by dissolving NIPAM monomer (unit g) shown in Table 1 in 43 g of methyl ethyl ketone and further dissolving 0.12 g of lauryl peroxide was dropped into the flask over 2 hours. Thereafter, when the temperature was raised over 1 hour to reach a reflux state, a solution obtained by dissolving 0.17 g of lauryl peroxide in 33 g of methyl ethyl ketone was dropped into the flask over 2 hours and reacted at the same temperature for further 3 hours. . Thereafter, a solution in which 0.33 g of methylhydroquinone was dissolved in 107 g of methyl ethyl ketone was added and cooled to obtain polymer solutions A to L having a polymer content of 30% by mass. Molecular weight was calculated | required as a weight average molecular weight of polystyrene conversion by GPC.

In the following,
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)
The above are all made from Japanese fats and oils.
NIPAM: N-isopropylacrylamide (manufactured by Kojin)
DEAA: N-diethylacrylamide (manufactured by Kojin)
DAAM: Diacetone acrylamide (Kyowa Hakko)
BMA: Butyl methacrylate (Tokyo Chemicals)
(1.2) Evaluation of lower limit critical temperature of temperature-responsive polymers A to L After gradually adding 300 g of the polymer / MEK solution prepared in (1.1) above to 1000 g of distilled water at room temperature (polymer solution C A uniform solution is formed except for D and D), and when the temperature is raised to 45 ° C., it precipitates. This was separated and washed thoroughly with warm water of 40 ° C. to remove residual monomers. Thereafter, 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.

From Table 1, by changing the copolymer composition ratio of the acrylic resin, it was possible to change the lower critical temperature of the polymer that generates the lower critical temperature near the body temperature at which cells are easily cultured.

(1.3) Inkjet Application of Monomers and Polymerization Method Each monomer is made to have the composition ratio shown in Table 2 below so as to have the same composition as the polymer / MEK (30% by mass solution) prepared in (1.1) above. Similarly, it was dissolved in the MEK solution so as to have a solid content of 30% by mass. Further, instead of lauryl peroxide described in (1.1), IRGACURE 184 (manufactured by Ciba Japan) as a UV polymerization initiator was contained in an amount of 1% by mass based on the monomer.

Further, 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 μm, 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). On the commercially available polystyrene cell culture dish (Falcon 3001 Petri dish (diameter: 3.5 cm) manufactured by Becton Dickinson Labware) Each one is pudding so that one side becomes a thin line of 100μm width It was.

Each ink was continuously ejected, and 0.1 seconds after landing, a 120 W / cm metal halide lamp (manufactured by Nippon Battery Co., Ltd., MAL 400 NL, power supply power 3 kW · hr) was irradiated to polymerize the monomers.

Thus, the temperature-responsive polymer was adsorbed on the surface of the polystyrene cell culture dish.

Thereafter, in order to remove the residual monomer and MEK as a solvent, it was washed with a large amount of water to obtain a cell culture support.

(1.4) Control polymerization No. As a control, instead of inkjet coating, the M solution described in Table 2 was dropped into a 0.1 ml dish, and the coating solution was leveled (1.3) and polymerized in the same manner.

(1.5) Application of polymerized polymer by ink jet No. 1 in Table 3 Nos. 19 to 25 were adsorbed in the same manner except that the polymer / MEK solution shown in Table 1 (type shown in Table 3) was used instead of the monomer / MEK solution of (1.3).

(1.6) Application and polymerization by screen printing method Nos. 26 to 31 use a fine line mask with a width of 100 μm so as to form a pattern similar to that of the ink jet coating described in (1.3), and the monomer / MEK solution of the type described in Table 3 (M (Prepared with X), and the same patterning as the inkjet coating described in (1.3) was performed. 0.1 seconds later, a monomer was polymerized by irradiation with a 120 W / cm metal halide lamp (MAL 400NL, manufactured by Nippon Battery Co., Ltd., power supply power 3 kW · hr).

(1.7) Cell Culture 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.

One week after seeding, 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.

(1.8) Bleed resistance The cells were visually observed, and the bleed resistance of the cells was evaluated according to the following criteria.
◎: The boundary between the fine line and the solid is clear. ○: There is a part where the boundary is slightly blurred, but the quality has no problem in practical use. X: The occurrence of clear bleeding at the boundary is recognized, the line width is about 1.5 times, and this is a quality that is a problem in practice. XX: The boundary between the thin line and the solid part is unclear (1.9) Lamination of cells 14 days after culturing with the cells prepared in (1.7) above, the polyvinylidene difluoride (PVDF with a diameter of 3.5 cm) was cultured on the cultured cells. ) 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.

(1.10) Evaluation of cell detachability The cell state when the cells were detached from the cell culture support in (1.9) above was visually observed and evaluated according to the following criteria. The results shown in Table 3 were averaged from the first sheet to the 20th layer.
◎: Completely peeled without problems ○: Slightly cells remained on the support, but no problem level △: Level at which some cells remained on the support and holes were confirmed in the sheet ×: Peeled from the support However, the sheet is crumpled. XX: Cells are not peeled off from the support. (1.11) Evaluation of stacked functionality An important function in liver cells is ammonia metabolism. Therefore, the rate of ammonia metabolism was evaluated. The ratio of cells collected from the swine in vivo was calculated as 100%, and the results are shown in Table 3. The method of the present invention was equivalent to or better than that obtained directly from the living body with respect to the comparative example.

Using the cell culture support of the present invention, 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.

Claims

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. A cell culture support.
2. The cell culture support according to claim 1, wherein at least one of the temperature-responsive polymers containing the acrylic resin is represented by the following general formula (1).

(In the general formula (1), 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 oxyalkylene group, wherein n represents 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, and R 4 represents a carbon atom. 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 ≦ z ≦ 40, where x + y + z = 100 .)
3. The cell culture support according to claim 1, wherein at least one of the temperature-responsive polymers is a homopolymer of N-isopropylacrylamide or a copolymer with other monomer components.
The cell culture support according to any one of claims 1 to 3, wherein the temperature-responsive polymer is patterned on a substrate by an inkjet method.
A cell culture method comprising culturing two or more different types of cells on the cell culture support according to any one of claims 1 to 4.
6. The cell culture method according to claim 5, wherein two or more kinds of different cells are exfoliated at a temperature lower than the critical temperature of the temperature-responsive polymer.
A cell culture method comprising superposing cells cultured by the cell culture method according to claim 6.