WO2018198495A1

WO2018198495A1 - Temperature-responsive cell culture substrate, and method for producing same

Info

Publication number: WO2018198495A1
Application number: PCT/JP2018/005836
Authority: WO
Inventors: 礼奈森安; 美子茂原; 綱之奥村; 淳史坂倉; 正道森田
Original assignee: ダイキン工業株式会社
Priority date: 2017-04-25
Filing date: 2018-02-20
Publication date: 2018-11-01
Also published as: JPWO2018198495A1; JP6879365B2

Abstract

Provided is a temperature-responsive cell culture substrate having superior cell detachability. A temperature-responsive cell culture substrate containing (A) a temperature-responsive layer and (B) a base layer, wherein the temperature-responsive layer (A) contains a temperature-responsive polymer having a nitrogen atom, the temperature-responsive layer (A) is arranged on at least one surface of the base layer (B), the base layer (B) has no nitrogen atom, and the nitrogen element concentration N1s and the carbon element concentration C1s both measured by an X-ray photoelectron spectroscopy at an emission angle of 45° meet mathematical formula (1): 100×(N1s/C1s)/(N/C) ≥ 80 (wherein N/C represents a theoretical value of the content ratio of the elements in the temperature-responsive polymer) in a temperature-responsive layer (A) side surface of the base layer (B).

Description

Temperature-responsive cell culture substrate and method for producing the same

The present invention relates to a temperature-responsive cell culture substrate and a method for producing the same.

As a cell culture substrate, a substrate having a temperature-responsive surface is used. These substrates are mainly used for the cultivation of adherent cells. Adherent cells grow by adhering to the surface of the carrier. Adhesive cells are generally considered to have the property of being easily adhered to an appropriate hydrophobic surface and not adhering to a hydrophilic surface. For this reason, it becomes possible to control adhesion of adherent cells to the surface by temperature change by using a base material having a surface that changes from moderate hydrophobicity to hydrophilicity by the temperature response or vice versa. .

Such a cell culture substrate is referred to as a “temperature-responsive cell culture substrate”. Until now, cultured cells “chemically disrupt” the protein between the substrate and cells using proteolytic enzymes (pepsin, trypsin, peptidase, etc.), EDTA (ethylenediaminetetraacetic acid = metal chelating agent), etc. The protein could be peeled off from the substrate surface by a method or a method of “physically destroying” the protein using pipetting (injecting the culture solution with a pipette), rubber policeman, cell scraper, or the like. If a temperature-responsive cell culture substrate is used, all these operations are unnecessary, and the cultured cells can be peeled off simply by changing the temperature.
Cells collected from a temperature-responsive cell culture substrate (not limited to a sheet) are characterized by being not chemically or physically damaged. In addition, the cell sheet collected from the temperature-responsive cell culture substrate retains the adhesive protein as it is, and can quickly adhere to a living tissue. In addition, if cell sheets are laminated, they can be organized and positioned as a platform technology for future development of regenerative medicine technology.

A variety of temperature-responsive cell culture substrates have been developed. For example, a culture substrate has been developed in which poly-N-isopropylacrylamide (PNIPAM) having a lower critical solution temperature (Low Critical Solution temperature (LCST)) of 32 ° C. is immobilized on the surface (Patent Document 1). The polymer chain on the surface of the base material exposed to water having a temperature of 32 ° C. or more is condensed, and the property of the base material surface is strongly the property of the NIPAM polymer chain itself, and is relatively hydrophobic. On the other hand, when the substrate surface comes into contact with water having a temperature of 32 ° C. or lower, a temperature response occurs, and the polymer chains on the surface hydrate with water molecules. Thereby, as a property of the substrate surface, hydrophilicity is exhibited as compared with that before the temperature response, and the adhered cells can be peeled off.

JP-A-2-21865

The present inventors have found that there is a point to be improved in the cell detachability at the time of temperature response in the conventional temperature-responsive cell culture substrate. An object of the present invention is to provide a temperature-responsive cell culture substrate having superior cell detachability.

The inventors of the present invention have made extensive studies, and in the temperature-responsive cell culture substrate whose surface is covered with the temperature-responsive polymer immobilized on the surface of the substrate, the surface coverage by the temperature-responsive polymer is higher than the conventional one. The present inventors have found that the above problems can be solved by further improvement. Furthermore, the present inventors have developed a method for evaluating the surface coverage of the temperature-responsive polymer based on the element concentration measured by the X-ray photoelectron spectroscopy (XPS) method. The surface properties that can solve the problem are defined. The present invention has been completed by further trial and error based on such findings, and includes the following aspects.

Item 1.
(A) a temperature-responsive layer; and (B) a temperature-responsive cell culture substrate containing a substrate layer,
The temperature responsive layer (A) contains a temperature responsive polymer having nitrogen atoms,
The temperature responsive layer (A) is disposed on at least one surface of the base material layer (B),
The substrate layer (B) does not have nitrogen atoms, and the surface on the temperature response layer (A) side has a nitrogen element concentration N _1s and carbon measured by X-ray photoelectron spectroscopy at an emission angle of 45 °. The element concentration C _1s is _expressed by the following formula (1):
(1) 100 × (N _1s / C _1s ) / (N / C) ≧ 80
(In the formula, N / C represents a theoretical value of each element ratio in the temperature-responsive polymer)
Satisfying a temperature-responsive cell culture substrate.
Item 2.
The surface on the temperature-responsive layer (A) side is etched from the surface side with an argon gas cluster ion beam, and N _1s and C _1s measured by X-ray photoelectron spectroscopy satisfy the following condition (A): The temperature-responsive cell culture substrate according to Item 1,
(A) The measured value at an etching depth of 2 nm satisfies the following formula (2):
(2) 100 × (N _1s / C _1s ) / (N / C) ≧ 50
(In the formula, N / C represents the same meaning as described above).
Item 3.
Item 3. The temperature-responsive cell culture substrate according to

Item

1 or 2, wherein the temperature-responsive polymer is fixed to 1 to 10 μg / cm ² on the surface of the temperature-responsive layer (A).
Item 4.
Item 4. The temperature-responsive cell culture substrate according to any one of Items 1 to 3, wherein the temperature-responsive layer (A) contains a block polymer in which the temperature-responsive polymer is bonded to the end of a dendritic polymer.
Item 5.
Item 5. The temperature-responsive cell culture substrate according to Item 4, wherein the dendritic polymer is a dendritic polymer having a styrene skeleton or a siloxane skeleton.
Item 6.
At least one of the temperature-responsive polymers is
A temperature-responsive polymer obtainable by polymerizing a monomer composition containing at least one selected from the group consisting of (meth) acrylamide, N- (or N, N-di) -substituted (meth) acrylamide and vinyl ether, or Item 6. The temperature-responsive cell culture substrate according to any one of Items 1 to 5, which is a partially acetylated polyvinyl alcohol.
Item 7.
The N- (or N, N-di) -substituted (meth) acrylamide is poly-N-isopropyl (meth) acrylamide, poly-N, N-diethyl (meth) acrylamide, and poly-N, N-dimethyl (meta). Item 7. The temperature-responsive cell culture substrate according to Item 6, which is at least one selected from the group consisting of acrylamide.
Item 8.
Item 8. The temperature-responsive cell culture substrate according to any one of Items 1 to 7, wherein the substrate layer (B) contains polystyrene.
Item 9.
Item 9. The temperature-responsive cell culture substrate according to any one of Items 1 to 8, which is for reuse.
Item 10.
(A) A block polymer, a solution containing a good solvent and a poor solvent of polystyrene, in which a block polymer having a temperature-responsive polymer bonded to a terminal of a dendritic polymer having a styrene skeleton or a siloxane skeleton is dissolved, A step of dropping on the surface of a polystyrene substrate and developing the surface;
(B) placing the surface obtained in step (a) under the vapor of the solvent for 2 hours or more; and (c) drying the surface obtained in step (b). A method for producing a temperature-responsive cell culture substrate.

According to the present invention, it is possible to provide a temperature-responsive cell culture substrate with more excellent cell detachability during temperature response.

The temperature-responsive cell culture substrate of the present invention, which is a temperature responsiveness at a total of 5 points of the surface of the temperature-responsive layer (A), that is, a central point and four specific points equally spaced from the central point. It is drawing which showed the example of how to take these measurement points in one mode of the present invention characterized by there being no big variation between each measured value of polymer immobilization amount. It is drawing which showed how to take the five measurement points in another mode of the present invention. It is the graph which showed the measured value of Numerical formula (2) left side for every etching depth in the temperature-responsive cell culture substratum of this invention.

1. Temperature response layer (A)
1.1 Parameters relating to distribution of temperature-responsive polymer The temperature-responsive cell culture substrate of the present invention comprises:
(A) a temperature-responsive layer; and (B) a temperature-responsive cell culture substrate containing a substrate layer,
The temperature responsive layer (A) contains a temperature responsive polymer having nitrogen atoms,
The temperature responsive layer (A) is disposed on at least one surface of the base material layer (B), the base material layer (B) does not have nitrogen atoms, and the temperature responsive layer (A) ) Side surface has a nitrogen element concentration N _1s and a carbon element concentration C _1s measured by the XPS method at an emission angle of 45 °, the following formula (1):
(1) 100 × (N _1s / C _1s ) / (N / C) ≧ 80
(In the formula, N / C represents a theoretical value of each element ratio in the temperature-responsive polymer)
It is a temperature-responsive cell culture substrate that satisfies the above.

The temperature responsive layer (A) includes at least a temperature responsive polymer, and the surface exhibits temperature responsiveness. The temperature-responsive cell culture substrate of the present invention uses a surface exhibiting this temperature responsiveness as a surface for cell culture.

The temperature responsive layer (A) includes at least one temperature responsive polymer. The temperature responsive layer (A) may contain a temperature responsive polymer in the form of a single molecule, or may contain in the form of a complex containing a temperature responsive polymer. In such composites, the temperature responsive polymer is bound in some manner to other structural moieties. The bonding mode is not limited, but a covalent bond is preferable in that the temperature-responsive polymer is stably immobilized on the surface of the base material layer (B).

The temperature-responsive cell culture substrate of the present invention has more excellent cell detachability during temperature response because the surface on the temperature-responsive layer (A) side has the above characteristics.

Specifically, the surface of the temperature responsive layer (A) side has a feature that the coating amount of the temperature responsive polymer is higher than that in the conventional temperature responsive cell culture substrate. Formula (1) is one of the indicators of the coating amount of the temperature-responsive polymer obtained by measuring the surface on the temperature-responsive layer (A) side by the XPS method. When the surface is completely covered with the temperature-responsive polymer, since N _1s / C _1s is equal to N / C, the value on the left side of Equation (1) is 100 (%). Here, N _1s represents the nitrogen atom concentration measured by the XPS method, and N / C represents the theoretical value of the ratio of nitrogen atom (N) to the amount of carbon atoms (C) contained in the temperature-responsive polymer. . When the temperature-responsive polymer is polyisopropylacrylamide, the repeating unit isopropylacrylamide is composed of one nitrogen atom and six carbon atoms, so the theoretical value of N / C is 1/6 = 0.167. .

On the other hand, when the surface is not covered with the temperature-responsive polymer at all, N _1s is not detected because the base material layer (B) does not have the nitrogen atom of the temperature-responsive polymer. Therefore, the value on the left side of Equation (1) is 0 (%).

When the value on the left side of the mathematical formula (1) is 80 (%) or more, the surface coverage of the temperature-responsive polymer is sufficiently high, and the cell detachability at the time of temperature response becomes more excellent. In view of this effect, the left side of Formula (1) is preferably 85 (%) or more, more preferably 90 (%) or more, and even more preferably 95 (%) or more.

After immersing the temperature-responsive cell culture substrate of the present invention in 20 ° C. distilled water and allowing it to stand for 24 hours, the value on the left side of Equation (1) calculated based on the value measured by the above method is It is preferable to be within the above range.

Specifically, XPS is measured as follows.
Using a surface analysis device PHI5000 VersaProbe II manufactured by ULVAC-PHI, Inc. or its equivalent, it is irradiated with monochromatic X-ray AlKα and measured at an emission angle of 45 °.

The temperature-responsive cell culture substrate of the present invention preferably has N _1s and C _1s measured by XPS while the surface on the temperature-responsive layer (A) side is etched from the surface side with an argon gas cluster ion beam. Satisfy the following conditions (A) and / or (B):
(A) The measured value at an etching depth of 2 nm satisfies the following formula (2):
(2) 100 × (N _1s / C _1s ) / (N / C) ≧ 50
(In the formula, N / C represents the same meaning as described above.)
(B) The etching depth that satisfies the following formula (3) is 5 nm or more:
(3) 100 × (N _1s / C _1s ) / (N / C) ≦ 5
(In the formula, N / C represents the same meaning as described above).

Equation (2) is a temperature-responsive polymer at a position of 2 nm depth from the surface obtained by measuring by XPS while etching from the surface side of the temperature-responsive cell culture substrate with an argon gas cluster ion beam. Is an indicator of the abundance of. When this position is completely occupied by the temperature-responsive polymer, since N _1s / C _1s is equal to N / C, the value on the left side of Equation (2) is 100 (%).

When the value on the left side of Formula (2) is 50% or more, the amount of the temperature-responsive polymer at a position 2 nm deep from the surface is sufficiently large, and the cell detachability during temperature response is more excellent. It will be a thing. In view of this effect, the left side of the formula (2) is preferably 60 (%) or more, more preferably 70 (%) or more, and further preferably 80 (%) or more.

Equation (3) shows that the amount of the temperature-responsive polymer obtained by measuring by X-ray photoelectron spectroscopy while etching from the surface side of the temperature-responsive cell culture substrate with an argon gas cluster ion beam is sufficiently small. It is an index of the depth position. That is, the amount of the temperature-responsive polymer is sufficiently reduced at the point where the mathematical formula (3) is satisfied. If the depth position where the mathematical formula (3) is satisfied is 5 nm or more, it can be said that a sufficient amount of the temperature-responsive polymer exists from the surface side to a deeper position. It will be better. In view of this effect, the etching depth that satisfies the left side of Equation (3) is preferably 4 nm or more, more preferably 5 nm or more, and even more preferably 6 nm or more.

The method of measuring XPS while etching with an argon gas cluster ion beam is specifically performed as follows.
Using a surface analyzer PHI5000 VersaProbe II manufactured by ULVAC-PHI, Inc. or an equivalent thereof, elements in the depth direction are measured while etching with argon gas cluster ions under conditions of 2.5 kV, 10 nA (Area 2 mm □). The etching rate is calculated to be 2.0 nm / min by etching a 61 nm thick PNIPAM thin film (substrate: silicon wafer) with argon gas cluster ions until Si is detected.

In the temperature-responsive cell culture substrate of the present invention, it is preferable that the surface coverage of the temperature-responsive polymer is more uniformly coated on the surface. In this regard,
The center point of the surface on the temperature response layer (A) side, and each end point in a cross line with the center point as an intersection, which is set so that the sum of the distances from the center point to each end point is maximized The coefficient of variation [(standard deviation / average value) × 100] calculated from each measured value of the temperature-responsive polymer immobilization amount at five points including the center point of the line connecting the center point is expressed by the following formula ( 4):
(4) Coefficient of variation ≦ 10
It is preferable to satisfy.

In the formula (4), there is no large variation between the measured values of the temperature-responsive polymer immobilization amount at a total of five points, that is, the center point and the specific four points that are equally spaced from the center point on the surface. It is guaranteed. When there is no variation, the standard deviation is 0, so the left side of Equation (4) is 0. In terms of a surface with less variation, the left side of the formula (4) is preferably 10 or less, more preferably 7 or less, and even more preferably 5 or less.

Examples of how to take specific 5 points are shown in FIGS. FIG. 1 shows an example when the surface is circular, and FIG. 2 shows an example when the surface is substantially square. In both FIG. 1 and FIG. 2, the point A indicates the center point, and the point B indicates the center point of the line connecting the end points and the center point in the cross line (shown by a broken line). The area of the substrate used for the measurement is, for example, 9.6 to 78.5 cm ² .

In the present invention, the amount of the temperature-responsive polymer immobilized on the substrate surface can be calculated from the amount of the temperature-responsive polymer applied to the substrate surface. However, if necessary, the amount of the temperature-responsive polymer immobilized can be measured according to a conventional method. Examples of such a measuring method include Fourier transform infrared spectroscopic total reflection attenuation method (FT-IR-ATR method), elemental analysis method, XPS method and the like. Any measurement method may be selected as long as the measurement result does not vary. However, in the case where variation occurs, the measurement result by the FT-IR-ATR method is adopted in the present invention.

The measurement by the FT-IR-ATR method is specifically performed as follows. A case where a polystyrene cell culture dish is used as a base material and a case where PNIPAM is used as a temperature-responsive polymer will be described as an example, but other base materials and / or polymers may be used in the same manner by applying the following examples. It can be measured.

A temperature-responsive cell culture substrate in which a cell culture dish made of polystyrene is used as a substrate and PNIPAM is immobilized as a temperature-responsive polymer is prepared. When the same substrate FT-IR-ATR measuring, represented by the following formula (5), for the absorption intensity of the benzene ring stretching derived from polystyrene (1600 cm ^-1), amide stretch derived from PNIPAM (1650 cm ^-1 ) Absorption intensity ratio can be obtained.
(5) Absorption intensity ratio = I ₁₆₅₀ / I ₁₆₀₀

A known amount of PNIPAM (1 to 10 μg / cm ² ) is applied to a polystyrene substrate, and a calibration curve is prepared in advance from the absorption intensity ratio obtained by the formula (5). The amount of unknown PNIPAM can be determined. The polymer immobilization layer on the polystyrene substrate is assumed to be sufficiently thin with respect to the penetration depth (on the order of 1 μm) of infrared light (evanescent wave) on the sample (reference document: Langmuir 2004, 20, 5506-5511). ).

In the temperature-responsive cell culture substrate of the present invention, the temperature-responsive polymer is preferably immobilized on the surface by 0.5 μg / cm ² or more. In the temperature-responsive cell culture substrate of the present invention, when the temperature-responsive polymer is immobilized on the surface as a complex with another structure, the complex is 0.5 μg / cm ^{2 in} terms of temperature-responsive polymer. As described above, it is preferably 1.0 μg / cm ² or more, more preferably 1.5 μg / cm ² or more, and more preferably 1.5 μg / cm ² or more. In the temperature-responsive cell culture substrate of the present invention, when the temperature-responsive polymer is immobilized in the above amount or more, the cultured cells on the polymer are not easily detached even if the temperature is changed.

In the temperature-responsive cell culture substrate of the present invention, when the temperature-responsive polymer or the temperature-responsive polymer is immobilized on the surface as a complex with another structure, the complex is the temperature-responsive polymer. If converted to 10 μg / cm ² or less, preferably 5 μg / cm ² or less, more preferably 4 μg / cm ² or less, the cells are likely to adhere to the surface in the state before the temperature response. , It becomes easy to attach the cells sufficiently.

In summary, the temperature-responsive cell culture substrate of the present invention has a temperature-responsive polymer, or when the temperature-responsive polymer is immobilized on the surface as a complex with another structure, the complex is In terms of responsive polymer, 0.5 to 10 μg / cm ² is preferably immobilized on the surface, preferably 1 to 5 μg / cm ² , more preferably immobilized on the surface, and 1.5 to 4 μg. / cm ^2, more preferably immobilized on the surface.

1.2 Temperature-responsive polymer The temperature-responsive polymer is not particularly limited as long as it is a temperature-responsive polymer having a nitrogen atom, and can be widely selected. Specific examples of the temperature-responsive polymer include a polymer having a lower critical solution temperature (LCST) or a polymer having an upper critical solution temperature (UCST), and a block polymer having a specific structure is used. It is preferable. The block polymer may contain a kind of temperature-responsive polymer as a block, or may contain a plurality of kinds of temperature-responsive polymers as a block.

Examples of the temperature-responsive polymer include polymers described in Japanese Patent Publication No. 06-104061. Specific examples include polymers having structural units based on at least one of the following monomers. Examples of the monomer include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, vinyl ether derivatives, and the like.

Examples of temperature-responsive polymers include polyvinyl alcohol partial acetylated products and nitrogen-containing cyclic polymers.

Examples of the temperature-responsive polymer include alkyl-substituted cellulose derivatives, polyalkylene oxide block copolymers, and polyalkylene oxide block copolymers.

Since culturing of the cultured cells is usually preferably performed in the range of 5 ° C. to 50 ° C., a temperature-responsive polymer having LCST or UCST within this range is preferable. Polymers obtained by polymerizing poly (N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives having such temperature responsiveness (poly (N- (or N, N-di) alkyl-substituted) Specific examples of (meth) acrylamide)) are poly-Nn-propylacrylamide (lower critical solution temperature 21 ° C.), poly-Nn-propyl methacrylamide (27 ° C.), poly-N-isopropylacrylamide. (32 ° C), poly-N-isopropylmethacrylamide (43 ° C), poly-N-cyclopropylacrylamide (45 ° C), poly-N-ethoxyethylacrylamide (about 35 ° C), poly-N- Ethoxyethyl methacrylamide (about 45 ° C), poly-N-tetrahydrofurfurylacrylamide (about 28 ° C), poly- -Tetrahydrofurfuryl methacrylamide (about 35 ° C), poly-N, N-ethylmethylacrylamide (56 ° C), poly-N, N-diethylacrylamide (32 ° C), poly (N-ethylacrylamide), Examples include poly (N-isopropylmethacrylamide), poly (N-cyclopropylacrylamide), and poly (N-cyclopropylmethacrylamide).

Other specific examples of the polymer having LCST or UCST in the same range as described above are as follows. Examples of the polyvinyl ether include polymethyl vinyl ether. Examples of the nitrogen-containing cyclic polymer include poly (N-acryloylpyrrolidine) and poly (N-acryloylpiperidine). Examples of the alkyl-substituted cellulose derivative include methyl cellulose, ethyl cellulose, and hydroxypropyl cellulose. Examples of the polyalkylene oxide block copolymer include a block copolymer of polypolypropylene oxide and polyethylene oxide.

As the temperature-responsive polymer, a copolymer of at least one of the above-mentioned monomers whose homopolymer exhibits temperature-responsiveness and at least one monomer other than the above-mentioned monomer can be used. As such other monomers, for example, charged monomers and / or hydrophobic monomers can be used.

Examples of the charged monomer include a monomer having an amino group, a monomer having an ammonium salt, a monomer having a carboxyl group, and a monomer having a sulfonic acid group.

Examples of the monomer having an amino group include dialkylaminoalkyl (meth) acrylamide, dialkylaminoalkyl (meth) acrylate, aminoalkyl (meth) acrylate, aminostyrene, aminoalkylstyrene, aminoalkyl (meth) acrylamide, and the like. .

Examples of the monomer having an ammonium salt include [2- (2-methylacryloyloxy) ethyl] trimethylammonium salt and 3-acrylamidopropyltrimethylammonium chloride which is a (meth) acrylamide alkyltrimethylammonium salt.

Examples of the monomer having a carboxyl group include acrylic acid and methacrylic acid.

In addition, examples of the monomer having sulfonic acid include (meth) acrylamide alkyl sulfonic acid.

Examples of the hydrophobic monomer include alkyl acrylate and alkyl methacrylate. Examples of the alkyl acrylate include n-butyl acrylate and t-butyl acrylate. Examples of the alkyl methacrylate include n-butyl methacrylate, t-butyl methacrylate, methyl methacrylate and the like. As the hydrophobic monomer, the following fluorine-containing monomers can also be used.

As the fluorine-containing monomer, for example, an acrylate ester having a fluoroalkyl group that is ester-bonded or amide-bonded directly or via a divalent organic group to the carboxyl group, and may have a substituent at the α-position (hereinafter referred to as “a”) , “Fluoroalkyl group-containing acrylate ester”), or “fluoroalkyl group-containing acrylamide”.

Preferable specific examples of the fluoroalkyl group-containing acrylate ester or the fluoroalkyl group-containing acrylamide include the following general formula (1):
CH ₂ = C (-X) -C (= O) -YZ-Rf (1)

[Wherein, X represents a hydrogen atom, a linear or branched alkyl group having 1 to 21 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a CFX ¹ X ² group (where X ¹ and X ² is a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom), a cyano group, a linear or branched fluoroalkyl group having 1 to 21 carbon atoms, a substituted or unsubstituted benzyl group or A substituted or unsubstituted phenyl group;
Y is —O— or —NH—;
Z is an aliphatic group having 1 to 10 carbon atoms, an aromatic group having 6 to 10 carbon atoms or a cyclic aliphatic group,
—CH ₂ CH ₂ N (R ¹ ) SO ₂ — group (where R ¹ is an alkyl group having 1 to 4 carbon atoms).
), —CH ₂ CH (OZ) ¹ ) CH ₂ — group (wherein Z ¹ is a hydrogen atom or an acetyl group), — (CH ₂ ) _m —SO ₂ — (CH ₂ ) _n — group, — (CH ₂ ) _m —S— (CH ₂ ) _n — group (where m is 1 to 10 and n is 0 to 10) or — (CH ₂ ) _m —COO— group (m is 1 to 10) Is));
Rf is a linear or branched fluoroalkyl group having 1 to 20 carbon atoms which may have a hetero atom. An acrylate ester and acrylamide represented by the formula:

In the general formula (1), the fluoroalkyl group represented by Rf is an alkyl group which may have a hetero atom, in which at least one hydrogen atom is substituted with a fluorine atom, and all the hydrogen atoms are A perfluoroalkyl group which may be substituted with a fluorine atom and which may have a hetero atom is also included.

In the acrylic acid ester and acrylamide represented by the general formula (1), Rf is preferably a linear or branched fluoroalkyl group having 1 to 6 carbon atoms, and particularly a straight chain having 1 to 3 carbon atoms. It is preferably a chain or branched perfluoroalkyl group. In recent years, it has been pointed out by the EPA (US Environmental Protection Agency) that a compound having a fluoroalkyl group having 8 or more carbon atoms is a high environmental load that may decompose and accumulate in the environment and living organisms. However, in the acrylic ester and acrylamide represented by the general formula (1), when Rf is a linear or branched fluoroalkyl group having 1 to 6 carbon atoms, such environmental problems are pointed out. Because it is not.

In the above formula (1), examples of the Rf group include —CF ₃ , —CF ₂ CF ₃ , —CF ₂ CF ₂ H, —CF ₂ CF ₂ CF ₃ , —CF ₂ CFHCF ₃ , —CF (CF ₃ ) ₂ , -CF ₂ CF ₂ CF ₂ CF ₃ , -CF ₂ CF (CF ₃ ) ₂ , -C (CF ₃ ) ₃ ,-(CF ₂ ) ₄ CF ₃ ,-(CF ₂ ) ₂ CF (CF ₃ ) ₂ , -CF ₂ C (CF ₃ ) ₃ , -CF (CF ₃ ) CF ₂ CF ₂ CF ₃ ,-(CF ₂ ) ₅ CF ₃ ,-(CF ₂ ) ₃ CF (CF ₃ ) ₂ .

Further, the fluorine-containing monomer is preferably a non-telomer, and in this respect, the Rf group includes a fluoroalkyl group having 1 to 2 carbon atoms, or two or more carbon atoms having 1 to 3 carbon atoms interposed by a hetero atom. A fluoroalkyl group is preferred. Specific examples include C ₃ F ₇ OCF (CF ₃ ) CF ₂ OCF (CF ₃ ) —, (CF ₃ ) ₂ NC _p F _2p — (p = 1 to 6), and the like.

Specific examples of the acrylate ester and acrylamide represented by the general formula (1) are as follows.

CH ₂ ═C (—H) —C (═O) —O— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—H) —C (═O) —O—C ₆ H ₄ —Rf
CH ₂ ═C (—Cl) —C (═O) —O— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—H) —C (═O) —O— (CH ₂ ) ₂ N (—CH ₃ ) SO ₂ —Rf
CH ₂ ═C (—H) —C (═O) —O— (CH ₂ ) ₂ N (—C ₂ H ₅ ) SO ₂ —Rf CH ₂ ═C (—H) —C (═O) —O —CH ₂ CH (—OH) CH ₂ —Rf
CH ₂ ═C (—H) —C (═O) —O—CH ₂ CH (—OCOCH ₃ ) CH ₂ —Rf CH ₂ ═C (—H) —C (═O) —O— (CH ₂ ) ₂ -S-Rf
CH ₂ ═C (—H) —C (═O) —O— (CH ₂ ) ₂ —S— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—H) —C (═O) —O— (CH ₂ ) ₃ —SO ₂ —Rf
CH ₂ ═C (—H) —C (═O) —O— (CH ₂ ) ₂ —SO ₂ — (CH ₂ ) ₂ —Rf CH ₂ ═C (—H) —C (═O) —NH— (CH ₂ ) ₂ -Rf
CH ₂ ═C (—CH ₃ ) —C (═O) —O— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CH ₃ ) —C (═O) —O—C ₆ H ₄ —Rf
CH ₂ ═C (—CH ₃ ) —C (═O) —O— (CH ₂ ) ₂ N (—CH ₃ ) SO ₂ —Rf
CH ₂ ═C (—CH ₃ ) —C (═O) —O— (CH ₂ ) ₂ N (—C ₂ H ₅ ) SO ₂ —Rf
CH ₂ ═C (—CH ₃ ) —C (═O) —O—CH ₂ CH (—OH) CH ₂ —Rf
CH ₂ ═C (—CH ₃ ) —C (═O) —O—CH ₂ CH (—OCOCH ₃ ) CH ₂ —Rf

CH ₂ ═C (—CH ₃ ) —C (═O) —O— (CH ₂ ) ₂ —S—Rf
CH ₂ ═C (—CH ₃ ) —C (═O) —O— (CH ₂ ) ₂ —S— (CH ₂ ) ₂ —Rf CH ₂ ═C (—CH ₃ ) —C (═O) —O — (CH ₂ ) ₃ —SO ₂ —Rf
CH ₂ ═C (—CH ₃ ) —C (═O) —O— (CH ₂ ) ₂ —SO ₂ — (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CH ₃ ) —C (═O) —NH— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—F) —C (═O) —O— (CH ₂ ) ₂ —S—Rf
CH ₂ ═C (—F) —C (═O) —O— (CH ₂ ) ₂ —S— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—F) —C (═O) —O— (CH ₂ ) ₂ —SO ₂ —Rf
CH ₂ ═C (—F) —C (═O) —O— (CH ₂ ) ₂ —SO ₂ — (CH ₂ ) ₂ —Rf CH ₂ ═C (—F) —C (═O) —NH— (CH ₂ ) ₂ -Rf
CH ₂ ═C (—Cl) —C (═O) —O— (CH ₂ ) ₂ —S—Rf
CH ₂ ═C (—Cl) —C (═O) —O— (CH ₂ ) ₂ —S— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—Cl) —C (═O) —O— (CH ₂ ) ₂ —SO ₂ —Rf
CH ₂ ═C (—Cl) —C (═O) —O— (CH ₂ ) ₂ —SO ₂ — (CH ₂ ) ₂ —Rf

CH ₂ ═C (—Cl) —C (═O) —NH— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CF ₃ ) —C (═O) —O— (CH ₂ ) ₂ —S—Rf
CH ₂ ═C (—CF ₃ ) —C (═O) —O— (CH ₂ ) ₂ —S— (CH ₂ ) ₂ —Rf CH ₂ ═C (—CF ₃ ) —C (═O) —O — (CH ₂ ) ₂ —SO ₂ —Rf
CH ₂ ═C (—CF ₃ ) —C (═O) —O— (CH ₂ ) ₂ —SO ₂ — (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CF ₃ ) —C (═O) —NH— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CF ₂ H) —C (═O) —O— (CH ₂ ) ₂ —S—Rf
CH ₂ ═C (—CF ₂ H) —C (═O) —O— (CH ₂ ) ₂ —S— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CF ₂ H) —C (═O) —O— (CH ₂ ) ₂ —SO ₂ —Rf
CH ₂ ═C (—CF ₂ H) —C (═O) —O— (CH ₂ ) ₂ —SO ₂ — (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CF ₂ H) —C (═O) —NH— (CH ₂ ) ₂ —Rf

CH ₂ ═C (—CN) —C (═O) —O— (CH ₂ ) ₂ —S—Rf
CH ₂ ═C (—CN) —C (═O) —O— (CH ₂ ) ₂ —S— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CN) —C (═O) —O— (CH ₂ ) ₂ —SO ₂ —Rf
CH ₂ ═C (—CN) —C (═O) —O— (CH ₂ ) ₂ —SO ₂ — (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CN) —C (═O) —NH— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CF ₂ CF ₃ ) —C (═O) —O— (CH ₂ ) ₂ —S—Rf
CH ₂ ═C (—CF ₂ CF ₃ ) —C (═O) —O— (CH ₂ ) ₂ —S— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CF ₂ CF ₃ ) —C (═O) —O— (CH ₂ ) ₂ —SO ₂ —Rf
CH ₂ ═C (—CF ₂ CF ₃ ) —C (═O) —O— (CH ₂ ) ₂ —SO ₂ — (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CF ₂ CF ₃ ) —C (═O) —NH— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—F) —C (═O) —O— (CH ₂ ) ₃ —S—Rf
CH ₂ ═C (—F) —C (═O) —O— (CH ₂ ) ₃ —S— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—F) —C (═O) —O— (CH ₂ ) ₃ —SO ₂ —Rf
CH ₂ ═C (—F) —C (═O) —O— (CH ₂ ) ₃ —SO ₂ — (CH ₂ ) ₂ —Rf CH ₂ ═C (—F) —C (═O) —NH— (CH ₂ ) ₃ -Rf

CH ₂ ═C (—Cl) —C (═O) —O— (CH ₂ ) ₃ —S—Rf
CH ₂ ═C (—Cl) —C (═O) —O— (CH ₂ ) ₃ —S— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—Cl) —C (═O) —O— (CH ₂ ) ₃ —SO ₂ —Rf
CH ₂ ═C (—Cl) —C (═O) —O— (CH ₂ ) ₃ —SO ₂ — (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CF ₃ ) —C (═O) —O— (CH ₂ ) ₃ —S—Rf
CH ₂ ═C (—CF ₃ ) —C (═O) —O— (CH ₂ ) ₃ —S— (CH ₂ ) ₂ —Rf CH ₂ ═C (—CF ₃ ) —C (═O) —O — (CH ₂ ) ₃ —SO ₂ —Rf
CH ₂ ═C (—CF ₃ ) —C (═O) —O— (CH ₂ ) ₃ —SO ₂ — (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CF ₂ H) —C (═O) —O— (CH ₂ ) ₃ —S—Rf
CH ₂ ═C (—CF ₂ H) —C (═O) —O— (CH ₂ ) ₃ —S— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CF ₂ H) —C (═O) —O— (CH ₂ ) ₃ —SO ₂ —Rf
CH ₂ ═C (—CF ₂ H) —C (═O) —O— (CH ₂ ) ₃ —SO ₂ — (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CN) —C (═O) —O— (CH ₂ ) ₃ —S—Rf
CH ₂ ═C (—CN) —C (═O) —O— (CH ₂ ) ₃ —S— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CN) —C (═O) —O— (CH ₂ ) ₃ —SO ₂ —Rf
CH ₂ ═C (—CN) —C (═O) —O— (CH ₂ ) ₃ —SO ₂ — (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CF ₂ CF ₃ ) —C (═O) —O— (CH ₂ ) ₃ —S—Rf
CH ₂ ═C (—CF ₂ CF ₃ ) —C (═O) —O— (CH ₂ ) ₃ —S— (CH ₂ ) ₂ —Rf
CH ₂ ═C (—CF ₂ CF ₃ ) —C (═O) —O— (CH ₂ ) ₃ —SO ₂ —Rf
CH ₂ ═C (—CF ₂ CF ₃ ) —C (═O) —O— (CH ₂ ) ₂ —SO ₂ — (CH ₂ ) ₂ —Rf
[In the above formula, Rf is a fluoroalkyl group having 1 to 6, preferably 1 to 3 carbon atoms. ]

_{_{_{_{C 3 F 7 OCF (CF 3}}}} ) CF 2 OCF (CF 3) CH 2 -MAc
C ₃ F ₇ OCF (CF ₃ ) CF ₂ O—CF (CF ₃ ) CH ₂ —Ac
(CF ₃ ) ₂ CH-Ac
C ₂ F ₅ CH ₂ -MAc
C ₂ F ₅ CH ₂ -Ac
[In the above formula, Ac represents acrylate, and MAc represents methacrylate, respectively. ]

The above-mentioned fluoroalkyl group-containing acrylic ester and fluoroalkyl group-containing acrylamide can be used alone or in combination of two or more.

The temperature-responsive polymer is obtained by polymerizing a monomer composition containing at least one selected from the group consisting of (meth) acrylamide, N- (or N, N-di) -substituted (meth) acrylamide and vinyl ether. Preferred is a temperature-responsive polymer or polyvinyl alcohol partially acetylated product.

Further, a block polymer having the above-described temperature-responsive polymer as a segment may be used. Moreover, you may use what cross-linked temperature-responsive polymer in the range which does not impair the original property of a polymer. The crosslinkable monomer used at that time is not particularly limited and can be selected widely. An example is N, N'-methylenebis (meth) acrylamide.

1.3 Dendritic Block Copolymer In the present invention, the temperature-responsive layer (A) is a block polymer in which the temperature-responsive polymer is bonded to the end of the dendritic polymer (hereinafter referred to as “dendritic block copolymer”). ) Is preferably contained. This makes it easier to achieve the parameters relating to the preferred temperature-responsive polymer distribution.

In the present invention, when the temperature responsive layer (A) contains a dendritic block copolymer, the temperature responsive polymer is more easily fixed to the temperature responsive layer (A). Specifically, in the said aspect, the residual rate to the temperature-responsive layer (A) of the temperature-responsive polymer after washing the temperature-responsive layer (A) with water is excellent. For this reason, the temperature-responsive cell culture substrate of this aspect is preferably used for reuse.
In the temperature-responsive cell culture substrate of this embodiment, the temperature-responsive layer (A) preferably has a temperature-responsive polymer residual amount of 1 μg / cm ² after being immersed in distilled water at 20 ° C. and allowed to stand for 24 hours. That's it. Although not particularly limited, in the above, the upper limit of the temperature-responsive polymer remaining amount is usually 10 μg / cm ² , preferably 5 μg / cm ² , more preferably 4 μg / cm ² . The remaining amount of the temperature-responsive polymer can be measured by the same method as described above as the method for measuring the immobilized amount of the temperature-responsive polymer.

In particular, the core dendritic polymer portion excluding the temperature-responsive polymer portion (in this specification, this portion is simply referred to as “dendritic polymer” in order to distinguish it from the entire dendritic block copolymer. However, it is preferably a dendritic polymer having a styrene skeleton or a siloxane skeleton. According to the study by the present inventors, by having such a skeleton, the dendritic polymer portion is easily arranged regularly on the surface of the base material, thereby stably fixing to the surface of the base material. As a result, an effect that it is difficult to release not only when culturing cells but also when detaching cells by temperature response can be obtained.

In addition, the dendritic block copolymer in which the temperature-responsive polymer is bonded to the terminal of the dendritic polymer having a styrene skeleton has a water-insoluble styrenic dendritic polymer portion and a temperature-responsive polymer portion having affinity for water. It is a combination. Therefore, it is expected that a fine phase separation structure is formed on the substrate surface by coating the substrate surface with this dendritic block copolymer and drying it. When cells adhere to the surface of the substrate, it is preferable to have a phase separation structure on the surface of the substrate because cell denaturation can be suppressed.

The dendritic polymer is preferably a dendritic polymer having 15 or more terminals. When the number of terminals is 15 or more, the density per unit volume of the temperature-responsive polymer bonded to the terminals can be within a preferable range, which contributes to the improvement of cell detachability during temperature response. In this respect, the number of terminals of the dendritic polymer having a styrene skeleton is preferably 15 or more, and more preferably 20 or more.

Further, the number of terminals of the dendritic polymer is preferably 100 or less, and more preferably 50 or less, in that the reaction time for adding the temperature-responsive polymer can be shortened.

In summary, the preferred range of the number of terminals of the dendritic polymer is 15 to 50, of which 20 to 50 is preferred, and 30 to 50 is more preferred.

The molecular weight of the dendritic polymer is not particularly limited, and can be selected from a wide range. In particular, when the molecular weight of the dendritic polymer is 2,000 or more, the dendritic block copolymer is easily immobilized on the polystyrene base material, and the possibility of elution into the medium or the like is reduced. In this respect, the molecular weight of the dendritic polymer is preferably 3,000 or more, more preferably 4,000 or more, and most preferably 5,000 or more.

The molecular weight of the dendritic polymer of the present invention is measured by GPC under the following conditions.
In addition, it may replace with the apparatus, reagent, etc. which are listed below, and may use the thing equivalent to them.
Apparatus: Not particularly limited.
Detector: Differential refractive index detector RI column: LF-604 (2), KF-601 (2) (Shodex)
Solvent: Tetrahydrofuran flow rate: 0.6 ml / min
Column temperature: 40 ° C
Sample preparation: A sample was dissolved in tetrahydrofuran to prepare 0.5 mass%. After dissolution, it was filtered using a 0.45 μm filter.
Injection volume: 0.2ml
Standard sample: standard polystyrene made by shodex

Further, when the molecular weight of the dendritic polymer is 1,000,000 or less, the temperature-responsive polymer introduction rate can be kept within a preferable range. In this respect, the molecular weight of the dendritic polymer is preferably 500,000 or less, more preferably 300,000 or less, and most preferably 100,000 or less.

If necessary, a group having a positive or negative charge such as a hydroxyl group, a carboxyl group, an amino group, a carbonyl group, an aldehyde group, or a sulfonic acid group may be added to the end of the dendritic block copolymer. The application of these groups can be performed by a conventional method.

Further, if necessary, in the dendritic block copolymer in which the temperature-responsive polymer is bonded, the dendritic polymer portion has a positive or negative group such as a hydroxyl group, a carboxyl group, an amino group, a carbonyl group, an aldehyde group, or a sulfonic acid group. A charged group may remain. A group having a positive or negative charge such as a hydroxyl group, a carboxyl group, an amino group, a carbonyl group, an aldehyde group, or a sulfonic acid group may be imparted to or at the terminal of the temperature-responsive polymer.

In the dendritic block copolymer, at least one kind of temperature-responsive polymer is bonded to the end of the dendritic polymer, but at least one other polymer may be further bonded.

As long as the dendritic block copolymer has a temperature-responsive polymer having a molecular weight of 3000 or more bonded to 50 to 99.5% by mass with respect to the whole dendritic block copolymer at the end of a dendritic polymer having 15 or more terminals. preferable. In this dendritic block copolymer, since the temperature-responsive polymer is sufficiently bonded to the end of the dendritic polymer, the cultured cells on the polymer are not easily detached even when the temperature is changed. In this respect, in the dendritic block copolymer of the present invention, it is preferable that the temperature-responsive polymer is bonded to the terminal of the dendritic polymer at 70% by mass or more with respect to the entire dendritic block copolymer, and 80% by mass or more is bonded. It is more preferable.

The dendritic block copolymer is preferably one in which the temperature-responsive polymer is bonded to the end of the dendritic polymer at 99.5% by mass or less with respect to the entire dendritic block copolymer. In this respect, in the dendritic block copolymer, it is preferable that the temperature-responsive polymer is bonded to the end of the dendritic polymer with 98% by mass or less, and 97% by mass or less with respect to the entire dendritic block copolymer. More preferred.

The molecular weight of the dendritic block copolymer of the present invention is measured by GPC under the following conditions. In addition, it may replace with the apparatus, reagent, etc. which are listed below, and may use the thing equivalent to them.
Apparatus: Not particularly limited.
Detector: Differential refractive index detector RI
Column: TSKgel-M (1), TSKgel-3000 (1) (Tosoh)
Solvent: Dimethylformamide (50 mM LiBr added)
Flow rate: 0.8mL / min
Column temperature: 23 ° C
Sample preparation: 5 mL of DMF solvent is added to 10 mg of sample, dissolved by gently stirring at room temperature, and then filtered using a 0.45 μm filter.
Injection volume: 0.2 mL
Standard sample: Tosoh monodisperse polystyrene

The molecular weight (weight basis) of the dendritic block copolymer of the present invention is preferably 100,000 to 500,000. When the measured molecular weight is less than 100,000, the temperature-responsive polymer is not introduced into the end of the dendritic polymer, and the cell detachability is lowered because the blended ratio is large.

In the dendritic block copolymer of the present invention, the method for bonding the temperature-responsive polymer to the end of the dendritic polymer is not particularly limited and can be selected widely.

Examples of the bonding method include a method in which a RAFT agent is introduced at the end of the dendritic polymer, and various monomers are grown using the RAFT agent as a starting point.
The initiator for RAFT polymerization is not particularly limited and can be selected widely. For example, 2,2′-azobis (isobutyronitrile), 2,2′-azobis (4-methoxy-2,4-dimethylvaleronitrile) (V-70) and 2,2′-azobis [(2- Carboxyethyl) -2- (methylpropionamidine) (V-057) and the like.

The solvent used in the RAFT polymerization is not particularly limited and can be selected widely. For example, benzene, tetrahydrofuran, 1,4-dioxane, dimethylformaldehyde (DMF) and the like are preferable, and can be appropriately selected depending on the types of monomers, RAFT agent, and polymerization initiator used in the polymerization reaction. The initiator concentration, the amount of RAFT agent, the reaction temperature, the reaction time, and the like at the time of polymerization are not particularly limited and can be appropriately set according to the purpose. During the polymerization, the reaction solution may be allowed to stand or be stirred.

The structure of the styrene skeleton dendritic polymer can be selected widely. The structure of the styrene skeleton dendritic polymer can be represented, for example, by the following general formula (2).

In the above formula, R ² is a group into which the temperature-responsive polymer can be introduced by a covalent bond. n represents the degree of polymerization.

R ² is not particularly limited and can be selected widely. In particular, a group that can act as a reversible addition-cleavage chain transfer (RAFT) agent is preferable because a temperature-responsive polymer can be introduced by RAFT polymerization. Such a group that can act as a RAFT agent is not particularly limited and can be selected widely. Examples include a thiocarbonylthio group.
Examples of the thiocarbonylthio group include a dithioester group, a dithiocarbamate group, a trithiocarbonate group, a xanthate group, and a dithiobenzoate group.

R ² may have an optionally substituted hydrocarbon group having 3 to 12 carbon atoms at the terminal. The hydrocarbon group is preferably a branched hydrocarbon group. Thereby, an appropriate steric hindrance can be given to the temperature-responsive portion in the dendritic block copolymer, and the effect that the substrate surface can be coated more effectively can be obtained. Specific examples of R ² at this time include the following trithiocarbonate groups.

In the above formula, R ³ represents an optionally substituted hydrocarbon group having 3 to 12 carbon atoms. R ³ is preferably a branched hydrocarbon group. Specifically, methyl, ethyl, propyl, branched propyl, butyl, branched butyl, hexyl, branched hexyl, pentyl, branched pentyl, heptyl, branched heptyl, octyl, branched There are an octyl group, a 2-ethylhexyl group, a nonyl group, a branched nonyl group, a decyl group, a branched decyl group, a dodecyl group, and a branched dodecyl group, preferably an isopropyl group, an ethylhexyl group, and a butyloctyl group.

The method for producing the styrene skeleton dendritic polymer is not particularly limited, and can be widely selected. For example, it can be obtained by an atom transfer radical polymerization (ATRP) method in the presence of copper chloride in chlorobenzene, which is conventionally performed. Alternatively, it can also be obtained by radical polymerization in dry toluene in the presence of azobisisobutyronitrile (AIBN) under heating.

More specifically, for the purpose of introducing R ² , it is preferable to perform polymerization using at least a styrene derivative having a functional group as a monomer. Examples of such styrene derivatives include halogenated methylstyrene. As the halogenated methylstyrene, chloromethylstyrene, bromomethylstyrene, or the like is used. A styrene derivative may be used individually by 1 type, and 2 or more types may be mixed and used for it.

As described above, when the styrene skeleton dendritic polymer is produced by the polymerization reaction, the ratio of the styrene derivative having a functional group to the total monomer used is preferably 5% or more. When the ratio of the styrene derivative having a functional group is 5% or more, the efficiency of introducing the temperature-responsive polymer chain becomes good, and the target temperature-responsive polymer introduction rate in the present invention is easily achieved. In this respect, the proportion of the styrene derivative having a functional group is more preferably 10% or more, further preferably 15% or more, and most preferably 20% or more.

When the proportion of the styrene derivative having a functional group is 90% or less, the resulting dendritic block copolymer is hardly soluble in water while maintaining the efficiency of introducing the temperature-responsive polymer chain within a good range, and the dendritic block The possibility of the copolymer eluting into the medium or the like is reduced. In this respect, the ratio of the styrene derivative having a functional group is more preferably 80% or less, further preferably 70% or less, and most preferably 60% or less.

In summary, the proportion of the styrene derivative having a functional group is preferably 5% to 90%, more preferably 10% to 80%, further preferably 15% to 70%, and most preferably 20% to 60%.

Siloxane skeleton dendritic polymer can be widely selected. The siloxane skeleton dendritic polymer is, for example, at least one selected from the group consisting of bis (dimethylvinylsiloxane) methylsilane, tris (dimethylvinylsiloxane) silane, bis (dimethylallylsiloxane) methylsilane, and tris (dimethylallylsiloxane) silane. The thing etc. which can be obtained by superposing | polymerizing a monomer are mentioned. Further, it can be obtained by polymerizing at least one monomer selected from the group consisting of bis (dimethylsiloxy) methylvinylsilane, tris (dimethylsiloxy) vinylsilane, bis (dimethylsiloxy) methylallylsilane and tris (dimethylsiloxy) allylsilane. Examples are also included. Such a siloxane skeleton dendritic polymer can be obtained by, for example, the method described in WO 2004/074177 pamphlet or the like.

1.4. Arrangement method of temperature response layer (A) on surface of base material layer (B)

The temperature-responsive cell culture substrate of the present invention can be obtained by disposing the temperature-responsive layer (A) on at least one surface of the substrate layer (B). Specifically, for example, the temperature responsive polymer (A) is disposed on the surface of the base material layer (B) by directly or indirectly fixing the temperature responsive polymer to the surface of the base material layer (B). it can.

The temperature response layer (A) may have two or more regions having different UCST or LCST, and these regions may be arranged so as to form a two-dimensional pattern.

The temperature response layer (A) is disposed on a part of at least one surface of the base material layer (B), and the region and the region not temperature-responsive are arranged so as to form a two-dimensional pattern. May be.

The method for fixing the temperature-responsive polymer to the surface of the base material layer (B) is not particularly limited and can be selected widely. For example, the temperature-responsive polymer can be directly fixed by dissolving or dispersing it in a solvent and then applying it so that the substrate surface is uniformly coated.

Further, the temperature-responsive polymer can be indirectly fixed by dissolving or dispersing the complex containing the temperature-responsive polymer in a solvent and then applying the complex to the surface of the base material layer (B). Examples of such a complex include the above-mentioned dendritic block copolymer of 1.3.

In this case, the solvent is not particularly limited as long as it can dissolve or disperse the temperature-responsive polymer or the complex containing the polymer, and can be selected widely. Examples thereof include N, N-dimethylacrylamide; isopropyl alcohol; and a mixture of acetonitrile and N, N-dimethylformamide.

A plurality of types of solvents may be mixed and used. In this case, the mixing ratio is not particularly limited and can be selected widely. In the case of a mixed solvent of tetrahydrofuran and methanol, for example, tetrahydrofuran: methanol = 0.5 to 2: 4 can be set. In the case of a mixed solvent of acetone and ethanol, for example, acetone: ethanol = 0.5 to 1: 4 can be set.
In the case of a mixed solvent of dioxane and normal propanol, for example, dioxane: normal propanol can be set to 0.5 to 2: 4. In the case of a mixed solvent of toluene and normal butanol, for example, toluene: normal butanol can be set to 0.5 to 2: 4. In the case of a mixed solvent of acetonitrile and N, N-dimethylformamide, for example, acetonitrile: N, N-dimethylformamide can be set to 4: 1 to 6: 1.

As the solvent, a solution containing a good solvent and a poor solvent of polystyrene is preferable. When such a solvent is used, particularly when the material of the base material layer (B) is polystyrene, while the polystyrene on the surface of the base material layer (B) is swollen, the styrene skeleton dendritic block copolymer of the above 1.3 or The siloxane skeleton dendritic block copolymer can be fixed, and as a result, the styrene skeleton dendritic block copolymer or the siloxane skeleton dendritic block copolymer is preferably embedded in the surface of the base material layer (B). In this case, it is preferable that the good solvent and the poor solvent are tetrahydrofuran and methanol, respectively. Further, the tetrahydrofuran content in the mixed solvent of tetrahydrofuran and methanol is more preferably 10 to 35% by volume.

In immobilizing the temperature-responsive polymer or the composite containing the same to the surface of the base material layer (B), the solution containing the temperature-responsive polymer or the composite containing the same is uniformly applied to the surface of the base material layer (B). It is preferable to apply to. The method is not particularly limited and can be selected widely. For example, the method of using a dispenser, the method of leaving a base material layer (B) on a horizontal stand, etc. are mentioned.

The temperature-responsive cell culture substrate of the present invention is obtained by applying a solution containing a temperature-responsive polymer or a complex containing the same to the surface of the substrate layer (B) and then removing the solvent. The method for removing the solvent is not particularly limited, and can be selected widely. For example, a method of slowly evaporating over time in the atmosphere at room temperature, a method of evaporating slowly over time in a solvent-saturated environment at room temperature, a method of evaporating under heating, and evaporating under reduced pressure Methods and the like. In particular, a method of slowly evaporating at room temperature and in a solvent saturated environment is preferable in that a uniform surface of the temperature responsive layer (A) can be obtained. Specifically, it is preferable to put it under the vapor of the solvent for 2 hours or more.

In terms of obtaining a uniform surface of the temperature-responsive layer (A), it is preferable to slowly evaporate the solvent over time in a solvent-saturated environment and then wash the surface once and then dry.

2. Base material layer (B)
The material of the base material layer (B) is not particularly limited as long as it does not have a nitrogen atom. Usually, any material can be used as long as it is used for cell culture. For example, glass, modified glass, various resins, etc. are mentioned. In addition to these, it may be made of a material that can generally be given a form. Such a material is not particularly limited and can be selected widely. For example, a graft polymer, ceramics, metals, etc. are mentioned.

Examples of the resin include polystyrene, polyethylene, polypropylene, cycloolefin, polyethylene terephthalate, polymethyl methacrylate, polycarbonate, fluororesin, polyvinyl chloride, polysulfone, and polyphenylene sulfide.

Of these, polystyrene, polymethyl methacrylate and the like are particularly preferably used.

The surface of the base material layer (B) on the side where the temperature responsive layer (A) is arranged may be subjected to a surface treatment as necessary. Examples of the surface treatment include plasma treatment and corona treatment.

Further, the surface of the base material layer (B) on the side where the temperature response layer (A) is arranged may be smooth, and a three-dimensional structure such as a hole shape, a protrusion shape, or a wall shape is formed. May be. Examples of the substrate layer (B) having such a three-dimensional structure on the surface include, for example, commercially available three-dimensional structure cell culture substrates, SCIVAX NanoCulture Plate, Hitachi High-Technologies Nanopillar Plate, 3D Biomatrix Perfecta3D or Kuraray ELPLASIA can be used.

The temperature response layer (A) may be disposed on the surface of the base material layer (B) via at least one other layer. Examples of the other layers include polystyrene, polyethylene, polypropylene, cycloolefin, polyethylene terephthalate, polymethyl methacrylate, polycarbonate, fluororesin, polyvinyl chloride, polysulfone, and polyphenylene sulfide.

3. Other configurations, characteristics and applications of temperature-responsive cell culture substrate

The temperature-responsive cell culture substrate of the present invention may further include other layers in addition to the temperature-responsive layer (A) and the substrate layer (B) as necessary. Examples of other layers include a support layer used for the purpose of shape retention.

The shape of the temperature-responsive cell culture substrate of the present invention is not particularly limited. It may be a cell culture dish such as a Petri dish, or a plate, fiber or particle. The particles may be porous. Further, it may have another container shape generally used for cell culture or the like. As such a container shape, a flask, a bag, etc. are mentioned, for example.

The temperature-responsive cell culture substrate of the present invention can be used for all cells. Examples thereof include cells such as animals, insects and plants, and bacteria. Examples of the origin of animal cells include humans, monkeys, dogs, cats, rabbits, rats, nude mice, mice, guinea pigs, pigs, sheep, Chinese hamsters, cattle, marmoset, and African green monkeys.

The temperature-responsive cell culture substrate of the present invention can be preferably used for adherent cells. Adhesive cells can be widely selected and include, for example, endothelial cells, epidermis cells, epithelial cells, muscle cells, nerve cells, bone cells, fat cells, etc., as well as dendritic cells and macrophages. Examples of the endothelial cells include hepatocytes, Kupffer cells, vascular endothelial cells, and corneal endothelial cells. Examples of epidermal cells include fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells and epidermal keratinocytes. Examples of epithelial cells include tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, and corneal epithelial cells. Examples of muscle cells include mammary gland cells, pericytes, smooth muscle cells, and cardiomyocytes. Examples of nerve cells include kidney cells, pancreatic islets of Langerhans, peripheral nerve cells, and optic nerve cells. Examples of bone cells include osteoclasts and chondrocytes.

¡Various stem cells can be used as adherent cells. Adhesive stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), induced pluripotent stem cells (IPS cells; induced pluripotent stem cells) and other pluripotent stem cells; mesenchymal stem cells; hematopoietic stem cells; Stem cells such as unipotent stem cells (progenitor cells) such as cells, dermal fibroblasts, skeletal muscle myoblasts, osteoblasts, and odontoblasts.

In the temperature-responsive cell culture substrate of the present invention, a medium usually used for culturing target cells can be used as it is.

In the temperature-responsive base material for cell culture of the present invention, the temperature of the whole or part of the culture base material is made to be not less than UCST or not more than LCST of the temperature-responsive polymer, so that the cultured cells are detached without enzyme treatment. be able to. The peeling due to the temperature change may be performed in a culture solution or in another isotonic solution. In addition, the substrate can be tapped or swayed for the purpose of detaching and collecting the cells faster and more efficiently. If necessary, the medium may be stirred using a pipette or the like. Advantages of changing the temperature of only a part of the substrate include, for example, that it is possible to selectively detach only differentiated cell colonies in induction of iPS cell differentiation.

In the temperature-responsive substrate for cell culture of the present invention, the temperature-responsive polymer is preferably firmly fixed on the surface, and in this case, it can be used for reuse. In particular, the temperature-responsive base material for cell culture having a surface on which the above-mentioned 1.3 dendritic block copolymer is fixed is particularly preferably used for reuse because the temperature-responsive polymer is firmly fixed on the surface. be able to.

When used for reuse, a series of steps are performed in which cell culture is performed by adding a liquid medium, the cells are detached by temperature response, and the substrate surface is washed with an appropriate washing solution such as phosphate buffered saline. As a cycle, the same temperature-responsive substrate for cell culture is used in two or more cycles, that is, used for two or more reuses. The temperature-responsive substrate for cell culture of the present invention is preferably used for reuse three times or more.

Hereinafter, the present invention will be described in detail, but the present invention is not limited to the following examples.

<Production Example 1>
[Synthesis of S- (4-vinyl) benzyl S′-alkyltrithiocarbonate]
Sodium methoxide (5.6 ml, 0.028 mol) and methanol (MeOH, 28 ml) were put into a 100 ml three-necked flask under nitrogen substitution and stirred for 2 minutes. Thereto was added 2-ethyl-1-hexanethiol (4.10 g, 0.028 mol) dissolved in MeOH (22 ml), and the mixture was stirred at room temperature for 2 hours. Carbon disulfide (CS ₂ ) (2.1 ml, 0.035 mol) was added thereto, and the mixture was stirred at room temperature for 5 hours. Further, 4-vinylbenzyl chloride (4.3 ml, 0.028 mol) was added and the mixture was stirred as it was at room temperature for 20 hours. After the reaction, water was added and the mixture was extracted with dichloromethane. The organic layer was washed with saturated brine and dried over magnesium sulfide, and the solvent was distilled off under reduced pressure. Purification was performed by column chromatography using hexane as a developing solvent. The target product as an orange liquid was obtained in a yield of 5.6 g.
Reference: Macromolecules 2011, 44, 2034-2049

[Synthesis of Dendritic Polymer 1]
S- (4-vinyl) benzyl S ′-(2-ethyl-1-hexyl) trithiocarbonate (1.20 g, 3.61 × 10 ⁻³ mol) into a 10 ml branch tube flask under nitrogen substitution, Dehydrated toluene (1 ml) was added and dissolved by stirring. Further, 2,2′-azodiisobutyronitrile (AIBN, 0.0745 g, 4.50 × 10 ⁻⁴ mol) was added, and the mixture was stirred at 80 ° C. for 10 hours. After the reaction, 2 ml of toluene was added and diluted, and then dropped and reprecipitated into hexane cooled in an ice bath to recover 0.58 g of an orange oily polymer.

[Synthesis of dendritic block copolymer (temperature-responsive polymer content 85%)]
Dendritic polymer 1 (0.028 g), N-isopropylacrylamide (NIPAM, 1 g, 8.85 × 10 ⁻³ mol), dehydrated tetrahydrofuran (THF, 3 ml) described above into a 10 ml branch tube flask under nitrogen substitution And dissolved by stirring.
Further, AIBN (4.0 × 10 ⁻³ g, 4.6 × 10 ⁻⁶ mol) was added, and the mixture was stirred at 70 ° C. for 10 hours. After the reaction, 3 ml of THF was added for dilution, and then dropped and reprecipitated into diethyl ether to recover an egg-colored solid. It was again dissolved in 5 ml of THF and reprecipitated into diethyl ether to obtain 0.706 g of a white powdery solid.

<Production Example 2>
[Synthesis of Dendritic Polymer 2]
S- (4-vinyl) benzyl S ′-(2-ethyl-1-hexyl) trithiocarbonate (1.20 g, 3.61 × 10 ⁻³ mol) into a 10 ml branch tube flask under nitrogen substitution, Dehydrated toluene (1 ml) was added and dissolved by stirring. Further, AIBN (0.0745 g, 4.50 × 10 ⁻⁴ mol) was added, and the mixture was stirred at 80 ° C. for 24 hours. After the reaction, 2 ml of toluene was added and diluted, and then dropped and reprecipitated into hexane cooled in an ice bath to recover an orange oily polymer. The yield was 0.58g.

[Synthesis of dendritic block copolymer (temperature-responsive polymer content 95%)]
Add the above-mentioned dendritic polymer 2 (0.028 g), NIPAM (1 g, 8.85 × 10 ⁻³ mol), dehydrated THF (3 ml) into a 10 ml branch tube flask under nitrogen substitution, and dissolve by stirring. I let you. Further, AIBN (4.0 × 10 ⁻³ g, 4.6 × 10 ⁻⁶ mol) was added, and the mixture was stirred at 70 ° C. for 24 hours. After the reaction, 3 ml of THF was added for dilution, and then dropped and reprecipitated into diethyl ether to recover an egg-colored solid. It was again dissolved in 5 ml of THF and reprecipitated into diethyl ether to obtain 0.730 g of a white powdery solid.

<Comparative Production Example 1>
[Synthesis of poly-N-isopropylacrylamide (PNIPAM)]
NIPAM (5 g, 0.044 mol) and 2-propanol (25 ml) were added to a 50 ml three-necked flask under nitrogen substitution, and dissolved by stirring. Further, AIBN (0.0726 g, 4.4 × 10 ⁻⁴ mol) was added thereto, and the mixture was stirred at 80 ° C. for 6 hours. After the reaction, the solvent was distilled off under reduced pressure to obtain 4.95 g of a white solid.

<Example 1>
[Preparation of coating solution]
10 mg of the dendritic block copolymer synthesized in Production Example 1 was dissolved in a mixed solvent (4 ml) of THF / MeOH = 1/4 (v / v). This is called mother liquor. THF / MeOH = 1/4 (v / v) so that the applied amount in terms of PNIPAM is 3.5 μg / cm ² when 50 μl of this mother liquor is applied to a cell culture dish having a culture area of 9.6 cm ^2. Further dilution with a mixed solvent of This is referred to as a coating solution.

[Polymer immobilization on polystyrene substrate, no water washing]
The coating solution (50 μl) obtained by the above method was applied to a polystyrene cell culture dish (Corning Falcon 3001, culture area 9.6 cm ² ) and covered. The mixture was allowed to stand for 2.5 hours and then dried to obtain a temperature-responsive cell culture substrate.

[Polymer immobilization on polystyrene substrate, with water washing]
The coating solution (50 μl) was applied to a cell culture dish made of polystyrene (Falcon 3001, Corning, culture area 9.6 cm ² ) and covered. The mixture was allowed to stand for 4 hours and then dried. After drying, it was washed with pure water for 1 hour. After washing, the membrane was naturally dried overnight after draining to obtain a temperature-responsive cell culture substrate.

<Example 2>
[Preparation of coating solution]
The mixed solvent used for the preparation of the coating solvent was THF / MeOH = 2/5 (v / v). Other operations were performed in the same manner as in Example 1.

[Other operations]
The immobilization of the polymer on the polystyrene substrate and the cell evaluation method were carried out in the same manner as in Example 1.

<Example 3>
[Preparation of coating solution]
It was prepared in the same manner as in Example 1, except that the dendritic block copolymer was replaced with that synthesized in Production Example 2.

<Example 4>
[Preparation of coating solution]
The mixed solvent used for the preparation of the coating solution was THF / MeOH = 2/5 (v / v). In addition, the coating solution preparation method was the same as in Example 3.

<Comparative Example 1>
Corning Falcon 3001 (culture area 9.6 cm ² ) was used as Comparative Example 1.

<Comparative Examples 2 and 3>
[Preparation of coating solution]
A dendritic block copolymer was prepared in the same manner as in Example 1, except that the PNIPAM synthesized in Comparative Production Example 1 was replaced.

[Other operations]
The immobilization of the polymer on the polystyrene substrate and the cell evaluation method were carried out in the same manner as in Example 1. After the polymer application, the case without water washing was designated as Comparative Example 2, and the case with water washing was designated as Comparative Example 3.

<Comparative example 4>
As a comparative example 4, a commercially available temperature-responsive cell culture substrate A in which PNIPAM was chemically imparted to the polystyrene substrate surface by electron beam irradiation by graft polymerization was used.

[Measurement by XPS]
Using a surface analyzer PHI5000 VersaProbe II manufactured by ULVAC-PHI, monochromatic X-ray AlKα was irradiated and measured at an emission angle of 45 °.

X-ray photoelectron spectroscopy measurement while etching with an argon gas cluster ion beam was performed as follows.
The elements in the depth direction were measured while etching with argon gas cluster ions under the conditions of 2.5 kV and 10 nA (Area 2 mm) using a surface analyzer PHI5000 VersaProbe II manufactured by ULVAC-PHI. The etching rate was calculated to be 2.0 nm / min by etching a 61 nm thick PNIPAM thin film (substrate: silicon wafer) with argon gas cluster ions until Si was detected.

[Coverage with temperature-responsive polymer]
The coverage with the temperature-responsive polymer at the surface or a specific etching depth was evaluated as follows.
The value (%) of the following formula (1) regarding the nitrogen element concentration N _1s and the carbon element concentration C _1s measured by the XPS method at an emission angle of 45 ° was defined as the coverage.
(1) 100 × (N _1s / C _1s ) / (N / C) (%)
(In the formula, N / C represents the theoretical value of each element ratio in the temperature-responsive polymer)

[FT-IR-ATR method]
A temperature-responsive cell culture substrate was prepared using a polystyrene cell culture dish as a substrate and PNIPAM immobilized as a temperature-responsive polymer. By performing FT-IR-ATR measurement on the substrate, amide stretch (1650 cm) derived from PNIPAM with respect to the absorption strength of benzene ring stretch (1600 cm ⁻¹ ) derived from polystyrene represented by the following formula (5): A ratio of absorption intensity of ^-1 ) was obtained.
(5) Absorption intensity ratio = I ₁₆₅₀ / I ₁₆₀₀
A known amount of PNIPAM (1 to 10 μg / cm ² ) is immobilized on a polystyrene substrate, and a calibration curve is prepared in advance from the absorption intensity ratio obtained by the formula (5), thereby immobilizing on the polystyrene substrate. The amount of unknown PNIPAM was determined. It was assumed that the polymer immobilization layer on the polystyrene substrate was sufficiently thin with respect to the penetration depth of IR light into the sample (reference document: Langmuir 2004, 20, 5506-5511).

[Cell evaluation method of temperature-responsive cell culture substrate after culture]
1 ml of medium [Dulbecco's modified Eagle medium (DMEM), 10% calf serum, 1% antibiotics] is added to a 3.5 cmφ temperature-responsive cell culture substrate, and 1 × 10 ⁵ 3T3 mouse fibroblasts are dispersed. 1 ml of the prepared medium was added and cultured in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for 4 days.

After completion of the culture, the state of attachment of the cultured cells and whether the cultured cells were growing until they became full in the petri dish were observed using an inverted microscope. Thereafter, the temperature-responsive petri dish containing the cultured cells was allowed to stand in a low-temperature CO ₂ incubator (20 ° C., 5% CO ₂ ) and cooled for 15 minutes. After cooling, the peeled state of the cultured cells and whether the cultured cells were peeled in a sheet form were observed with an inverted microscope, and the peelability of the cell sheets was evaluated according to the following criteria.
1. Does not peel. Partial peeling 2. 4. The whole cultured cell peels off, but it takes about 15 minutes to peel off. 4. Good peeling within 15 minutes. It peels very well in a short time

[Evaluation of Example 1 (without washing) and Comparison with Comparative Example 4]
The results are shown in Table 1. The base material of Example 1 (without water washing) had a PNIPAM coverage of 99% or more on the outermost surface at an etching depth of 0 nm. On the other hand, the base material obtained in Comparative Example 4 (commercial product A) had an outermost surface PNIPAM coverage of less than 80%, suggesting that the base material was exposed. Furthermore, when the PNIPAM coverage with respect to the etching depth was measured, it was suggested that the base material obtained in Example 1 was twice or more deeper than the base material obtained in Comparative Example 4. It was. From the above, it was confirmed that the product of the present invention was able to effectively embed the polymer in the base material and that the surface of the base material was almost covered with PNIPAM.

The results of Examples and Comparative Examples are summarized in Table 1.
[Evaluation of Example 1 (with water washing) and Comparison with Comparative Example 4]
In the base material of Example 1 (without water washing), there was a possibility that the polymer that could not be immobilized on the base material might inhibit the cell sheet peelability. Therefore, the temperature-responsive cell culture substrate obtained in Example 1 was washed with water, and before and after washing with water, the etching depth at which the PNIPAM coverage was 5% or less (= depth of PNIPAM) was compared. It was confirmed that the existence depth of PNIPAM was about 3 nm shallow. In the FT-IR-ATR measurement, it was confirmed that the PNIPMA coating amount was reduced to about half by washing with water, and that the coefficient of variation was reduced to 5% or less. Therefore, it was confirmed that a very uniform PNIPAM layer can be obtained by removing excess polymer. As in Comparative Example 4, there was concern about exposure of the substrate surface, but when the PNIPAM coverage at an etching depth of 0 nm was confirmed, it was found that the surface was coated in the same manner as before washing.

The cell sheet peelability was evaluated using mouse fibroblasts for the substrate without washing in Example 1, the substrate with washing in Example 1, and the substrate in Comparative Example 4. As a result, good peelability was exhibited in the order of Comparative Example 4 <Example 1 (without water washing) <Example 1 (with water washing). This is considered to be due to the effect that the surface was effectively covered with PNIPAM by embedding the polystyrene dendritic polymer site in the base material, and the excess polymer was removed by washing with water. On the other hand, in Comparative Example 4, it was considered that the peeling time was longer than that in Example 1 because both the coating amount and the coverage of PNIPAM were insufficient.

In the above [Evaluation of Example 1 (with water washing)], it was confirmed that about half of the initial coating amount of polymer was removed by water washing. Therefore, with the aim of better cell sheet peelability, an attempt was made to increase the amount of polymer immobilized on the substrate as follows.

[Evaluation of Example 2]
In Example 2, the THF content in the coating solvent was increased from 20% to 29% by volume. The effect was evaluated from both the PNIPAM coverage and coverage. As a result, it was confirmed by FT-IR-ATR measurement that the amount of PNIPAM coating after washing was increased by about 20% compared to Example 1, and accordingly, the PNIPAM coverage with respect to the etching depth of 2 nm and the PNIPAM coverage was 5 nm. The following etching depth also increased slightly.

As a result of evaluating the cell sheet peelability of the substrate of Example 2 obtained above using mouse fibroblasts, the peel was better than that of Example 1 in both “without water wash” / “with water wash” Showed sex. In particular, “with water” exhibited very good peelability, and evaluation 5 was obtained.

[Evaluation of Examples 3 and 4]
For further exfoliation, in Examples 3 and 4, a temperature-responsive cell culture substrate using a dendritic block copolymer having a PNIPAM content of 95% by weight in the polymer was prepared. When compared with the results of Examples 1 and 2 in which the PNIPAM content in the polymer was 85%, the PNIPAM coating amount, the coating rate at each etching depth, and PNIPAM, especially in the base material of “THF content 29%, with water washing” There was an increasing trend in the depth of existence. This is thought to be due to the effect that the PNIPAM chain in the dendritic block copolymer is made longer.

For each of the substrates of Examples 3 and 4 obtained above, cell sheet peelability was evaluated using mouse fibroblasts. As a result, the “without water washing” base material had the same results as in Examples 1 and 2, but the “with water washing” base material showed better peelability than Examples 1 and 2. In particular, the substrate having a "THF content of 29% and water washing" could be peeled off in 8 minutes, which is an extremely short time.

[Evaluation of Comparative Example 1]
In the base material of Comparative Example 1, since the surface was not coated with the temperature-responsive polymer, the cell detachability as described above was not expressed.

[Evaluation of Comparative Examples 2 and 3]
In Comparative Example 2, the substrate coated with PNIPAM obtained in Comparative Production Example 1 was evaluated, but cell detachability was not expressed. This PNIPAM does not have a means for binding to the base material, so it is considered that it was dissolved and removed when the medium (water) was added. In Comparative Example 3, the base material of Comparative Example 2 was washed with water, and the surface coating with PNIPAM could not be confirmed. This is probably because all the applied PNIPAM was already removed at the time of water washing because PNIPAM was not bonded to the substrate. Therefore, in Comparative Example 3, cell detachability was not expressed.

[Evaluation of Example 1 (with water washing)]
About the base material with water washing of Example 1, the polymer elution test with respect to room temperature water was done. Specifically, the degree of change in the PNIPAM coverage on the surface after immersion of the sample in room temperature water was measured using the XPS (analysis depth of about 10 nm) and FT-IR-ATR (analysis depth of 1 to several). μm) was used for analysis. As Comparative Example 4, a commercially available temperature-responsive cell culture substrate in which PNIPAM was chemically applied to a polystyrene substrate was measured in the same manner by a graft polymerization method using electron beam irradiation.

Evaluation using XPS was performed according to the following procedure.
1. Each base material was cut into an appropriate size and used as a sample.
2. The back of each sample was marked with a magic pen. At this time, in order to perform measurement at n = 2, marks were added at two locations.
3. 2. XPS analysis was performed on the mark portion.
4). The sample after measurement was taken out from the apparatus and immersed in a 50 ml screw tube bottle filled with distilled water. At this time, the water temperature was 20 ° C.
5. After 24 hours 4. After the sample was taken out and dried, XPS analysis was performed again on the mark portion.
6). From the measured values before and after immersion, the PNIPAM coverage was calculated as described above. Moreover, the change rate calculated from each value was shown as PNIPAM residual rate after a dissolution test.

The calculation results are shown in Table 2.

As a result of the evaluation, at the analysis depth of about 10 nm, the base material of Example 1 showed no change in the PNIPAM residual rate before and after the dissolution test. On the other hand, in Comparative Example 4 in which PNIPAM was chemically bonded to the substrate surface, the coverage was clearly reduced, suggesting that the polymer was eluted.

Next, evaluation using FT-IR-ATR was performed according to the following procedure.
1. Each base material was cut into an appropriate size to prepare a sample (two about 1.5 cm squares were prepared).
2. A 50 ml screw tube was filled with distilled water. At this time, the water temperature was 20 ° C.
3. 2. In the distilled water of 1. above. One of these samples was immersed and taken out after 24 hours.
4). FT-IR-ATR analysis was performed for each sample with and without immersion, and the amount of PNIPAM on each surface was analyzed by a calibration curve method (measured at 5 points for each sample).
5). The PNIPAM residual rate was calculated according to the following formula.
(PNIPAM amount with immersion / PNIPAM amount without immersion) x 100 (%)

Table 3 shows the calculation results.

In contrast to Comparative Example 4 in which PNIPAM was chemically bonded to the surface by graft polymerization, it was revealed that the PNIPAM was less eluted in the base material of Example 1.

Claims

(A) a temperature-responsive layer; and (B) a temperature-responsive cell culture substrate containing a substrate layer,
The temperature responsive layer (A) contains a temperature responsive polymer having nitrogen atoms,
The temperature responsive layer (A) is disposed on at least one surface of the base material layer (B),
The substrate layer (B) does not have nitrogen atoms, and the surface on the temperature response layer (A) side has a nitrogen element concentration N 1s and carbon measured by X-ray photoelectron spectroscopy at an emission angle of 45 °. The element concentration C 1s is expressed by the following formula (1):
(1) 100 × (N 1s / C 1s ) / (N / C) ≧ 80
(In the formula, N / C represents a theoretical value of each element ratio in the temperature-responsive polymer)
Satisfying a temperature-responsive cell culture substrate.
The surface on the temperature-responsive layer (A) side is etched from the surface side with an argon gas cluster ion beam, and N 1s and C 1s measured by X-ray photoelectron spectroscopy satisfy the following condition (A): The temperature-responsive cell culture substrate according to claim 1:
(A) For the nitrogen atom, the measured value at an etching depth of 2 nm satisfies the following formula (2):
(2) 100 × (N 1s / C 1s ) / (N / C) ≧ 50
(In the formula, N / C represents the same meaning as described above).
The temperature-responsive cell culture substrate according to claim 1 or 2, wherein the temperature-responsive polymer is fixed to 1 to 10 µg / cm 2 on the surface of the temperature-responsive layer (A).
The temperature-responsive cell culture substrate according to any one of claims 1 to 3, wherein the temperature-responsive layer (A) contains a block polymer in which the temperature-responsive polymer is bonded to an end of a dendritic polymer.
The temperature-responsive cell culture substrate according to claim 4, wherein the dendritic polymer is a dendritic polymer having a styrene skeleton or a siloxane skeleton.
At least one of the temperature-responsive polymers is
A temperature-responsive polymer obtainable by polymerizing a monomer composition containing at least one selected from the group consisting of (meth) acrylamide, N- (or N, N-di) -substituted (meth) acrylamide and vinyl ether, or The temperature-responsive cell culture substrate according to any one of claims 1 to 5, which is a polyvinyl alcohol partially acetylated product.
The N- (or N, N-di) -substituted (meth) acrylamide is poly-N-isopropyl (meth) acrylamide, poly-N, N-diethyl (meth) acrylamide, and poly-N, N-dimethyl (meta). The temperature-responsive cell culture substrate according to claim 6, which is at least one selected from the group consisting of acrylamide).
The temperature-responsive cell culture substrate according to any one of claims 1 to 7, wherein the substrate layer (B) contains polystyrene.
The temperature-responsive cell culture substrate according to any one of claims 1 to 8, which is for reuse.
(A) A block polymer, a solution containing a good solvent and a poor solvent of polystyrene, in which a block polymer having a temperature-responsive polymer bonded to a terminal of a dendritic polymer having a styrene skeleton or a siloxane skeleton is dissolved, A step of dropping on the surface of a polystyrene substrate and developing the surface;
(B) placing the surface obtained in step (a) under the vapor of the solvent for 2 hours or more; and (c) drying the surface obtained in step (b). A method for producing a temperature-responsive cell culture substrate.