JP4430123B1 - Cell culture substrate and method for producing the same - Google Patents

Abstract

（式中、Ｒ_１は水素原子またはメチル基、Ｒ_２は炭素原子数２〜３のアルキレン基、Ｒ_３は水素原子または炭素原子数１〜２のアルキル基を表し、ｎは１〜９の整数を表す。）
【選択図】図1PROBLEM TO BE SOLVED: To provide a cell culture substrate in which a polymer (B) having a lower critical solution temperature contained in the substrate is not cross-linked, has good adhesion to a support, and is hydrophobic with respect to environmental temperature. A cell culture substrate and a method for producing the same are provided. The cell culture substrate can be easily detached from the surface of the culture substrate and collected.
One or more selected from a polymer (A) of a monomer (a) represented by the following formula (1), a polymer (B) having a lower critical solution temperature, a water-swellable clay mineral and silica. A cell culture substrate containing the inorganic material (C).

Wherein R ₁ represents a hydrogen atom or a methyl group, R ₂ represents an alkylene group having 2 to 3 carbon atoms, R ₃ represents a hydrogen atom or an alkyl group having 1 to 2 carbon atoms, and n represents 1 to 9 Represents an integer.)
[Selection] Figure 1

Description

  The present invention relates to a cell culture technique, and specifically to a cell culture substrate that can easily peel and collect cultured cells, and a method for producing the same.

  Conventionally, plastic (for example, polystyrene) containers have been mainly used as cell culture substrates for animal tissues and the like. These containers are subjected to surface treatment such as plasma treatment or coating of silicon, cell adhesion factor, or the like in order to effectively perform cell culture. When these cell culture vessels are used as a culture substrate, cultured (growth) cells adhere to the vessel surface, and in order to isolate and recover the cells, trypsin or other protein hydrolase or chemical It was necessary to peel from the container surface using chemicals. The operation of detaching cells with such enzymes and chemicals is not only complicated, but there is a risk that impurities such as bacteria and DNA or RNA may be mixed. In addition, not only the binding part of the cell and the base material is cut, but also the bond between the cells is cut, so that the cell cannot be taken out in a proliferating shape (for example, a sheet shape) There has been a problem that the properties of the material will change.

  In recent years, using a base material in which a polymer having a lower critical solution temperature such as poly-N-isopropylacrylamide is coated on the surface of a cell culture vessel very thinly, the polymer shows a hydrophobic state at the cell culture temperature, and the cell becomes a polymer. Adhesion and low temperature treatment of the polymer after culturing to make it hydrophilic, reducing the adhesion between the cell and the polymer, making the cell a sheet from the substrate without using hydrolytic enzymes or chemicals A technique for peeling is reported (for example, see Patent Documents 1 and 2 and Non-Patent Document 1).

  However, a polymer such as poly-N-isopropylacrylamide has low adhesiveness with a plastic surface such as polystyrene, and when applied to water, the applied polymer layer easily peels off. In order to prevent such a polymer layer from being peeled off from the plastic surface even when it is exposed to water, it is necessary to fix the polymer. As one of the methods, there is a method in which a solution of N-isopropylacrylamide (monomer) is applied to the surface of a cell culture substrate and graft polymerization is performed by electron beam irradiation (see, for example, Patent Document 3).

  Graft polymerization by electron beam irradiation always causes a cross-linking reaction between the polymers at the same time as the polymerization, and the temperature response speed of the polymer greatly decreases as the degree of cross-linking progresses, and the low temperature is maintained to make the polymer hydrophilic. There is a problem that it takes a long time, and in the meantime, the cells are also exposed to a low temperature state for a long time and damaged. In addition, when the cell culture substrate produced by this method is subjected to radiation (eg, γ-ray) sterilization treatment, the temperature responsiveness of the polymer is greatly reduced, and the original cells are not easily detached. there were.

  On the other hand, it comprises a polymer hydrogel obtained by polymerizing a water-soluble organic monomer by irradiation with radiation in the presence of a water-swellable clay mineral uniformly dispersed in water. A cell culture substrate having a three-dimensional network structure composed of a water-swellable clay mineral and a polymer having a lower critical solution temperature as described above is disclosed (for example, see Patent Document 4).

  In the field of biochemistry, there has been a demand for a cell culture substrate in which a cell culture substrate is integrated with a container such as a plastic culture dish in terms of cell culture operations and the like. However, the above-mentioned conventional literature does not disclose specific means for such an integrated cell culture container.

Japanese Patent Publication No. 6-104061 JP-A-5-192138 JP-A-5-192130 JP 2006-288251 A

Masayuki Yamato, Mitsuo Okano, “Frontiers of Nanobiotechnology”, Chapter 6, P.A. 340-P. 347, CMC Publishing (published in 2003)

  The problem to be solved by the present invention is a cell culture substrate in which the polymer (B) having a lower critical solution temperature contained in the substrate is not crosslinked, and further has hydrophobicity and hydrophilicity with respect to environmental temperature. Rapid change, without using a protease such as trypsin when separating and recovering cultured cells, without damaging the cells, quickly cultivating rapidly cultured cells from the culture substrate surface, The object is to provide a cell culture substrate that can be recovered.

  Another object of the present invention is a method for producing the above cell culture substrate, wherein the polymer (B) having a lower critical solution temperature contained in the substrate is not crosslinked, and Without using a method such as electron beam irradiation, the cell culture substrate can be easily adhered to the surface of a plastic container. Further, depending on the type (adhesiveness) of cells to be cultured, An object of the present invention is to provide a production method using a simple apparatus and process, in which the length and density of the combined body (B) can be easily adjusted.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the polymer (A) of the (meth) acrylic acid ester monomer (a), one or more selected from water-swellable clay minerals and silica The cell culture substrate containing the inorganic material (C) and the polymer (B) having a lower critical solution temperature has good culturing properties for various cells, and reduces the temperature of the cultured cells. The inventors have found that the properties that can be easily peeled and the culturing properties and the peelability can be easily prepared according to the cell type, and the present invention has been completed.

That is, the present invention is a polymer of monomers (a) which is the table by the following formula (1) and (A), a polymer having a lower critical solution temperature (B), and is selected from water-swellable clay mineral and silica containing one or more inorganic materials (C) and that,
The mass ratio ((C) / (A)) between the polymer (A) and the inorganic material (C) is in the range of 0.01 to 10,
Provided is a cell culture substrate in which the content of the polymer (B) in the whole cell culture substrate is 0.0001% by mass to 40% by mass .

（式中、Ｒ_１は水素原子またはメチル基、Ｒ_２は炭素原子数２〜３のアルキレン基、Ｒ_３は水素原子または炭素原子数１〜２のアルキル基を表し、ｎは１〜９の整数を表す。）

Wherein R ₁ represents a hydrogen atom or a methyl group, R ₂ represents an alkylene group having 2 to 3 carbon atoms, R ₃ represents a hydrogen atom or an alkyl group having 1 to 2 carbon atoms, and n represents 1 to 9 Represents an integer.)

In the present invention, the polymer (A) of the monomer (a) represented by the formula (1) and at least one inorganic material (C) selected from water-swellable clay mineral and silica are mutually A complex formed by acting;
Containing a polymer (B) having a lower critical solution temperature ,
The mass ratio ((C) / (A)) between the polymer (A) and the inorganic material (C) is in the range of 0.01 to 10,
A cell culture substrate having a content of the polymer (B) of 0.0001% by mass to 40% by mass with respect to the entire cell culture substrate ,
Provided is a cell culture substrate characterized in that the polymer (B) is exposed on the cell culture surface of the cell culture substrate.

In the present invention, the polymer (B) is a polymer of at least one monomer (b) selected from the group consisting of N-substituted (meth) acrylamide derivatives and N, N-disubstituted (meth) acrylamide derivatives. A method for producing a cell culture substrate,
The monomer (a) and the inorganic material (so that the concentration of the water-swellable inorganic material (C) in the aqueous medium (W) falls within the range represented by the following formula (2) or formula (3): C) and a polymerization initiator (D) are mixed in an aqueous medium (W), and then the monomer (a) is polymerized to form a composite (X) of the polymer (A) and the inorganic material (C). A first step of producing a dispersion (L) of
A second step of forming a thin layer of the composite (X) by applying the dispersion (L) to a support and then drying;
A third step in which a solution obtained by dissolving a water-insoluble polymerization initiator (D) in a solvent (E) is applied to the surface (S) of the thin layer of the composite (X), and the solvent (E) is volatilized. ,
Provided is a method for producing a cell culture substrate, comprising: applying an aqueous solution of the monomer (b) to the surface (S), and then sequentially performing a fourth step of polymerizing the monomer (b) by ultraviolet irradiation. To do.
Formula (2) When Ra <0.19
Concentration (% by mass) of inorganic material (C) <12.4Ra + 0.05

Formula (3) When Ra ≧ 0.19
Concentration (% by mass) of inorganic material (C) <0.87Ra + 2.17
(In the formula, the concentration (% by mass) of the inorganic material (C) is a value obtained by dividing the mass of the inorganic material (C) by the total mass of the aqueous medium (W) and the inorganic material (C) and multiplying by 100, Ra Is a mass ratio ((C) / (A)) between the inorganic material (C) and the polymer (A).)

In the present invention, the polymer (B) is a polymer of at least one monomer (b) selected from the group consisting of N-substituted (meth) acrylamide derivatives and N, N-disubstituted (meth) acrylamide derivatives. A method for producing a cell culture substrate,
An aqueous medium (W) in which the monomer (a), the inorganic material (C) and the polymerization initiator (D) are mixed is applied to a support,
A first step of forming a thin layer of a composite (X) of the polymer (A) and the inorganic material (C) by polymerizing the monomer (a);
A second step in which a solution in which a water-insoluble polymerization initiator (D) is dissolved in a solvent (E) is applied to the surface (S) of the thin layer of the composite (X), and the solvent (E) is volatilized;
Provided is a method for producing a cell culture substrate, comprising applying the aqueous solution of the monomer (b) to the surface (S) and then sequentially performing a third step of polymerizing the monomer (b) by ultraviolet irradiation. To do.

Furthermore, the present invention provides the polymer (B) comprising at least one monomer (b) selected from the group consisting of N-substituted (meth) acrylamide derivatives and N, N-disubstituted (meth) acrylamide derivatives. A method for producing a cell culture substrate,
The monomer (a), the inorganic material (C), and the concentration of the inorganic material (C) in the aqueous medium (W) are in a range represented by the following formula (2) or formula (3): After mixing the polymerization initiator (D) with the aqueous medium (W), the monomer (a) is polymerized to disperse the complex (X) of the polymer (A) and the inorganic material (C). A first step of producing a liquid (L);
The polymer (B), which is a polymer of the monomer (b), is added to the dispersion (L), mixed, applied to a support, and then dried in order. A method for producing a cell culture substrate is provided.
Formula (2) When Ra <0.19
Concentration (% by mass) of inorganic material (C) <12.4Ra + 0.05
Formula (3) When Ra ≧ 0.19
Concentration (% by mass) of inorganic material (C) <0.87Ra + 2.17
(In the formula, the concentration (% by mass) of the inorganic material (C) is a value obtained by dividing the mass of the inorganic material (C) by the total mass of the aqueous medium (W) and the inorganic material (C) and multiplying by 100, Ra Is a mass ratio ((C) / (A)) between the inorganic material (C) and the polymer (A).)

  The greatest feature of the cell culture substrate of the present invention is that the constituent parts of the polymer (A) and the inorganic material (C) are responsible for cell growth, and the polymer (B) having LCST peels off cells due to temperature changes. The two parts can be controlled independently according to the cell type. For example, since the culture temperature (37 ° C.) is higher than the LCST (32 ° C.) of poly-N-isopropylacrylamide at the time of culture, poly-N-isopropylacrylamide is in a water-insoluble (hydrophobic) state, and the cell is the base material. Although it grows on the surface, when the temperature is lowered to 32 ° C. or lower (for example, 20 ° C.), poly-N-isopropylacrylamide becomes water-soluble and extends from the substrate surface to the aqueous solution. Peel while releasing.

  The polymer (A) and the polymer (B) interact and bond with the inorganic material (C) mainly through ionic bonds and hydrogen bonds. This bonding force is strong, and the polymer and the inorganic material (C) cannot be easily separated. For example, a hydrogel having a three-dimensional network structure composed of poly-N-isopropylacrylamide and clay mineral (water content 90%) exhibits a tensile strength at break of 95 kPa (see Patent Document 4) JP-A-2006-288251. ).

  The cell culture substrate of the present invention comprises a thin layer of a composite (X) in which an inorganic material (C) and a polymer (A) have a substantially uniform layered structure, and from the thin layer toward the surface. It is comprised from the extending | stretching polymer (B).

  By appropriately adjusting the length (molecular weight) and density (content) of the polymer (B), the thin layer surface of the composite (X) is appropriately exposed without being completely covered by the polymer (B). Thus, good cell proliferation and cell detachability can be maintained.

  The cell culture substrate of the present invention has a high rate of change in hydrophobicity and hydrophilicity with respect to environmental temperature, and the length of the polymer (B) having a lower critical solution temperature depending on the type of cell to be cultured (adhesiveness). The sheath density can be easily adjusted, and the cultured cells can be quickly detached and recovered from the surface of the culture substrate without using a drug (such as trypsin).

  In addition, the production method of the present invention is such that the polymer (B) having a lower critical solution temperature contained in the base material is not cross-linked (a more rapid temperature responsiveness can be maintained), and electron beam irradiation Without using such a method, the cell culture substrate can be easily adhered to a support (such as a plastic culture vessel), and further, depending on the type of cells to be cultured (adhesiveness). The length and density of the polymer (B) can be easily adjusted, and the apparatus and the process are simple.

It is the photograph image | photographed with the optical microscope of the cell-culture base material (Example 8) which apply | coated the dispersion liquid (L5) in circular pattern shape.

  The monomer (a) used in the present invention can be suitably used as long as the polymer interacts with a clay mineral and can form an organic-inorganic composite hydrogel by polymerization. Among them, (meth) acrylic acid polypropylene glycol And a polyethylene glycol ester monomer are preferably used, and a monomer (a) of the following formula (1) is particularly preferably used.

（式中、Ｒ_１は水素原子またはメチル基、Ｒ_２は炭素原子数２〜３のアルキレン基、Ｒ_３は水素原子または炭素原子数１〜２のアルキル基であり、ｎは１〜９である。）

Wherein R ₁ is a hydrogen atom or a methyl group, R ₂ is an alkylene group having 2 to 3 carbon atoms, R ₃ is a hydrogen atom or an alkyl group having 1 to 2 carbon atoms, and n is 1 to 9 is there.)

  By using these monomers (a), the initial cell adhesion can be easily adjusted, and a cell culture substrate having good cell growth and detachability can be obtained. Moreover, when this cell culture substrate is laminated on the surface of a support such as a plastic substrate such as polystyrene, the adhesiveness between the two is strong and the production can be simplified.

  One or more of the above-mentioned monomers (a) may be mixed and used depending on required mechanical properties and surface properties. In addition, as necessary, for example, an acrylic monomer having an anion group such as a sulfone group or a carboxyl group, quaternary, etc., as long as it does not affect the culture properties and physical properties of the cell culture substrate. Acrylic monomer having a cationic group such as an ammonium group, an acrylic monomer having a zwitterionic group having a quaternary ammonium group and a phosphate group, an acrylic monomer having an amino acid residue having a carboxyl group and an amino group, sugar Acrylic monomer having a residue, acrylic monomer having a hydroxyl group, polyethylene glycol, acrylic monomer having a polypropylene glycol chain, and also having a hydrophilic chain such as polyethylene glycol and a hydrophobic group such as a nonylphenyl group Amphiphilic acrylic monomer, polyethylene Glycol diacrylate, N- substituted (meth) acrylamide derivatives, N, N-di-substituted (meth) acrylamide derivatives, N, may be used in combination such as N'- methylenebisacrylamide.

  The inorganic material (C) used in the present invention is one or more inorganic materials selected from water-swellable clay minerals and silica. Examples of water-swellable clay minerals include water-swellable clay minerals that can be peeled in layers, preferably clay minerals that can swell and uniformly disperse in water or a mixed solution of water and an organic solvent, particularly preferably water. An inorganic clay mineral that can be uniformly dispersed at a molecular level (single layer) or at a level close thereto is used. Specific examples include water-swellable hectorite containing sodium as an interlayer ion, water-swellable montmorillonite, water-swellable saponite, and water-swellable synthetic mica. You may mix and use these clay minerals.

Examples of the silica (SiO ₂ ) used in the present invention include colloidal silica, preferably colloidal silica that can be uniformly dispersed in an aqueous solution and has a particle size of 10 nm to 500 nm, and particularly preferably a colloidal particle having a particle size of 10 to 50 nm. Silica is used.

  In the cell culture substrate of the present invention, the mass ratio ((C) / (A)) of the polymer (A) to the inorganic material (C) is preferably 0.01 to 10, and preferably 0.03 to 0.03. 5 is more preferable, and 0.05 to 3 is particularly preferable. When the mass ratio ((C) / (A)) is within this range, a composite structure of a clay mineral or silica and a polymer (A) (for example, a shell portion composed mainly of a clay mineral and a polymer) (A) A core-shell structure consisting of a core (inner) part composed mainly of (A), a uniform structure in which clay mineral and polymer (A) are uniformly combined, etc.) can be easily designed, and the surface characteristics of the resulting coating film (For example, the degree of hydrophilicity / hydrophobicity and cell culture properties) and physical properties of the coating film are good, a uniform coating film is obtained, adhesion to the support is good, and there is no brittleness, which is preferable.

  In the cell culture substrate of the present invention, the content of the polymer (B) with respect to the entire substrate is preferably 0.0001% by mass to 40% by mass, and preferably 0.01 to 30% by mass. More preferably, the content is 1 to 20% by mass.

  When the content of the polymer (B) is 0.0001% by mass to 40% by mass, the cell adhesion and proliferation of the culture substrate and the peelability when the temperature is lowered are good, and the surface of the culture substrate is smooth. In addition, the coating property is good and the coating property when being laminated on the surface of the plastic substrate and the adhesion to the surface of the substrate are good.

  The polymer (B) having a lower critical solution temperature (hereinafter abbreviated as LCST) used in the present invention can be suitably used as long as it has a portion showing LCST in the molecule. Among them, a polymer of at least one monomer (b) selected from the group consisting of N-substituted (meth) acrylamide derivatives and N, N-disubstituted (meth) acrylamide derivatives is preferable. Examples of such monomers (b) include N-isopropyl (meth) acrylamide, Nn-propyl (meth) acrylamide, N-cyclopropyl (meth) acrylamide, N-ethoxyethyl (meth) acrylamide, N- Tetrahydrofurfuryl (meth) acrylamide, N-ethyl (meth) acrylamide, N-ethyl-N-methyl (meth) acrylamide, N, N-diethyl (meth) acrylamide, N-methyl-Nn-propyl (meth) Examples include acrylamide, N-methyl-N-isopropyl (meth) acrylamide, N- (meth) acryloylpiperidine, and N- (meth) acryloylpyrrolidine.

  These monomers may be used alone, or a plurality of monomers may be mixed and used as necessary. Furthermore, a copolymer of the monomer (b) and other water-soluble organic monomer or organic solvent-soluble organic monomer should also be used as long as the obtained polymer exhibits both hydrophilicity and hydrophobicity. I can do it.

  The above-mentioned lower critical solution temperature (LCST) is a temperature at which the polymer becomes insoluble in water (indicating hydrophobicity) above this temperature, and becomes water-soluble (indicating hydrophilicity) below this temperature. That is. For example, the LCST of poly-N-isopropylacrylamide is 32 ° C.

Next, the production method of the present invention will be described.
The cell culture substrate of the present invention can be produced by the following three methods.
That is, as a first production method, the monomer (C) is adjusted so that the concentration of the inorganic material (C) in the aqueous medium (W) is in the range represented by the following formula (2) or formula (3). a), the inorganic material (C) and the polymerization initiator (D) are mixed in an aqueous medium (W), and then the monomer (a) is polymerized to polymer (A) and the inorganic material (C). A first step of producing a dispersion (L) of a complex (X) with
A second step of forming a thin layer of the composite (X) by applying the dispersion (L) to a substrate and then drying;
A third step in which a solution obtained by dissolving a water-insoluble polymerization initiator (D) in a solvent (E) is applied to the surface (S) of the thin layer of the composite (X), and the solvent (E) is volatilized. ,
A method for producing a cell culture substrate, comprising: applying an aqueous solution of the monomer (b) to the surface (S); and sequentially performing a fourth step of polymerizing the monomer (b) by irradiation with ultraviolet rays. .
Formula (2) When Ra <0.19
Concentration (% by mass) of inorganic material (C) <12.4Ra + 0.05
Formula (3) When Ra ≧ 0.19
Concentration (% by mass) of inorganic material (C) <0.87Ra + 2.17
(In the formula, the concentration (% by mass) of the inorganic material (C) is a value obtained by dividing the mass of the inorganic material (C) by the total mass of the aqueous medium (W) and the inorganic material (C) and multiplying by 100, Ra Is a mass ratio ((C) / (A)) between the inorganic material (C) and the polymer (A).)

  The monomer (a), the inorganic material (C) and the monomer (b) used in this production method can be the same as those described in the description of the cell culture substrate, and will be omitted.

  The aqueous medium (W) used in the production method of the present invention can contain the monomer (a), the inorganic material (C), etc., and it is sufficient that an organic-inorganic composite dispersion liquid having good physical properties is obtained by polymerization. It is not limited. For example, it may be water or an aqueous solution containing a solvent miscible with water and / or other compounds, and further includes antiseptics and antibacterial agents, coloring agents, fragrances, enzymes, proteins, collagen, sugars, Amino acids, cells, DNAs, salts, water-soluble organic solvents, surfactants, polymer compounds, leveling agents and the like can be included.

As the polymerization initiator (D) used in the present invention, a known radical polymerization initiator can be appropriately selected and used. Preferably, those having water solubility or water dispersibility and uniformly contained in the entire system are preferably used. Specifically, as a polymerization initiator, a water-soluble peroxide such as potassium peroxodisulfate or ammonium peroxodisulfate, a water-soluble azo compound such as VA-044, V-50, V-501 (all of which are Wako Pure Chemical Industries, Ltd.) In addition to Yaku Kogyo Co., Ltd., a mixture of Fe ²⁺ and hydrogen peroxide is exemplified.

  As the catalyst, tertiary amine compounds such as N, N, N ′, N′-tetramethylethylenediamine are preferably used. However, the catalyst is not necessarily used. The polymerization temperature is, for example, 0 ° C. to 100 ° C. according to the type of polymerization catalyst or initiator. The polymerization time can also be carried out for several tens of seconds to several tens of hours.

  On the other hand, the photopolymerization initiator is less susceptible to oxygen inhibition and has a high polymerization rate, and therefore is suitably used as the polymerization initiator (D). Specifically, acetophenones such as p-tert-butyltrichloroacetophenone, benzophenones such as 4,4′-bisdimethylaminobenzophenone, ketones such as 2-methylthioxanthone, benzoin ethers such as benzoin methyl ether, hydroxy Examples include α-hydroxy ketones such as cyclohexyl phenyl ketone, phenyl glyoxylates such as methyl benzoyl formate, and metallocenes.

  The photopolymerization initiator is water-insoluble. The term “water-insoluble” as used herein means that the amount of polymerization initiator dissolved in water is 0.5% by mass or less. By using a water-insoluble polymerization initiator, the initiator is more likely to be present in the vicinity of the clay mineral, the number of initiation reaction points from the vicinity of the clay mineral is increased, and the resulting polymer (A) and inorganic material (C The particle size distribution of the complex (X) with N) is narrow, and the dispersion (L) has high stability, which is preferable.

  It is preferable that a solution prepared by dissolving the photopolymerization initiator in a solvent (E) compatible with the aqueous medium (W) is added to the aqueous medium (W). By this method, the photopolymerization initiator can be more uniformly dispersed, and a composite (X) having a more uniform particle diameter can be obtained.

  The mass ratio (D) / (E) of the photopolymerization initiator (D) to the solvent (E) in the solution obtained by dissolving the photopolymerization initiator (D) in the solvent (E) is 0.001 to 0.1. Preferably, 0.01 to 0.05 is more preferable. When the amount is 0.001 or more, a sufficient amount of radicals are generated by irradiation with ultraviolet rays, so that the polymerization reaction can be suitably performed. The cost can be reduced.

  The addition amount of the solution obtained by dissolving the photopolymerization initiator (D) in the solvent (E) is monomer (a), inorganic material (C), aqueous medium (W), polymerization initiator (D), and solvent (E). The total mass is preferably 0.1% by mass to 5% by mass, and more preferably 0.2% by mass to 2% by mass. When the dispersion amount is 0.1% by mass or more, the polymerization is sufficiently started, and when it is less than 5% by mass, odor is generated due to an increase in the polymerization initiator in the composite (X), and further, once dispersed. This is preferable because problems such as aggregation of the resulting photopolymerization initiator can be reduced and a uniform dispersion (L) of the composite (X) can be obtained.

  By using the water-insoluble polymerization initiator (D), in the fourth step, when the aqueous solution of the monomer (b) is applied, there is no elution of the initiator, and the starting reaction point from the vicinity of the clay mineral or silica is The interaction between the obtained polymer (B) and the inorganic material (C) becomes stronger, which is preferable.

  The greatest characteristic of the cell culture substrate production of the present invention is that the concentration (mass%) of the inorganic material (C) in the aqueous medium is in the range represented by the formula (2) or the formula (3). When the concentration (% by mass) of the inorganic material (C) in the aqueous medium is within the above range, a good dispersion (L) of the composite (X) can be obtained, and the coating onto the support is easy and smooth. A uniform thin coating film is obtained, which is preferable.

  The dispersion (L) produced by the production method of the present invention may be used as it is, or may be used after undergoing a purification step such as washing with water. Further, a leveling agent, a surfactant, a peptide, a protein, collagen, an amino acid, a polymer compound or the like may be added to the dispersion (L).

  The method for applying the dispersion liquid (L) to the support in the second step of the production method may be a known and conventional method. For example, a method of casting the dispersion on a support, a coater method using a bar coater or a spin coater, or a spraying method such as spraying, a method of transferring a dispersion to a patterned rubber plate and then transferring it to the support, Examples thereof include a pattern-like coating in which a portion not coated on the support is shielded in advance and the shielded portion is removed after coating, and a dispersion coating method using an ink jet printer method.

  When the dispersion (L) is applied in a pattern on the surface of a support on which cells do not adhere or proliferate (for example, polystyrene that has not been subjected to corona discharge treatment), the cells are not applied when the distance between the applied parts is sufficiently narrow Can grow over the part and finally cultivate the cells across the entire surface of the support, furthermore, the cultured cell layer has less adhesion points with the support, and detachment occurs more easily, preferable. The distance between the boundary between the coated portions (the width of the uncoated portion) is preferably 300 μm or less, more preferably 200 μm or less, and most preferably 100 μm or less. If it is 300 μm or less, the cells can sufficiently grow beyond this uncoated width, and a good cell layer can be cultured.

  The drying method may be any method as long as the volatile components in the dispersion (L) are volatilized and a thin layer of the composite (X) is formed. For example, room temperature natural drying, room temperature air or heating or hot air drying, far infrared drying, and the like can be mentioned. Alternatively, a method of applying hot air or heating the dispersion while rotating it with a spin coater can also be mentioned.

  In the third step of the production method, a solution obtained by dissolving the water-insoluble polymerization initiator (D) in the solvent (E) is applied to the surface (S) of the thin layer of the complex (X) or the solvent ( As the volatilization method of E), a known and commonly used method may be used according to the second step.

  The solution (D + E) applied to the surface (S) penetrates into the thin layer of the composite (X), and the initiator is uniformly distributed throughout the thin layer of the composite (X) by volatilization of the solvent (E). It will be in a state that exists. As the water-insoluble polymerization initiator (D), those according to the above photopolymerization initiator can be used. By using the water-insoluble polymerization initiator (D), there is little elution of the initiator when applying the aqueous solution of the monomer (b) in the fourth step, and there are many initiation reaction points from the vicinity of the clay mineral or silica. This is preferable because the interaction between the resulting polymer (B) and the inorganic material (C) becomes stronger.

  The mass ratio (D) / (E) of the polymerization initiator (D) to the solvent (E) in a solution in which the water-insoluble polymerization initiator (D) is dissolved in the solvent (E) is 0.001 to 0.00. 1 is preferable, and 0.01 to 0.05 is more preferable. When the amount is 0.001 or more, a sufficient amount of radicals are generated by irradiation with ultraviolet rays, so that the polymerization reaction can be suitably performed. The cost can be reduced.

  As the solvent (E) of the present invention, a water-soluble solvent that can dissolve the photopolymerization initiator (D) or the water-insoluble polymerization initiator (D), or a photopolymerization initiator (D) and a water-insoluble polymerization initiator. An acrylic monomer (a ′) that dissolves (D) and has an HLB (hydrophilic / hydrophobic balance) value of 8 or more can be used. The HLB value here is a value determined according to the Davis formula (“surfactant—physical properties / application / chemical ecology”, edited by Fumio Kitahara, Kodansha, 1979, p. 24-27). For example, polypropylene glycol diacrylates such as tripropylene glycol diacrylate, polyethylene glycol diacrylates, polypropylene glycol acrylates such as pentapropylene glycol acrylate, polyethylene glycol acrylates, methoxyethyl acrylate, methoxytriethylene glycol acrylate Examples thereof include non-methyl polyethylene glycol acrylates, nonyl phenoxy polyethylene glycol acrylates, N-substituted acrylamides such as dimethyl acrylamide, hydroxyethyl acrylate, hydroxypropyl acrylate, and the like. It is preferable that the acrylic monomer as the solvent (E) has an HLB value of 8 or more because it is excellent in solubility or dispersibility in the aqueous medium (W). These acrylic monomers can be used by mixing one or more.

  In addition, as the solvent (E) of the present invention, a solvent that can dissolve the photopolymerization initiator (D) and the water-insoluble polymerization initiator (D) and has a certain level of water solubility can be used. The water-soluble solvent mentioned here is preferably a solvent that can dissolve 50 g or more with respect to 100 g of water. When the solubility in water is 50 g or more, the water-insoluble polymerization initiator (D) has good dispersibility in the aqueous medium (W), and the resulting composite (X) has a uniform particle size. The stability of the dispersion (L) is high and preferable.

For example, examples of the water-soluble solvent include amides such as dimethylacetamide and dimethylformamide, alcohols such as methanol, ethanol, and 2-propanol, dimethyl sulfoxide, and tetrahydrofuran. A mixture of these solvents may be used. In the fourth step of the production method, the aqueous solution of the monomer (b) is applied to the surface (S) using a known and conventional method according to the second step. Good.

  As for the density | concentration of the monomer (b) in the aqueous solution of a monomer (b), 1-20 mass% is preferable, and it is still more preferable that it is 5-18 mass%. If it is 1% by mass or more, a sufficiently long polymer (B) can be obtained, and the cell detachability can be maintained. If it is 20% by mass or less, sufficient cell proliferation can be maintained, and the performance is good. A cell culture substrate can be produced.

  The monomer (b) applied to the surface (S) penetrates into the thin layer of the composite (X) and is polymerized by irradiation with ultraviolet rays. The surface of the cell culture substrate obtained by this production method is not completely covered with the polymer of the monomer (b) (polymer (B)), and the polymer (B) is complex (X). The structure extends from the thin layer and the surface of the thin layer is appropriately exposed. The polymer (B) is bonded to the clay mineral by ionic bond or hydrogen bond from the thin layer of the complex (X) to the surface, and the bond is not broken even in physical force or water, and is stable. It has a structure. Further, depending on the type of cells to be cultured, the length (molecular weight) and density (content in the thin layer of the complex (X)) of the polymer (B) are determined depending on the aqueous solution concentration of the monomer (b). And the coating amount can be adjusted as appropriate.

As the light used in this step, electron beam, γ-ray, X-ray, ultraviolet ray, visible light, etc. can be used. Among them, the apparatus and handling are easy and the viewpoint of not causing crosslinking simultaneously with polymerization of the monomer (b). It is preferable to use ultraviolet rays. The intensity of the irradiated ultraviolet light is preferably 10 to 500 mW / cm ² and the irradiation time is generally about 0.1 to 200 seconds. In radical polymerization by normal heating, oxygen works as an inhibitor of polymerization. However, in the present invention, it is not always necessary to prepare a solution in an atmosphere in which oxygen is blocked and to perform polymerization by ultraviolet irradiation, and these are performed in an air atmosphere. It is possible. However, it may be desirable that the polymerization rate can be further increased by performing ultraviolet irradiation in an inert gas atmosphere.

  In the first production method, by adjusting the ratio of the monomer (a) and the inorganic material (C), the cell growth rate can be widely adjusted, and the type, concentration and application of the monomer (b) can be adjusted. By adjusting the amount, it is possible to control the cell peeling rate due to temperature change.

As a second production method of the present invention, an aqueous medium (W) obtained by mixing the monomer (a), the inorganic material (C) and the polymerization initiator (D) is applied to a support, and the monomer (a a first step of forming a thin layer of the composite (X) of the polymer (A) and the inorganic material (C) by polymerizing a);
A second step in which a solution in which a water-insoluble polymerization initiator (D) is dissolved in a solvent (E) is applied to the surface (S) of the thin layer of the composite (X), and the solvent (E) is volatilized;
A method for producing a cell culture substrate, comprising applying an aqueous solution of the monomer (b) to the surface (S) and then sequentially performing a third step of polymerizing the monomer (b) by irradiation with ultraviolet rays. .

  The concentration of the coating method, the ultraviolet ray, the polymerization initiator (D) and the monomer (b) in this production method is all in accordance with the first production method. In the first step of this production method, since the thin layer of the composite (X) is produced directly from the reaction solution, the concentration of the inorganic material (C) in the aqueous medium (W) is expressed by the formula (2) or the formula (3). It is not necessary to be in the range represented by. The surface structure of the cell culture substrate obtained by this production method is almost the same as that obtained by the first production method.

As a third production method of the present invention, the monomer (C) is adjusted so that the concentration of the inorganic material (C) in the aqueous medium (W) falls within the range represented by the following formula (2) or formula (3). a), the inorganic material (C), and the polymerization initiator (D) are mixed in an aqueous medium (W), and then the monomer (a) is polymerized to polymer (A) and the inorganic material (C). A first step of producing a dispersion (L) of a complex (X) with
The polymer (B) is added to the dispersion liquid (L), mixed uniformly, coated on a support, and then subjected to a second step of drying, in order to produce a cell culture substrate Is the method.
Formula (2) When Ra <0.19
Concentration (% by mass) of inorganic material (C) <12.4Ra + 0.05
Formula (3) When Ra ≧ 0.19
Concentration (% by mass) of inorganic material (C) <0.87Ra + 2.17
(In the formula, the concentration (% by mass) of the inorganic material (C) is a value obtained by dividing the mass of the inorganic material (C) by the total mass of the aqueous medium (W) and the inorganic material (C) and multiplying by 100, Ra Is a mass ratio ((C) / (A)) between the inorganic material (C) and the polymer (A).)

  The polymer (B) in this production method preferably has a 10% by mass aqueous solution having a viscosity of 20 to 2000 mPa · s (using a DIGITAL VISCOMATE MODEL VM-100A viscometer manufactured by Yamaichi Electronics Co., Ltd.). The polymer having a viscosity of ˜1000 mPa · s is more preferred, and the polymer having a viscosity of 200 to 800 mPa · s is most preferred. If it is 20 mPa · s or more, sufficient cell detachability can be maintained, and if it is 1000 mPa · s or less, sufficient cell growth can be maintained, and a cell culture substrate with good performance can be produced.

Moreover, it is preferable that the weight average molecular weight Mw is 1 * 10 < ⁴ > -2 * 10 < ⁷ >, and, as for the polymer (B) in this manufacturing method, it is more preferable that it is 1 * 10 < ⁵ > -5 * 10 < ⁶ >. If it is 1 × 10 ⁴ or more, sufficient cell detachability can be maintained, and if it is 2 × 10 ⁷ or less, sufficient cell proliferation can be maintained, and a cell culture substrate with good performance can be produced.

  The surface of the cell culture substrate obtained by this production method is not covered with the polymer (B), but the polymer (B) extends from the thin layer of the complex (X), The surface of the thin layer is appropriately exposed. The polymer (B) is bonded to the clay mineral or silica from the thin layer of the complex (X) to the surface by ionic bond or hydrogen bond, and the bond does not break even in physical force or water. It has a stable structure. Further, the length (molecular weight) and content of the polymer (B) can be appropriately adjusted according to the type of cells to be cultured.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, the scope of the present invention is not limited only to these Examples.

Example 1
This example is an example of producing a cell culture substrate by the first production method.
[Preparation of reaction solution containing monomer (a), inorganic material (C) and aqueous medium (W)]
0.6 g of 2-methoxyethyl acrylate (manufactured by Toagosei Co., Ltd.) as monomer (a), 0.3 g of water-swellable clay mineral Laponite XLG (manufactured by Rockwood Additives Ltd.) as an inorganic material (C), aqueous medium (W The reaction solution (1) was prepared by uniformly mixing 20 g of water.

[Preparation of solution in which polymerization initiator (D) is dissolved in solvent (E)]
As a solvent (E), 9.8 g of 2-propanol and 0.2 g of 1-hydroxycyclohexyl phenyl ketone “Irgacure 184” (manufactured by Ciba Geigy) as a polymerization initiator (D) are mixed uniformly to obtain a solution (2) Was prepared.

[Preparation of dispersion (L) of complex (X) (first step)]
50 μl of the solution (2) is added to the total amount of the reaction solution (1) and dispersed uniformly, and then irradiated with ultraviolet rays having an ultraviolet intensity at 365 nm of 40 mW / cm ² for 180 seconds to give a milky white composite (X) dispersion. (L1) was produced.
Ra = 0.5 in this reaction system, concentration (mass%) of inorganic material (C) = 1.48 (%) <0.87Ra + 2.17 = 2.61

[Preparation of thin layer of composite (X) (second step)]
The dispersion (L1) of the composite (X) is placed in a polystyrene petri dish (Advantech Toyo Co., Ltd., PD-50K) having a diameter of 50 mm, and the dispersion is applied to the surface of the petri dish at 2000 revolutions using a spin coater. After thinly coating, it was dried in a hot air dryer at 80 ° C. for 10 minutes to prepare a thin layer of the composite (X).

[Coating of solution in which polymerization initiator (D) is dissolved in solvent (E) (third step)]
The solution (2) is placed in a petri dish, thinly applied at 2000 rpm with a spin coater, allowed to stand at room temperature for 5 minutes to evaporate ethanol, and a polymerization initiator (D) is deposited on the thin layer surface of the composite (X). It was applied to.

[Preparation of cell culture substrate (fourth step)]
2 ml of a 10% by mass aqueous solution of N-isopropylacrylamide (monomer (b), manufactured by Kojin Co., Ltd.) is placed in a petri dish and irradiated with ultraviolet rays having an ultraviolet intensity of 40 mW / cm ² at 365 nm for 60 seconds to give N-isopropylacrylamide. Polymerized. Next, after washing the petri dish with sterilized water, the petri dish was dried in a sterile bag at 40 ° C. for 5 hours to obtain a cell culture substrate 1.

[Culture of normal human dermal fibroblasts]
An appropriate amount of CS-C complete medium (Cell Systems Inc. medium) is added to the obtained cell culture substrate 1, and normal human dermal fibroblasts are seeded (seeding concentration is 1.2 × 10 ⁴ cells / cm 2). ² ) Culture was performed at 37 ° C. in 5% carbon dioxide. After confirming that the cells had sufficiently proliferated, the medium (at 37 ° C.) was sucked up, the medium at 4 ° C. was added, and allowed to stand for a certain period of time, and the proliferated cells were naturally detached. The ratio of the area of the detached cell part and the total area of the cells grown before detachment was determined. The time taken for this peeling was recorded. (Table 1, cell peeling recovery rate = 93%, required time was 18 minutes).

(Example 2)
This example is an example of producing a cell culture substrate by the first production method.
A cell culture substrate 2 was prepared in the same manner as in Example 1 except that a 17% by mass aqueous solution of N-isopropylacrylamide was used as the monomer (b) in the fourth step.

[Culture of normal human dermal fibroblasts]
In the same manner as in Example 1, normal human dermal fibroblasts were cultured. After confirming that the cells had sufficiently proliferated, the medium (at 37 ° C.) was replaced with a medium at 4 ° C. and allowed to stand for a certain period of time to allow the grown cells to spontaneously detach. The ratio of the area of the detached cell part and the total area of the cells grown before detachment was determined. The time taken for this peeling was recorded. (Table 1, cell peeling recovery rate = 98%, required time was 10 minutes).

  From Examples 1 and 2 above, an increase in cell detachability was observed by increasing the concentration of the monomer (b).

(Example 3)
This example is an example of producing a cell culture substrate by the second production method.
[Preparation of reaction solution containing monomer (a), inorganic material (C) and aqueous medium (W)]
2.28 g of 2-methoxyethyl acrylate (manufactured by Toagosei Co., Ltd.) as monomer (a), 0.24 g of water-swellable clay mineral Laponite XLG (manufactured by Rockwood Additives Ltd.) as an inorganic material (C), aqueous medium (W ) Was mixed uniformly with 10 g of water to prepare a reaction solution (3).

[Preparation of solution in which polymerization initiator (D) is dissolved in solvent (E)]
The same solution (2) as in Example 1 was used.

[Preparation of thin layer of composite (X) (first step)]
50 μl of the solution (2) is added to the total amount of the reaction solution (3) and dispersed uniformly, and then placed in a polystyrene petri dish having a diameter of 50 mm (manufactured by Advantech Toyo Co., Ltd., PD-50K) and 2000 using a spin coater. After thinly coating the surface of the petri dish by rotation, ultraviolet light having an ultraviolet intensity at 365 nm of 40 mW / cm ² was irradiated for 180 seconds to prepare a thin layer of the composite (X).

[Coating of solution in which polymerization initiator (D) is dissolved in solvent (E) (second step)]
The solution (2) was put in a petri dish, thinly applied at 2000 rpm with a spin coater, allowed to stand at room temperature for 5 minutes to evaporate ethanol, and a polymerization initiator (D) was applied.

[Production of cell culture substrate (third step)]
2 ml of a 15% by weight aqueous solution of N-isopropylacrylamide (monomer (b), manufactured by Kojin Co., Ltd.) is placed in a petri dish and irradiated with ultraviolet rays having an ultraviolet intensity at 365 nm of 40 mW / cm ² for 60 seconds to give N-isopropylacrylamide. Polymerized. Next, after washing the petri dish with sterilized water, the petri dish was dried in a sterile bag at 40 ° C. for 5 hours to obtain a cell culture substrate 3.

[Culture of normal human dermal fibroblasts]
Normal human dermal fibroblasts were cultured on the obtained cell culture substrate 3 in the same manner as in Example 1. After confirming that the cells had sufficiently proliferated, the medium (at 37 ° C.) was replaced with a medium at 4 ° C. and allowed to stand for a certain period of time to allow the grown cells to spontaneously detach. The ratio of the area of the detached cell part and the total area of the cells grown before detachment was determined. (Table 1, cell peeling recovery rate = 100%, required time was 12 minutes).

Example 4
This example is an example of producing a cell culture substrate by the second production method.
A cell culture substrate 4 was prepared in the same manner as in Example 3 except that a 3% by mass aqueous solution of N-isopropylacrylamide was used as the monomer (b) in the third step.

[Culture of normal human dermal fibroblasts]
Normal human dermal fibroblasts were cultured on the obtained cell culture substrate 4 in the same manner as in Example 1. After confirming that the cells had sufficiently proliferated, the medium (at 37 ° C.) was replaced with a medium at 4 ° C. and allowed to stand for a certain period of time to allow the grown cells to spontaneously detach. The ratio of the area of the detached cell part and the total area of the cells grown before detachment was determined. (Table 1, cell peeling recovery rate = 78%, required time was 30 minutes).

  From Examples 3 and 4 above, changes in cell detachability were observed by changing the monomer (b) concentration.

(Example 5)
In this example, a cell culture substrate is produced by the third production method.
[Preparation of reaction solution containing monomer (a), inorganic material (C) and aqueous medium (W)]
As monomer (a), 0.32 g of 2-methoxyethyl acrylate (manufactured by Toagosei Co., Ltd.), as inorganic material (C), 0.08 g of water-swellable clay mineral Laponite XLG (manufactured by Rockwood Additives Ltd.), as surfactant A reaction solution (5) was prepared by uniformly mixing 100 μl of a 20 wt% sodium dodecylbenzenesulfonate (Wako Pure Chemical Industries, Ltd.) aqueous solution and 10 g of water as an aqueous medium (W).

[Preparation of solution in which polymerization initiator (D) is dissolved in solvent (E)]
The same solution (2) as in Example 1 was used.
[Preparation of dispersion (L) of complex (X) (first step)]
30 μl of the solution (2) is added to the total amount of the reaction solution (5) and dispersed uniformly, and then irradiated with ultraviolet rays having an ultraviolet intensity of 40 mW / cm ² at 365 nm for 180 seconds, and a milky white composite (X) dispersion (L5) was produced.

  Ra = 0.25 in this reaction system, concentration (mass%) of inorganic material (C) = 0.79 (%) <0.87 Ra + 2.17 = 2.39

[Preparation of aqueous solution of polymer (B)]
After mixing 1.7 g of N-isopropylacrylamide (manufactured by Kojin Co., Ltd.), 10 g of water, and 140 μl of the solution (2) as the monomer (b), the surroundings of the glass container in which the solution is placed are cooled (about 10 ° C. ) Irradiation with ultraviolet rays having an ultraviolet intensity at 365 nm of 40 mW / cm ² was carried out for 180 seconds to prepare an aqueous solution (5) of poly N-isopropylacrylamide. After adding 5 g of water to this solution and mixing it uniformly, the viscosity of this solution was measured using a DIGITAL VISCOMATE viscometer (MODEL VM-100A, manufactured by Yamaichi Electronics Co., Ltd.). The viscosity was 368 mPa · s. Met. The solution temperature at the time of measurement was 24.2 ° C.

Moreover, as a result of measuring with Shodex GPC System-21 apparatus (made by Showa Denko KK), the weight average molecular weight Mw of this poly N-isopropylacrylamide was 3.40 * 10 < ⁶ >. An N, N-dimethylformamide (DMF) solution containing 10 mmol / L LiBr was used as a solvent for the measurement. STANDARD SH-75 and SM-105 kit (manufactured by Showa Denko KK) were used as polystyrene standard substances used for the calculation of molecular weight.

[Preparation of cell culture substrate (second step)]
To the total amount of the dispersion (L5), 1.0 g (0.1 g solid content) of the above poly N-isopropylacrylamide aqueous solution was added and mixed uniformly, and then a 60 mm polystyrene petri dish (60 mm / non-treated dish, Asahi Techno Glass Co., Ltd.) was applied thinly on the surface of the petri dish at 2000 revolutions using a spin coater and dried in a hot air drier at 80 ° C. for 10 minutes. Next, after washing the petri dish with sterilized water, the petri dish was dried at 40 ° C. for 5 hours in a sterilized bag to obtain the cell culture substrate 5.

[Culture of normal human dermal fibroblasts]
Normal human dermal fibroblasts were cultured on the obtained cell culture substrate 5 in the same manner as in Example 1. After confirming that the cells had sufficiently proliferated, the medium (at 37 ° C.) was replaced with a medium at 4 ° C. and allowed to stand for a certain period of time to allow the grown cells to spontaneously detach. The ratio of the area of the detached cell part and the total area of the cells grown before detachment was determined. (Table 1, cell peeling recovery rate = 100%, required time 7 minutes).

(Example 6)
In this example, a cell culture substrate is produced by the third production method.
A cell culture substrate 6 was prepared in the same manner as in Example 5 except that 0.7 g of the aqueous solution (5) of poly N-isopropylacrylamide in the second step was used.

[Culture of normal human dermal fibroblasts]
Normal human dermal fibroblasts were cultured on the obtained cell culture substrate 6 in the same manner as in Example 1. After confirming that the cells had sufficiently proliferated, the medium (at 37 ° C.) was replaced with a medium at 4 ° C. and allowed to stand for a certain period of time to allow the grown cells to spontaneously detach. The ratio of the area of the detached cell part and the total area of the cells grown before detachment was determined. (Table 1, cell peeling recovery rate = 100%, required time 15 minutes).

From Examples 5 and 6 above, changes in cell detachability (detachment time) were observed by changing the amount of poly N-isopropylacrylamide aqueous solution added.

(Example 7)
In this example, a cell culture substrate is produced by the third production method.
[Preparation of aqueous solution of polymer (B)]
After mixing 0.57 g of N-isopropylacrylamide (manufactured by Kojin Co., Ltd.) as a monomer (b) and 100 g of water and sufficiently removing oxygen in the aqueous solution by vacuum degassing, 0.1 g of K ₂ as an initiator is used. S ₂ O ₈ (potassium peroxodisulfate, manufactured by Wako Pure Chemical Industries, Ltd.), 80 μl of N, N, N ′, N′-tetramethylethylenediamine (manufactured by Kao Corporation) was added as a catalyst, The solution was allowed to stand for 20 hours to obtain an aqueous solution (6) of poly N-isopropylacrylamide. This aqueous solution is heated to 50 ° C. to precipitate poly N-isopropylacrylamide, further washed with ultrapure water at 50 ° C., and then dried at 80 ° C. for 6 hours to produce solid poly N-isopropylacrylamide. did.

As a result of measuring the molecular weight of this poly N-isopropylacrylamide using a Shodex GPC System-21 apparatus (manufactured by Showa Denko KK), the weight average molecular weight Mw was 6.0 × 10 ⁴ . An N, N-dimethylformamide (DMF) solution containing 10 mmol / L LiBr was used as a solvent for the measurement. STANDARD SH-75 and SM-105 kit (manufactured by Showa Denko KK) were used as polystyrene standard substances used for the calculation of molecular weight.

[Preparation of cell culture substrate (second step)]
0.1 g of the solid poly-N-isopropylacrylamide was added to the total amount of the dispersion liquid (L5) of Example 5 and mixed uniformly, and then a 60 mm polystyrene petri dish (60 mm / Non-Treated Dish, Asahi Techno Glass Co., Ltd.). And then thinly applied to the surface of the petri dish at 2000 rpm using a spin coater and dried in a hot air dryer at 80 ° C. for 10 minutes. Next, after washing the petri dish with sterilized water, the petri dish was dried at 40 ° C. for 5 hours in a sterilized bag to obtain a cell culture substrate 7.

[Culture of normal human dermal fibroblasts]
Normal human dermal fibroblasts were cultured on the obtained cell culture substrate 7 in the same manner as in Example 1. After confirming that the cells had sufficiently proliferated, the medium (at 37 ° C.) was replaced with a medium at 4 ° C. and allowed to stand for a certain period of time to allow the grown cells to spontaneously detach. The ratio of the area of the detached cell part and the total area of the cells grown before detachment was determined. (Table 1, cell peeling recovery rate = 79%, required time 29 minutes).

  From Examples 5 and 7, the addition amount of poly N-isopropylacrylamide (0.1 g) was the same, and the change in cell detachability (detachment time) was observed by changing the molecular weight of poly N-isopropylacrylamide. .

(Example 8)
In this example, a patterned cell culture substrate is produced by the third production method.
0.1 g of the aqueous solution (5) of poly N-isopropylacrylamide of Example 5 was added to the total amount of the dispersion (L5) of Example 5 and mixed uniformly, and then a single nozzle pulse ink injector (manufactured by Cluster Technology Co., Ltd.). ) Was applied to a 1 mm thick polystyrene plate in a circular (dot) shape with a diameter of 30 μm and a spacing of about 20 μm. Next, the polystyrene plate is heated for 10 minutes in a hot air dryer at 80 ° C., washed with sterilized water, and then dried in a sterile bag at 40 ° C. for 5 hours to obtain a cell culture substrate 8. It was.

  When this base material 8 was observed with the optical microscope, the pattern in which the point about 36 micrometers in diameter was formed in the whole surface on the polystyrene board was observed. The distance between the dots was about 21 μm (see Photo 1).

[Culture of Balb3T3 cells (mouse tumor fibroblasts)]
The obtained cell culture substrate 8 is placed in a 60 mm polystyrene petri dish (60 mm / Non-Treated Dish, manufactured by Asahi Techno Glass Co., Ltd.), and Doulbecco's modified Eagle's Medium (DMEM) medium (10% FBS added) (Nissui) A suitable amount was added, and Balb3T3 cells were seeded (seeding concentration was 1.0 × 10 ⁴ cells / cm ² ) and cultured at 37 ° C. in 5% carbon dioxide. When the cells grown for 46 hours were confirmed with a microscope, it was observed that the coated portion was almost entirely covered with cells. Subsequently, when the medium at 37 ° C. was replaced with a medium at 4 ° C. and left to stand for several minutes, it was observed that the cells were detached from the cell culture substrate 8. The ratio of the area of the detached cell part and the total area of the cells grown before detachment was determined. The time taken for this peeling was recorded. (Table 1, cell peeling recovery rate = 98%, required time was 9 minutes).

  On the other hand, when Balb3T3 cells were similarly cultured using the polystyrene plate, the cells hardly proliferated. From this result, it can be seen that this polystyrene plate is a base material on which seeded cells cannot be cultured.

  From Example 8 above, by applying a dispersion (L5) to which a small amount of poly-N-isopropylacrylamide was added in a dot pattern, the coated and uncoated surfaces were applied in the same manner as the cell culture substrate 5 coated on the entire surface. It can be understood that cells can be cultured over the entire surface, and that even when the amount of poly-N-isopropylacrylamide added is small, excellent cell detachability was exhibited.

Example 9
In this example, a cell culture substrate is produced by the third production method.
[Preparation of reaction solution containing monomer (a), inorganic material (C) and aqueous medium (W)]
0.91 g of polyoxypropylene monoacrylate “Blemma AP-400” (manufactured by Nippon Oil & Fats Co., Ltd.) as monomer (a), and Snowtex 20 (20% by weight colloidal silica aqueous solution, manufactured by Nissan Chemical Industries, Ltd.) as silica 4 g (solid content 0.08 g), 20% by weight sodium dodecylbenzenesulfonate (100 μl, manufactured by Wako Pure Chemical Industries, Ltd., 10 g of water as an aqueous medium (W) as a surfactant, and uniformly mixed with the reaction solution ( 9) was prepared.

[Preparation of solution in which polymerization initiator (D) is dissolved in solvent (E)]
The same solution (2) as in Example 1 was used.

[Preparation of dispersion (L) of complex (X) (first step)]
Disperse the pale milky white complex (X) by adding 30 μl of the solution (2) to the total amount of the reaction solution (7) and uniformly dispersing it, and then irradiating it with ultraviolet light having an ultraviolet intensity at 365 nm of 40 mW / cm ² for 180 seconds. A liquid (L9) was produced.

  Ra = 0.09 of this reaction system, concentration (mass%) of inorganic material (C) = 0.79 (%) <12.4Ra + 0.05 = 1.17

[Preparation of cell culture substrate (second step)]
After adding 0.7 g of the poly N-isopropylacrylamide aqueous solution (5) of Example 5 to the total amount of the dispersion (L9) and mixing it uniformly, a Petri dish made of 60 mm polystyrene (60 mm / Non-Treated Dish, Asahi Techno Glass Co., Ltd.) And then thinly applied to the surface of the petri dish at 2000 rpm using a spin coater and dried in a hot air dryer at 80 ° C. for 10 minutes. Next, after washing the petri dish with sterilized water, the petri dish was dried in a sterile bag to obtain a cell culture substrate (9).

[Culture of normal human dermal fibroblasts]
Normal human dermal fibroblasts were cultured on the obtained cell culture substrate 9 in the same manner as in Example 1. After confirming that the cells grew sufficiently, the medium (37 ° C.) was replaced with a medium at 4 ° C., the proliferated cells were naturally detached, and the area of the detached cell part and the total number of cells proliferated before detachment The ratio with the area was determined. (Table 1, cell peeling recovery rate = 95%, required time 10 minutes).

(Example 10)
It is an example which shows the performance of the cell culture substratum which carried out the radiation sterilization process.
[Radiation sterilization treatment of cell culture substrate]
The cell culture substrate 6 of Example 6 was irradiated with γ rays so that the absorbed dose was 10 kGy (irradiation treatment was performed by Nippon Irradiation Service Co., Ltd.).

  For comparison, γ-irradiation was performed in the same manner using a commercially available temperature-responsive cell culture equipment UpCell (6 cm dish, manufactured by Cellseed Co., Ltd.) for cell sheet collection.

[Culture of normal human umbilical vein endothelial cells]
An appropriate amount of Hu-media-EB2 medium (manufactured by Cell Systems) containing 10% FBS is added to the cell culture substrate 6 subjected to the above-mentioned γ-ray treatment, and normal human umbilical vein endothelial cells are seeded (seeding concentration). Was 1.2 × 10 ⁴ cells / cm ² ) and cultured at 37 ° C. in 5% carbon dioxide. After confirming that the cells had sufficiently proliferated, the medium (at 37 ° C.) was sucked up, the medium at 4 ° C. was added, and allowed to stand for a certain period of time, and the proliferated cells were naturally detached. The ratio of the area of the detached cell part and the total area of the cells grown before detachment was determined. The time taken for this peeling was recorded. (Table 1, cell peeling recovery rate = 96%, required time was 13 minutes).

  On the other hand, using the cell culture substrate 6 of Example 6 as it is (without radiation treatment), normal human umbilical vein vascular endothelial cells were cultured in the same manner, and spontaneous detachment was performed. The required time was 14%. Furthermore, when normal human umbilical vein endothelial cells were cultured in the same manner using a commercially available UpCell subjected to the above-mentioned γ-ray treatment and spontaneous detachment was performed, the detachment recovery rate of the cells was 30%, and the required time was It was 40 minutes.

  On the other hand, when normal human umbilical vein vascular endothelial cells were cultured in the same manner using a radiation-untreated UpCell and spontaneous detachment was performed, the detachment recovery rate of the cells was 100% and the required time was 15 minutes. .

  From the above Example (10), it can be seen that even if the cell culture substrate 6 is subjected to radiation sterilization treatment, the cell culture / peelability is not affected. On the other hand, in the case of a commercially available UpCell, it can be understood that the exfoliation property of the cells is greatly reduced by performing the radiation sterilization treatment.

(Comparative Example 1)
Cell culture substrate containing no polymer (B) [Preparation of cell culture substrate]
The dispersion liquid (L1) of Example 1 was placed in a petri dish made of 60 mm polystyrene (60 mm / Non-Treated Dish, manufactured by Asahi Techno Glass Co., Ltd.) and thinly applied to the surface of the petri dish at 2000 rotations using a spin coater. It dried for 10 minutes in an 80 degreeC hot-air dryer. Next, after washing the petri dish with sterilized water, the petri dish was dried in a sterilized bag to obtain a cell culture substrate (1 ′).

[Culture of normal human dermal fibroblasts]
After culturing normal human dermal fibroblasts on the obtained cell culture substrate 1 ′ in the same manner as in Example 1, the medium (at 37 ° C.) was replaced with a medium at 4 ° C. and proliferated cells Were naturally exfoliated, and the ratio of the area of the exfoliated cell portion to the total area of cells grown before exfoliation was determined. (Table 1, cell peeling recovery rate = 10%, required time was 30 minutes). The cell growth property of the cell culture substrate was almost the same as that of the cell culture substrate 1.

  From this comparative example, it can be understood that when the polymer (B) is not included, the cell proliferation does not change, but the peelability is greatly reduced.

(Comparative Example 2)
Cell culture substrate containing excess polymer (B) [Preparation of cell culture substrate]
6.3 g of the poly N-isopropylacrylamide aqueous solution (5) of Example 5 is added to the dispersion (L1) of Example 1 (content of poly N-isopropylacrylamide with respect to the total solid content is 41% by mass), A thin coat was applied to the surface of the petri dish at 2000 revolutions using a spin coater, and then dried in a hot air drier at 80 ° C. for 10 minutes. Next, after washing the petri dish with sterilized water, the petri dish was dried in a sterilized bag to obtain a cell culture substrate (2 ′).

[Culture of normal human dermal fibroblasts]
In the same manner as in Example 1, normal human dermal fibroblasts were cultured on the obtained cell culture substrate 2 ′. However, the seeded cells died and no growth was seen.

  From this comparative example, it can be understood that when the polymer (B) is contained excessively, cell growth is inhibited and cell culture becomes impossible.

(Comparative Example 3)
This is an example in which the concentration of the inorganic material (C) exceeds the range of the formula (3).
[Preparation of reaction liquid containing monomer (a), water-swellable inorganic material (C), and aqueous medium (W)]
As monomer (a), 1.32 g of 2-methoxyethyl acrylate (manufactured by Toagosei Co., Ltd.), as an inorganic material (C), 0.25 g of water-swellable clay mineral Laponite XLG (manufactured by Rockwood Additives Ltd.), water-insoluble 25 μl of the solution (2) as a polymerization initiator (d1) and 10 g of water as an aqueous medium (W) were uniformly mixed to prepare a reaction solution (3 ′).

When the reaction solution (3 ′) was stirred with a magnetic stirrer and irradiated with ultraviolet rays having an ultraviolet intensity at 365 nm of 40 mW / cm ² for 180 seconds, the entire reaction solution (3 ′) was gelled. Even when this gel was placed in a large amount of water, it did not dissolve or disperse and remained as a gel.

Ra = 0.19 of this reaction system, concentration (mass%) of inorganic material (C) = 2.42%> 0.87Ra + 2.17 = 2.34
From this comparative example, when the concentration (% by mass) of the inorganic material (C) exceeds the range of the formula (3), the entire reaction solution is gelled, and a dispersion (L) of the composite (X) is obtained. First, it can be understood that the cell culture substrate cannot be produced by coating the petri dish (of the first and third production methods).

(Comparative Example 4)
This is a production example of a cell culture substrate composed of a hydrogel having a three-dimensional network structure composed of poly N-isopropylacrylamide and an inorganic material (C).
[Preparation of reaction liquid containing monomer, inorganic material (C) and aqueous medium (W)]
1.13 g of N-isopropylacrylamide (manufactured by Kojin Co., Ltd.) as a monomer, 0.4 g of Laponite XLG (manufactured by Rockwood Additives Ltd.) as an inorganic material (C), and 10 g of water as an aqueous medium (W) are uniformly mixed. Thus, a reaction solution (4 ′) was prepared.

[Preparation of a solution in which the polymerization initiator (d ₁ ) is dissolved in the solvent (E)]
As a solvent (E), 98 g of polyoxypropylene monoacrylate “Blemma AP-400” (manufactured by NOF Corporation) and 1-hydroxycyclohexyl phenyl ketone “Irgacure 184” (manufactured by Ciba Geigy) as a polymerization initiator (d ₁ ) 2 g was uniformly mixed to prepare a solution (3).

[Preparation of cell culture substrate comprising hydrogel]
50 μl of the solution (3) is added to the total amount of the reaction solution (4 ′), and uniformly dispersed with an ultrasonic disperser, and then placed in a 60 mm polystyrene petri dish (60 mm / non-treated dish, manufactured by Asahi Techno Glass Co., Ltd.). Put it thinly on the surface of the petri dish at 2000 revolutions using a spin coater, and irradiate UV light with an ultraviolet intensity of 40 mW / cm ² at 365 nm for 180 seconds while cooling the area around the petri dish, polymerizing N-isopropylacrylamide. A thin layer of hydrogel was made.

  Subsequently, when the petri dish was washed with sterilized water, the thin layer of the hydrogel peeled off from the petri dish, and a cell culture substrate in which the thin layer of hydrogel was laminated on the petri dish was not obtained.

  The thin layer of the hydrogel peeled during the washing was dried as it was in a petri dish and subjected to cell culture.

[Culture of normal human dermal fibroblasts]
The dried thin layer of hydrogel is put in a petri dish, an appropriate amount of CS-C complete medium (Cell Systems medium) is added, and normal human dermal fibroblasts are seeded (seeding concentration is 1.2 × 10 ⁴ cells / cell). cm ² ) Incubation was performed at 37 ° C. in 5% carbon dioxide. After confirming that the cells grew sufficiently, the medium (37 ° C.) was replaced with a medium at 4 ° C., the proliferated cells were naturally detached, and the area of the detached cell part and the total number of cells proliferated before detachment The ratio with the area was determined. The time taken for this peeling was recorded. (Table 1, cell peeling recovery rate = 70%, required time was 30 minutes).

  From this comparative example, the cell culture substrate made of a hydrogel having a three-dimensional network structure of the polymer (B) having a lower critical solution temperature and a clay mineral has weak adhesion to a support such as plastic, and the support It can be understood that production of a cell culture substrate integrated with was not possible.

（注）（１）細胞Ｆ：正常ヒト真皮線維芽細胞
（２）細胞Ｔ：マウス腫瘍線維芽細胞(Balb3T3)
（３）細胞Ｈ：正常ヒト臍帯静脈血管内皮細胞

(Note) (1) Cell F: Normal human dermal fibroblast (2) Cell T: Mouse tumor fibroblast (Balb3T3)
(3) Cell H: Normal human umbilical vein endothelial cell

(Note) (1) Cell F: Normal human dermal fibroblast (2) Cell T: Mouse tumor fibroblast (Balb3T3)
(3) Cell H: Normal human umbilical vein endothelial cell

From the above Examples and Comparative Examples, the cell culture substrate of the present invention has good adhesiveness with a support of another material, and has an excellent cell culture and peeling function.
It was also clear that this cell culture substrate can be easily produced in a very short time without removing oxygen.

  The cell culture substrate of the present invention can be used for the preparation of colony-like cell groups, two-dimensional sheet-like cells, and three-dimensional three-dimensional cell proliferation in the field of regenerative medicine.

Claims (8)

Polymer of a monomer (a) which is the table by the following formula (1) and (A), a polymer having a lower critical solution temperature (B), 1 or more inorganic chosen from water-swellable clay mineral and silica Containing material (C) ,
The mass ratio ((C) / (A)) between the polymer (A) and the inorganic material (C) is in the range of 0.01 to 10,
The cell culture substrate whose content rate of the said polymer (B) with respect to the whole cell culture substrate is 0.0001 mass%-40 mass% .

Wherein R ₁ represents a hydrogen atom or a methyl group, R ₂ represents an alkylene group having 2 to 3 carbon atoms, R ₃ represents a hydrogen atom or an alkyl group having 1 to 2 carbon atoms, and n represents 1 to 9 Represents an integer.) It is formed by the interaction of the polymer (A) of the monomer (a) represented by the following formula (1) with one or more inorganic materials (C) selected from water-swellable clay minerals and silica. And the complex
Containing a polymer (B) having a lower critical solution temperature ,
The mass ratio ((C) / (A)) between the polymer (A) and the inorganic material (C) is in the range of 0.01 to 10,
A cell culture substrate having a content of the polymer (B) of 0.0001% by mass to 40% by mass with respect to the entire cell culture substrate ,
A cell culture substrate, wherein the polymer (B) is exposed on a cell culture surface of the cell culture substrate.

Wherein R ₁ represents a hydrogen atom or a methyl group, R ₂ represents an alkylene group having 2 to 3 carbon atoms, R ₃ represents a hydrogen atom or an alkyl group having 1 to 2 carbon atoms, and n represents 1 to 9 Represents an integer.) The water-swellable clay mineral is delaminated into 1 to 10 layers in an aqueous medium (W) selected from water-swellable hectorite, water-swellable montmorillonite, water-swellable saponite and water-swellable synthetic mica. 3. The cell culture substrate according to claim 1, wherein the cell culture substrate is a clay mineral of a seed or more, and the silica is water-dispersible colloidal silica. The polymer (B) is a polymer of at least one monomer (b) selected from the group consisting of N-substituted (meth) acrylamide derivatives and N, N-disubstituted (meth) acrylamide derivatives. 4. The cell culture substrate according to any one of 3. The monomer (b) is N-isopropyl (meth) acrylamide, Nn-propyl (meth) acrylamide, N-cyclopropyl (meth) acrylamide, N-ethoxyethyl (meth) acrylamide, N-tetrahydrofurfuryl (meth) ) Acrylamide, N-ethylacrylamide, N-ethyl-N-methylacrylamide, N, N-diethylacrylamide, N-methyl-Nn-propylacrylamide, N-methyl-N-isopropylacrylamide, N-acryloylpiperidine The cell culture substrate according to claim 4, which is at least one selected from the group consisting of N-acryloylpyrrolidine. A method for producing a cell culture substrate according to claim 4,
The monomer (a) and the inorganic material (C) are polymerized so that the concentration of the inorganic material (C) in the aqueous medium (W) is in the range represented by the following formula (2) or formula (3). After mixing the initiator (D) with the aqueous medium (W), the monomer (a) is polymerized to thereby disperse the composite (X) of the polymer (A) and the inorganic material (C) ( L) the first step of producing
A second step of forming a thin layer of the composite (X) by applying the dispersion (L) to a support and then drying;
A third step in which a solution obtained by dissolving a water-insoluble polymerization initiator (D) in a solvent (E) is applied to the surface (S) of the thin layer of the composite (X), and the solvent (E) is volatilized. ,
A method for producing a cell culture substrate, comprising sequentially applying a fourth step of polymerizing the monomer (b) by irradiation with ultraviolet rays after applying an aqueous solution of the monomer (b) to the surface (S).
Formula (2) When Ra <0.19
Concentration (% by mass) of inorganic material (C) <12.4Ra + 0.05
Formula (3) When Ra ≧ 0.19
Concentration (% by mass) of inorganic material (C) <0.87Ra + 2.17
(In the formula, the concentration (% by mass) of the inorganic material (C) is a value obtained by dividing the mass of the inorganic material (C) by the total mass of the aqueous medium (W) and the inorganic material (C) and multiplying by 100, Ra Is a mass ratio ((C) / (A)) between the inorganic material (C) and the polymer (A).) A method for producing a cell culture substrate according to claim 4,
An aqueous medium (W) in which the monomer (a), the inorganic material (C) and the polymerization initiator (D) are mixed is applied to a support,
A first step of forming a thin layer of a composite (X) of the polymer (A) and the inorganic material (C) by polymerizing the monomer (a);
A second step in which a solution in which a water-insoluble polymerization initiator (D) is dissolved in a solvent (E) is applied to the surface (S) of the thin layer of the composite (X), and the solvent (E) is volatilized;
A method for producing a cell culture substrate, comprising applying an aqueous solution of the monomer (b) to the surface (S) and then sequentially performing a third step of polymerizing the monomer (b) by irradiation with ultraviolet rays. A method for producing a cell culture substrate according to claim 4,
The monomer (a), the inorganic material (C), and the concentration of the inorganic material (C) in the aqueous medium (W) are in a range represented by the following formula (2) or formula (3): After mixing the polymerization initiator (D) with the aqueous medium (W), the monomer (a) is polymerized to disperse the complex (X) of the polymer (A) and the inorganic material (C). A first step of producing a liquid (L);
The polymer (B), which is a polymer of the monomer (b), is added to the dispersion (L), mixed, applied to a support, and then dried in order. A method for producing a cell culture substrate.
Formula (2) When Ra <0.19
Concentration (% by mass) of inorganic material (C) <12.4Ra + 0.05
Formula (3) When Ra ≧ 0.19
Concentration (% by mass) of inorganic material (C) <0.87Ra + 2.17
(In the formula, the concentration (% by mass) of the inorganic material (C) is a value obtained by dividing the mass of the inorganic material (C) by the total mass of the aqueous medium (W) and the inorganic material (C) and multiplying by 100, Ra Is a mass ratio ((C) / (A)) between the inorganic material (C) and the polymer (A).)

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