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

Abstract

The present disclosure pertains to a temperature-responsive block copolymer comprising: a block segment (A) having a lower critical solution temperature (LCST) with respect to water; and a water-insoluble block segment (B). When a film comprising the temperature-responsive block copolymer is formed, the amount of change in the bubble contact angle on the surface of the film as measured in water at 21°C and in water at 37°C is at least 5.0°.

Description

Temperature-responsive block copolymer, its production method, and cell culture substrate

[0001] The present invention relates to a temperature-responsive block copolymer, a method for producing the same, and a cell culture substrate.

[0002] The use of temperature-responsive polymers in various applications, such as cell culture substrates, has been investigated (see, for example, Patent Documents 1 to 3). When a cell culture substrate is used, the substrate surface is coated with a temperature-responsive polymer, and the sol transition of the temperature-responsive polymer due to a drop in the temperature of the surrounding environment weakens the adhesive force of the substrate surface, allowing cells to detach. This allows cells to be recovered while minimizing damage to the cells.

[0003] Japanese Patent Publication No. 2020014453 Japanese Patent Publication No. 6447787 Japanese Patent Publication No. 7293683

[0004] An object of the present invention is to provide a temperature-responsive block copolymer capable of forming a cell culture substrate with improved cell detachment properties, and a method for producing the same.An object of the present invention is to provide a cell culture substrate with improved cell detachment properties.

[0005] The present invention relates to the following inventions. [1] A temperature-responsive block copolymer comprising a block segment (A) having a lower critical solution temperature (LCST) in water and a water-insoluble block segment (B), wherein, when a film made of the temperature-responsive block copolymer is formed, the change in the air bubble contact angle on the film surface measured in water at 21°C and in water at 37°C is 5.0° or more. [2] The temperature-responsive block copolymer according to [1], wherein the change in the air bubble contact angle is 10° or more and 40° or less. [3] The temperature-responsive block copolymer according to [1] or [2], wherein at least one functional group selected from the group consisting of a hydroxyl group, a mercapto group, an amino group, a substituted amino group, a cyano group, an ester group, an amide group, a carbonyl group, and a carboxyl group is introduced into an end of at least one of the block segments (A) and (B). [4] The temperature-responsive block copolymer according to any one of [1] to [3], wherein at least one functional group selected from the group consisting of a hydroxyl group, a mercapto group, an amino group, a substituted amino group, a cyano group, an ester group, an amide group, a carbonyl group, and a carboxyl group is introduced into an end of the block segment (A). [5] The block copolymer according to any one of [1] to [4], wherein the block segment (B) contains a monomer unit represented by the following general formula (b1): [In the formula, R １ represents a hydrogen atom or a methyl group, Q represents an ester group, and R ２represents a hydrocarbon group having 1 to 30 carbon atoms.] [6] The temperature-responsive block copolymer according to any one of [1] to [5], wherein the degree of polymerization of the block segment (A) is 100 or more but less than 400. [7] A cell culture substrate comprising a substrate and a coating layer covering at least a part of the surface of the substrate, wherein the coating layer comprises a temperature-responsive block copolymer, the temperature-responsive block copolymer comprising a block segment (A) having a lower critical solution temperature (LCST) in water and a water-insoluble block segment (B), and wherein a change in bubble contact angle on the surface of the coating layer measured in water at 21°C and in water at 37°C is 5.0° or more. [8] The cell culture substrate according to [7], wherein the thickness of the coating layer is 5 to 1,000 nm. [9] The cell culture substrate according to any one of [7] and [8], wherein the substrate is any one of a plate, a film, a flask, and a bag.

[10] The cell culture substrate according to any one of [7] to [9], comprising a cell-adhesive region that is both cell-adhesive and temperature-responsively detachable, and a cell-non-adhesive region.

[11] The cell culture substrate according to

[10] , wherein the cell-non-adhesive region comprises a hydrophilic polymer.

[12] The cell culture substrate according to any one of [7] to

[11] , wherein the coating layer forms a phase-separated structure or a surface structure derived from phase separation at culture temperatures.

[13] The cell culture substrate according to any one of [7] to

[12] , wherein the coating layer has a thickness of 10 to 50 nm.

[14] The cell culture substrate according to any one of [7] to

[13] , wherein the substrate is made of polycarbonate.

[15] A method for producing a temperature-responsive block copolymer comprising a block segment (A) having a lower critical solution temperature (LCST) in water and a water-insoluble block segment (B), wherein a polar group is introduced at an end of the block segment (A) or (B), the method comprising: [In formula (I), the wavy line represents a bond on the block segment (A) or (B), and * represents a bond on the terminal side.], and a terminal group containing a thiocarbonylthio group, with a radical generator having a polar group to convert the terminal group containing the thiocarbonylthio group to a terminal group containing the polar group, thereby obtaining the temperature-responsive block copolymer.

[16] The method according to

[15] , wherein the radical generator having a polar group comprises at least one selected from the group consisting of azobisisobutyronitrile, 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 4,4'-azobis(4-cyanovaleric acid), and 2,2'-azobis(2-methylpropionic acid).

[17] A method for producing a temperature-responsive block copolymer comprising a block segment (A) having a lower critical solution temperature (LCST) in water and a water-insoluble block segment (B), wherein a mercapto group is introduced at an end of the block segment (A) or (B), the method comprising: and a terminal group containing a thiocarbonylthio group represented by formula (I): [in formula (I), the wavy line represents a bond on the block segment (A) or (B) side, and * represents a bond on the terminal side], and hydrolyzing the terminal group containing the thiocarbonylthio group to convert the terminal group containing the thiocarbonylthio group into a mercapto group, thereby obtaining the temperature-responsive block copolymer.

[0006] According to the present invention, it is possible to provide a temperature-responsive block copolymer capable of forming a cell culture substrate with improved cell detachment properties, and a method for producing the same. According to the present invention, it is possible to provide a cell culture substrate with improved cell detachment properties.

[0007] In this specification, a numerical range indicated using "to" indicates a range that includes the numerical values ​​before and after "to" as the minimum and maximum values, respectively. Furthermore, unless otherwise specified, the units of the numerical values ​​before and after "to" are the same. In the numerical ranges described in stages in this specification, the upper or lower limit of a certain numerical range may be replaced with the upper or lower limit of a numerical range of another stage. Furthermore, in the numerical ranges described in this specification, the upper or lower limit of that numerical range may be replaced with a value shown in the examples. Furthermore, the upper and lower limits individually described can be arbitrarily combined. Unless otherwise specified, the materials exemplified below may be used alone or in combination of two or more types. When multiple substances corresponding to each component are present in the composition, the content of each component in the composition refers to the total amount of the multiple substances present in the composition, unless otherwise specified. Furthermore, in this specification, "and / or" means one or both. For example, "A and / or B" means either A, B, or a combination of A and B.

[0008] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail below with reference to preferred embodiments thereof. However, the present invention is not limited to the following embodiments, and modifications of the following embodiments may be made.

[0009] [Block Copolymer] The temperature-responsive block copolymer according to this embodiment comprises a temperature-responsive block segment (A) having a lower critical solution temperature (LCST) in water and a water-insoluble block segment (B). In this specification, the term "temperature-responsive block copolymer" refers to a polymer whose degree of hydrophilicity and hydrophobicity changes with temperature. A temperature-responsive block copolymer is a polymer that becomes hydrophobic at a specific temperature when the temperature of the polymer is increased from a low temperature to a high temperature in the presence of water molecules.

[0010] When a film made of the temperature-responsive block copolymer is formed, the change in bubble contact angle on the film surface measured in water at 21°C and in water at 37°C is 5.0° or more. By forming a coating layer containing the temperature-responsive block copolymer on a cell culture substrate, a cell culture substrate with improved cell detachment properties can be obtained. As used herein, "cell detachment properties" refers to the ease with which cells grown on the cell culture substrate can be detached from the cell culture substrate. The film may be any film containing a temperature-responsive block copolymer. The film preferably contains 80% by mass or more of the temperature-responsive block copolymer, and more preferably is a film consisting solely of the temperature-responsive block copolymer.

[0011] The change in bubble contact angle is determined from the bubble contact angle measured by the following method. A polymer solution is prepared by diluting a temperature-responsive block copolymer with ethanol. The content of the temperature-responsive block copolymer in the polymer solution is 0.8% by mass based on the total amount of the polymer solution. 100 μL of the polymer solution is dropped onto a polycarbonate film, and a film made of the temperature-responsive block copolymer is formed on the polycarbonate film by spin coating at a rotation speed of 2000 rpm for 60 seconds. The polycarbonate film with the formed film is used as a film for measuring the bubble contact angle. The film for measuring the bubble contact angle is immersed in water at 21°C or 37°C for 10 minutes, and the bubble contact angle (°) is measured using a contact angle meter under conditions of 1 μL of air bubbles in water. The change in bubble contact angle is calculated from the bubble contact angle (θ) measured in water at 21°C. １ ) and the bubble contact angle (θ ２ ) and the difference (θ １ -θ ２ The detailed measurement conditions are as described in the Examples below.

[0012] The change in bubble contact angle may be 8.0° or more, and since cell detachment is further improved, it may be 10.0° or more, 12.0° or more, 14.0° or more, 16.0° or more, 18.0° or more, 20.0° or more, 22.0° or more, 24.0° or more, 26.0° or more, 28.0° or more, or 30.0° or more. The change in bubble contact angle may be 40.0° or less, 38.0° or less, 36.0° or less, 34.0° or less, 32.0° or less, 30.0° or less, 28.0° or less, 26.0° or less, 24.0° or less, 22.0° or less, 20.0° or less, 18.0° or less, 16.0° or less, 14.0° or less, or 12.0° or less. The change in bubble contact angle may be 10.0° or more and 40.0° or less, 14.0° or more and 36.0° or less, 18.0° or more and 32.0° or less, or 20.0° or more and 30.0° or less, thereby further improving cell detachment properties.

[0013] The bubble contact angle (θ １ ) may be, for example, 140° or more, 145° or more, 150° or more, 152° or more, or 154° or more, and may be 170° or less, 165° or less, or 161° or less.

[0014] The bubble contact angle (θ ２ ) may be, for example, 120° or more, 125° or more, or 130° or more, and may be 160° or less, 155° or less, or 150° or less.

[0015] The change in the bubble contact angle can be adjusted to satisfy the above-mentioned numerical range by, for example, adjusting the type and content of the block segment (A) in the block copolymer, adjusting the degree of hydrophilicity or hydrophobicity of the functional group present in the block segment (A) (for example, by introducing a polar group to the end of the block segment (A)), adjusting the ratio of the block segment (A) to the block segment (B), adjusting the layer thickness of the coating layer of the block copolymer, or a combination of these methods.

[0016] The temperature-responsive block copolymer may be a diblock copolymer containing at least a block segment (A) and a block segment (B). The block segment (A) and the block segment (B) may be bonded directly or via a spacer.

[0017] The temperature-responsive block copolymer may have a polar group introduced at one end of the block segment (A) and the block segment (B). Examples of the polar group include a hydroxyl group (—OH), a mercapto group (—SH), an amino group (—NH ２ ), a substituted amino group (a monosubstituted amino group (-NHR) or a disubstituted amino group (-NR ２ )), cyano group (-CN), ester group (-C(=O)-O-R or -O-C(=O)-R), amide group (-NH-C(=O)-R or -C(=O)-NH-R), carbonyl group (-C(=O)-R), carboxyl group (-COOH), nitro group (-NO ２ ), sulfo group (—SO ３ H). R represents a substituent in the polar group. R may be, for example, an alkyl group, and may be an alkyl group having 1 to 6 or 1 to 3 carbon atoms.

[0018] The polar group may be a nitrile group, a hydroxyl group, a carboxyl group, an ester group (for example, —C(═O)—O—CH ３ ), or mercapto group, and a nitrile group, a hydroxyl group, a carboxyl group, or an ester group is more preferred because reactions between the terminals or reactions with other substrates are less likely to occur (excellent terminal stability).

[0019] The introduction of polar groups at the terminals of the temperature-responsive block copolymer was confirmed by nuclear magnetic resonance spectroscopy ( １ H-NMR spectroscopy, and １３ This can be confirmed by C-NMR spectroscopy, Fourier transform infrared spectroscopy (FT-IR).

[0020] The number average molecular weight (Mn) of the temperature-responsive block copolymer may be 8,000 or more, 10,000 or more, 15,000 or more, 20,000 or more, 25,000 or more, 30,000 or more, or 35,000 or more, and may be 200,000 or less, 150,000 or less, 100,000 or less, 80,000 or less, 60,000 or less, or 45,000 or less.

[0021] The polydispersity (Mw / Mn) of the temperature-responsive block copolymer may be 1.30 or less, 1.25 or less, 1.20 or less, or 1.15 or less, since this further improves cell detachment. The polydispersity (Mw / Mn) of the temperature-responsive block copolymer may be, for example, 1.02 or more, 1.06 or more, 1.08 or more, 1.10 or more, 1.12 or more, or 1.14 or more.

[0022] The weight average molecular weight (Mw), number average molecular weight (Mn), and molecular weight distribution (Mw / Mn) are measured by gel permeation chromatography (GPC). The GPC device used is a Tosoh Corporation HLC-8320GPC, and two Tosoh Corporation TSKgel Super AWM-H columns are used. The column temperature is set to 40°C. The eluent used is N,N-dimethylformamide containing 10 mM lithium bromide. The measurement sample is prepared at 1.0 mg / mL. A molecular weight calibration curve is prepared using polymethyl methacrylate (Polymer Laboratories Ltd.) of known molecular weight.

[0023] The block segment (A) is a temperature-responsive block segment having a lower critical solution temperature (LCST) in water.

[0024] The LCST of block segment (A) refers to the LCST of a polymer (hereinafter also referred to as "polymer (A)") containing only the monomers constituting the monomer units of block segment (A) as monomer units. The LCST of block segment (A) is the temperature at which polymer (A) becomes insoluble in water. The LCST is a value determined by measuring the transmittance of light with a wavelength of 500 nm in an aqueous solution containing 0.6% by mass of polymer (A) while raising the temperature of the aqueous solution at a rate of 1°C / min. The transmittance is approximately constant from low temperatures until the LCST is reached. As the aqueous solution becomes cloudy near the LCST, the transmittance drops sharply, and then becomes approximately constant again. The LCST can be determined by determining the temperature at which the transmittance at temperatures below the LCST and the transmittance at temperatures above the LCST are averaged (midpoint method) in a curve obtained by plotting the change in transmittance with temperature. The temperature range for measurement includes a temperature range of 5°C or higher at which the transmittance becomes roughly constant at temperatures below the LCST, and also includes a temperature range of 5°C or higher at which the transmittance becomes constant at temperatures above the LCST. Specifically, the LCST can be determined by the method described in Patent Document 3.

[0025] The LCST of the block segment (A) is preferably 50°C or lower, more preferably 35°C or lower, because when a substrate coated with the block copolymer is used for cell culture, cells can be cultured at a temperature close to body temperature. From the viewpoint of further suppressing cell detachment due to operations such as changing the culture medium during culture, the LCST of the block segment (A) is preferably 25°C or lower. The LCST of the block segment (A) is preferably 0°C or higher, 5°C or higher, 10°C or higher, or 15°C or higher, because this enables cell detachment under conditions where damage to the cells is further suppressed.

[0026] The monomer constituting the monomer unit of the block segment (A) is not particularly limited, and examples thereof include (meth)acrylamide compounds such as acrylamide and methacrylamide; N-alkyl-substituted (meth)acrylamide derivatives such as N,N-diethylacrylamide, N-ethylacrylamide, N-n-propylacrylamide, N-n-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-t-butylacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, and N-tetrahydrofurfurylmethacrylamide; Examples include N,N-dialkyl-substituted (meth)acrylamide derivatives such as N,N-dimethyl(meth)acrylamide, N,N-ethylmethylacrylamide, and N,N-diethylacrylamide; (meth)acrylamide derivatives having a cyclic group such as 1-(1-oxo-2-propenyl)-pyrrolidine, 1-(1-oxo-2-propenyl)-piperidine, 4-(1-oxo-2-propenyl)-morpholine, 1-(1-oxo-2-methyl-2-propenyl)-pyrrolidine, 1-(1-oxo-2-methyl-2-propenyl)-piperidine, and 4-(1-oxo-2-methyl-2-propenyl)-morpholine; vinyl ethers such as methyl vinyl ether; and proline derivatives such as N-proline methyl ester acrylamide.

[0027] The monomer constituting the monomer unit of the block segment (A) is, for example, preferably at least one selected from the group consisting of N,N-diethylacrylamide, N-n-propylacrylamide, N-isopropylacrylamide, N-n-propylmethacrylamide, N-ethoxyethylacrylamide, N-tetrahydrofurfuryl acrylamide, and N-tetrahydrofurfuryl methacrylamide, more preferably at least one selected from the group consisting of N-n-propylacrylamide and N-isopropylacrylamide, and even more preferably N-isopropylacrylamide.

[0028] The block segment (A) may contain one or more types of monomers as monomer units. The arrangement of the block segment (A) containing two or more types of monomers as monomer units may be any of a random arrangement, an alternating arrangement, and a block arrangement.

[0029] The degree of polymerization of the block segment (A) in the temperature-responsive block copolymer may be 100 or more, 120 or more, 140 or more, 160 or more, 180 or more, 200 or more, 210 or more, 220 or more, or 230 or more, since this improves cell detachment. The degree of polymerization of the block segment (A) in the block copolymer may be less than 400, 350 or less, 300 or less, 280 or less, 260 or less, 250 or less, or 240 or less, since this reduces elution in water when used as a coating material for a cell culture substrate. The degree of polymerization of the block segment (A) in the temperature-responsive block copolymer is preferably 200 to 350, since this improves cell detachment. The degree of polymerization of the block segment (A) can be calculated, for example, by the following formula: Formula: Degree of polymerization of block segment (A) = (total molar amount of monomers dissolved for polymerization reaction) x (conversion rate of monomers to polymer) / (molar amount of chain transfer agent) Here, the conversion rate of monomers to polymer is the molar amount of the reaction solution immediately after the termination of the polymerization reaction. １ It is calculated by H-NMR measurement. Specifically, it can be calculated by the following formula using the integral value of protons at specific positions of the monomer or polymer: (Conversion rate of monomer to polymer) = (integral value of polymerized monomer) / {(integral value of polymerized monomer) + (integral value of unreacted monomer)} × 100

[0030] The degree of polymerization of the block segment (A) can also be determined by calculation from the copolymer composition ratio determined by analyzing the block copolymer by pyrolysis gas chromatography and the absolute molecular weight of the block copolymer.

[0031] The proportion of the block segment (A) in the temperature-responsive block copolymer may be 50% by mass or more, 55% by mass or more, 60% by mass or more, or 65% by mass or more, based on the total amount of the temperature-responsive block copolymer. The proportion of the block segment (A) in the block copolymer may be 95% by mass or less, 90% by mass or less, 85% by mass or less, or 80% by mass or less. The proportion of the block segment (A) can be calculated, for example, by the following formula: Formula: Proportion of block segment (A) = (Degree of polymerization of block segment (A)) x (Monomer molecular weight of block segment (A)) / {(Degree of polymerization of block segment (A)) x (Monomer molecular weight of block segment (A)) + (Degree of polymerization of block segment (B)) x (Monomer molecular weight of block segment (B))} x 100

[0032] It is preferable that a polar group be introduced into the end of the block segment (A). In this case, cell detachment properties are further improved. Since cell detachment properties are further improved, it is preferable that at least one functional group selected from the group consisting of a hydroxyl group, a mercapto group, an amino group, a substituted amino group, a cyano group, an ester group, an amide group, a carbonyl group, and a carboxyl group be introduced into the end of the block segment (A). When the degree of polymerization of the block segment (A) is 100 or more but less than 400, from the viewpoint of further improving cell detachment properties, it is preferable that the polar group at the end of the block segment (A) is at least one selected from the group consisting of a hydroxyl group, an amino group, a substituted amino group, a cyano group, an ester group, an amide group, a carbonyl group, and a carboxyl group. In this case, it is thought that the block segment (A) is more likely to be arranged on the opposite side (water side) of the substrate in water.

[0033] The block copolymer may contain, for example, a structure represented by the following general formula (a1) as a block segment (A) and an end group bonded to the block segment (A).

[0034] In formula (a1), R ａ１ represents a hydrogen atom or a methyl group. ａ２ represents an alkyl group. ａ２The alkyl group represented by R may be branched or linear. ａ２ The alkyl group represented by the formula (I) may be an alkyl group having 1 to 3 carbon atoms, or may be an isopropyl group. n represents an integer of 1 or more. * represents the bonding site with segment (B).

[0035] X represents a group containing a polar group. X may contain one or more polar groups. X may be a functional group consisting of a polar group and a linking group that links the block segment (A) and the polar group, or a functional group consisting of only a polar group. Examples of X include -C(CH ３ ) ２ -CN, -C(CH ３ ) ２ -C(=O)NHCH ２ CH ２ —OH, —C(CH ３ ) ２ -C(=O)OCH ３ , -C(CH ３ )(CN)-CH ２ CH ２ Examples include —COOH and —SH.

[0036] The block segment (B) is a water-insoluble block segment. The block segment (B) indicates that a polymer (hereinafter also referred to as "polymer (B)") containing only the monomers constituting the monomer units of the block segment (B) as monomer units is insoluble in water. "Water-insoluble" means that the amount of the polymer (B) that dissolves in 100 g of water at 25°C is less than 0.01 g. The amount of the polymer (B) that dissolves in 100 g of water is preferably 10 mg or less.

[0037] The block segment (B) may contain one or more types of monomers as monomer units. The arrangement of the block segment (B) containing two or more types of monomers as monomer units may be any of a random arrangement, an alternating arrangement, and a block arrangement.

[0038] The block segment (B) may contain, for example, a monomer unit represented by the following formula (b1) (hereinafter also referred to as "monomer unit (b1)").

[0039] In formula (b1), R１ represents a hydrogen atom or a methyl group.

[0040] Q represents a divalent group selected from the group consisting of an ester group (*1-C(=O)-O-*2), an amide group (*1-C(=O)-NH-*2), a urethane group (*1-NH-C(=O)-O-*2 or *1-O-C(=O)-NH-*2) and an ether group (-O-). *2 represents R ２ Q represents a bonding site with the carbon atom, and *1 represents a bonding site with the carbon atom. Q is preferably an ester group or an amide group, and more preferably an ester group.

[0041] R ２ represents a hydrocarbon group having 1 to 30 carbon atoms, a substituent represented by the following general formula (b2) (hereinafter also referred to as "substituent (b2)"), a substituent represented by the following general formula (b3) (hereinafter also referred to as "substituent (b3)"), or a hydrogen atom.

[0042] In formula (b2), A represents an ether group or an ester group, and R ３ represents a divalent hydrocarbon group having 1 to 5 carbon atoms, and R ４ represents a fluorine atom, and n represents an integer of 0 to 4.

[0043] In formula (b3), R ５ represents a divalent hydrocarbon group having 1 to 5 carbon atoms or a single bond. ６ , R ７ , R ８ , R ９ and R １０ each independently represents a hydrogen atom, a hydroxyl group, a carboxyl group, an amino group or a hydrocarbon group having 1 to 4 carbon atoms.

[0044] R ２ The hydrocarbon group having 1 to 30 carbon atoms represented by R may be linear or branched. ２ The lower limit of the number of carbon atoms in the hydrocarbon group having 1 to 30 carbon atoms represented by the formula (I) may be 2 or more, 3 or more, or 4 or more. The upper limit of the number of carbon atoms in the hydrocarbon group may be 25 or less, 20 or less, 15 or less, 12 or less, 9 or less, 7 or less, or 5 or less. ２is preferably a hydrocarbon group having 4 to 15 carbon atoms, as this provides good water insolubility, adhesion to the substrate, and cell proliferation.

[0045] R ２ is a hydrocarbon group having 1 to 30 carbon atoms, the monomer constituting the monomer unit (b1) is not particularly limited, and examples thereof include n-butyl acrylate, n-butyl methacrylate, isobutyl acrylate, isobutyl methacrylate, t-butyl acrylate, t-butyl methacrylate, n-hexyl acrylate, n-hexyl methacrylate, n-octyl acrylate, n-octyl methacrylate, n-decyl acrylate, n-decyl methacrylate, n-dodecyl acrylate, n-dodecyl methacrylate, n-tetradecyl acrylate, and n-tetradecyl methacrylate. The monomer constituting the monomer unit (b1) preferably includes at least one monomer selected from the group consisting of n-butyl acrylate and n-butyl methacrylate, because this improves water insolubility, adhesion to substrates, and cell proliferation, enabling a more stable coating of the block copolymer. When the monomer constituting the monomer unit (b1) is n-butyl methacrylate, a polymer having a smaller polydispersity index is likely to be obtained.

[0046] R ２ The monomer constituting the monomer unit (b1) having the substituent (b2) is not particularly limited, but examples thereof include 4-azidophenyl acrylate, 4-azidophenyl methacrylate, 2-((4-azidobenzoyl)oxy)ethyl acrylate, and 2-((4-azidobenzoyl)oxy)ethyl methacrylate.

[0047] R ２is the substituent (b3), the monomer constituting the monomer unit (b1) is not particularly limited, and examples thereof include 2-hydroxyphenyl acrylate, 2-hydroxyphenyl methacrylate, 3-hydroxyphenyl acrylate, 3-hydroxyphenyl methacrylate, 4-hydroxyphenyl acrylate, 4-hydroxyphenyl methacrylate, N-(2-hydroxyphenyl)acrylamide, N-(2-hydroxyphenyl)methacrylamide, N-(3-hydroxyphenyl)acrylamide, N-(3-hydroxyphenyl)methacrylamide, N-(4-hydroxyphenyl)acrylamide, N-(4-hydroxyphenyl)methacrylamide, and styrene.

[0048] The block segment (B) may contain a monomer unit other than the monomer unit (b1) (hereinafter also referred to as "other monomer unit"), or may not contain any other monomer unit. The monomers constituting the other monomer units are not particularly limited, and examples thereof include those having an amino group such as 2-dimethylaminoethyl acrylate, 2-dimethylaminoethyl methacrylate, 2-diethylaminoethyl acrylate, 2-diethylaminoethyl methacrylate, and N-[3-(dimethylamino)propyl]acrylamide; those having a betaine such as N-(3-sulfopropyl)-N-methacryloyloxyethyl-N,N-dimethylammonium betaine and N-methacryloyloxyethyl-N,N-dimethylammonium-α-N-methylcarboxybetaine; hydroxyethyl acrylate, hydroxyethyl methacrylate, N-(2-hydroxyethyl)acrylamide, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, polypropylene glycol monomethacrylate, methoxypolyethylene glycol monoacrylate, methoxypolyethylene glycol monomethacrylate, those having a polyethylene glycol group or a methoxyethyl group, such as acrylate, diethylene glycol monomethyl ether acrylate, diethylene glycol monomethyl ether methacrylate, diethylene glycol monoethyl ether acrylate, diethylene glycol monoethyl ether methacrylate, 2-methoxyethyl acrylate, 2-methoxyethyl methacrylate, 2-ethoxyethyl acrylate, 2-ethoxyethyl methacrylate, 3-butoxyethyl acrylate, 3-butoxyethyl methacrylate, 3-butoxyethyl acrylamide, furfuryl acrylate, furfuryl methacrylate, tetrahydrofurfuryl acrylate, and tetrahydrofurfuryl methacrylate; and those having an acrylate group, such as methoxymethyl acrylate, methoxymethyl methacrylate, 2-ethoxymethyl acrylate, 2-ethoxymethyl methacrylate, 3-butoxymethyl acrylate, 3-butoxymethyl methacrylate, and 3-butoxymethyl acrylamide;Examples of phosphorylcholine groups include those having a phosphorylcholine group, such as 2-methacryloyloxyethyl phosphorylcholine, 2-acryloyloxyethyl phosphorylcholine, 3-(meth)acryloyloxypropyl phosphorylcholine, 4-(meth)acryloyloxybutyl phosphorylcholine, 6-(meth)acryloyloxyhexyl phosphorylcholine, 10-(meth)acryloyloxydecyl phosphorylcholine, ω-(meth)acryloyl(poly)oxyethylene phosphorylcholine, 2-acrylamidoethyl phosphorylcholine, 3-acrylamidopropyl phosphorylcholine, 4-acrylamidobutyl phosphorylcholine, 6-acrylamidohexyl phosphorylcholine, 10-acrylamidodecyl phosphorylcholine, and ω-(meth)acrylamido(poly)oxyethylene phosphorylcholine;

[0049] The proportion of the other monomer units may be 10% by mass or less, 5% by mass or less, 3% by mass or less, or 1% by mass or less, or may be 1% by mass or more, 3% by mass or more, or 5% by mass or more, based on the total amount of the block segment (B).

[0050] The degree of polymerization of the block segment (B) in the temperature-responsive block copolymer may be 20 or more, 40 or more, or 60 or more. The degree of polymerization of the block segment (B) in the block copolymer may be 160 or less, 140 or less, 120 or less, or 100 or less. The degree of polymerization of the block segment (B) in the temperature-responsive block copolymer is preferably 60 to 100. The degree of polymerization of the block segment (B) can be calculated, for example, by the following formula: Degree of polymerization of block segment (B) = (total molar amount of monomers dissolved for the polymerization reaction) x (conversion rate of monomers to polymer) / (molar amount of chain transfer agent)

[0051] The proportion of the block segment (B) in the temperature-responsive block copolymer may be 5% by mass or more, 10% by mass or more, 15% by mass or more, or 20% by mass or more, based on the total amount of the temperature-responsive block copolymer. The proportion of the block segment (B) in the block copolymer may be 50% by mass or less, 45% by mass or less, 40% by mass or less, or 35% by mass or less, based on the total amount of the temperature-responsive block copolymer. The proportion of the block segment (B) can be calculated, for example, by the following formula: Formula: Proportion of block segment (B) = (Degree of polymerization of block segment (B)) x (Monomer molecular weight of block segment (B)) / {(Degree of polymerization of block segment (A)) x (Monomer molecular weight of block segment (A)) + (Degree of polymerization of block segment (B)) x (Monomer molecular weight of block segment (B))} x 100

[0052] The temperature-responsive block copolymer may contain a structure represented by the following formula (C):

[0053] R ａ１ , R ａ２ , n, R １ , R ２ , Q and X may have the same meanings as defined above, and m represents an integer of 1 or more.

[0054] [Method for Producing Temperature-Responsive Block Copolymer] The temperature-responsive block copolymer can be obtained, for example, by a method including adjusting the change in bubble contact angle of a block copolymer containing block segment (A) and block segment (B) to 5.0° or more. The method for adjusting the change in bubble contact angle can be, for example, the method described above.

[0055] The method for polymerizing the block copolymer is not particularly limited, and any conventional method for forming a copolymer can be used. The method for polymerizing the block copolymer may be radical polymerization, such as reversible addition-fragmentation chain transfer (RAFT) polymerization, atom transfer radical polymerization (ATRP), or nitroxide-mediated polymerization (NMP).

[0056] In the polymerization of the block copolymer, a chain transfer agent (RAFT agent), a polymerization initiator, a polymerization inhibitor, etc. may be used as necessary.

[0057] The RAFT agent is not particularly limited, and a commonly used agent can be suitably used. Examples of the RAFT agent include 4-cyano-4-[(dodecylsulfonylthiocarbonyl)sulfonyl]pentanoic acid, 4-[(2-carboxyethylsulfanylthiocarbonyl)sulfanyl]-4-cyanopentanoic acid, 2-{[(2-carboxyethyl)sulfanylthiocarbonyl]sulfanyl}propanoic acid, 2-cyano-2-[(dodecylsulfanylthiocarbonyl)sulfanyl]propane, 2-[(dodecylsulfanylthiocarbonyl)sulfanyl]propanoic acid, methyl 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoate, 4-cyano-4-[(thiobenzoyl)sulfanyl] Pentanoic acid, 4-chloro-3,5-dimethylpyrazole-1-carbodithioic acid 2'-cyanobutan-2'-yl, 3,5-dimethylpyrazole-1-carbodithioic acid 2'-cyanobutan-2'-yl, 3,5-dimethylpyrazole-1-carbodithioic acid cyanomethyl, S,S-dibenzyltrithiocarbonate, trithiocarbonate bis[4-(allyloxycarbonyl)benzyl], trithiocarbonate bis[4-(2,3-dihydroxypropoxycarbonyl)benzyl], trithiocarbonate bis{4-[ethyl-(2-acetyloxyethyl)carbamoyl]benzyl}, trithiocarbonate bis[4-(2-hydroxyethoxycarbonyl)benzyl], dithiobenzoate, trithiocarbonate, 2-cyanopropan-2-yl Examples include N-methyl-N-(pyridin-4-yl)carbamodithioate and methyl 2-propionate (4-pyridinyl)carbamodithioate.

[0058] The polymerization initiator is not particularly limited, and a commonly used one can be suitably used. Examples of the polymerization initiator include azobisisobutyronitrile, 1,1'-azobis(cyclohexanecarbonitrile), 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2-methylbutyronitrile), 2,2'-azobis(2-methylpropionate)dimethyl, 2,2'-azobis(2-methylbutyronitrile), diethyl 2,2'-azobis(2-methylbutyronitrile), and diethyl 2,2'-azobis(2-methylbutyronitrile). Examples of the azobis-based ester include methyl 1,1'-azobis(1-cyclohexanecarboxylate), 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine], 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 4,4'-azobis-(4-cyanovaleric acid), di-tert-butyl peroxide, tert-butyl hydroperoxide, hydrogen peroxide, potassium peroxodisulfate, benzoyl peroxide, triethylborane, and diethylzinc.

[0059] The polymerization method for the block copolymer is preferably RAFT polymerization, because it is easy to obtain the desired temperature-responsive block copolymer. RAFT polymerization is a radical-initiated polymerization reaction involving an exchange chain reaction using a thiocarbonylthio group (—S—C(═S)—), and the product of the RAFT polymerization reaction usually has a thiocarbonylthio group.

[0060] Specifically, the temperature-responsive block copolymer can be produced by a method including a modification step of modifying a thiocarbonylthio group-containing block copolymer to obtain the temperature-responsive block copolymer, in which the change in bubble contact angle is adjusted to fall within the above-mentioned range.

[0061] The thiocarbonylthio group-containing block copolymer comprises block segments (A) and (B) and a terminal group containing a thiocarbonylthio group, which is bonded to block segment (A) or (B). The terminal group containing a thiocarbonylthio group is represented by the following formula (I): In formula (I), the wavy line indicates a bond on the block segment (A) or (B) side, and * indicates a bond on the terminal side.

[0062] The thiocarbonylthio group-containing block copolymer can be obtained, for example, by a method including: a polymerization step (1) of obtaining a thiocarbonylthio group-containing intermediate by a polymerization reaction of a first monomer in a mixture containing the first monomer, a polymerization initiator, and a RAFT agent; and a polymerization step (2) of obtaining a thiocarbonylthio group-containing block copolymer by a reaction of the thiocarbonylthio group-containing intermediate with a second monomer in a mixture containing the thiocarbonylthio group-containing intermediate, a second monomer, and a polymerization initiator.

[0063] One of the first monomer and the second monomer is a monomer that forms a monomer unit of block segment (A), and the other is a monomer that forms a monomer unit of block segment (B). In this case, it is preferable that the first monomer is a monomer that forms a monomer unit of block segment (B), and the other is a monomer that forms a monomer unit of block segment (A), because this makes it easier to obtain the desired temperature-responsive block copolymer.

[0064] The amount of the RAFT agent used may be, for example, 0.001 mole or more, or 0.005 mole or more, and 0.050 mole or less, or 0.020 mole or less, relative to 1 mole of the first monomer.

[0065] The amount of the polymerization initiator used may be, for example, 0.0001 mol or more, or 0.0005 mol or more, and 0.0050 mol or less, or 0.0020 mol or less, relative to 1 mol of the first monomer.

[0066] The reaction temperature in the polymerization step (1) may be, for example, 30° C. or higher and 120° C. or lower. The time for which the reaction temperature is maintained may be, for example, 1 hour or higher and 48 hours or lower.

[0067] The thiocarbonylthio group-containing intermediate obtained in the polymerization step (1) contains the block segment (A) or (B) and a terminal group containing a thiocarbonylthio group bonded to the block segment (A) or (B).

[0068] The amount of the second monomer used in the polymerization step (2) may be 100 moles or more, 150 moles or more, or 250 moles or more, and may be 400 moles or less, or 350 moles or less, relative to 1 mole of the thiocarbonylthio group-containing intermediate.

[0069] The amount of the polymerization initiator used in the polymerization step (2) may be 0.0001 mol or more and 0.0010 mol or less per 1 mol of the second monomer, and 0.05 mol or more and 0.20 mol or less per 1 mol of the thiocarbonylthio group-containing intermediate.

[0070] The reaction temperature in the polymerization step (2) may be, for example, 30° C. or higher and 120° C. or lower. The time for which the reaction temperature is maintained may be, for example, 1 hour or higher and 48 hours or lower.

[0071] The reactions in the polymerization steps (1) and (2) may be carried out in the presence of an organic solvent. Examples of the organic solvent include 1,4-dioxane, 1,2-dimethoxyethane, tert-butyl alcohol, tetrahydrofuran, N,N-dimethylformamide, ethanol, and 2-propanol. The reactions in the polymerization steps (1) and (2) may be carried out in a nitrogen or argon atmosphere.

[0072] The modification step may be, for example, a step of adjusting the temperature-responsive block copolymer so that the change in bubble contact angle is 5.0° or more by introducing a polar group into the terminal of the thiocarbonylthio group-containing block copolymer.

[0073] A method for introducing a polar group into the terminal of the thiocarbonylthio group-containing block copolymer may be, for example, a reaction between the thiocarbonylthio group-containing block copolymer and a radical generator having a polar group.

[0074] As the radical generator, an azo-based or peroxide-based radical generator can be suitably used. Examples of radical generators include azobisisobutyronitrile, 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 4,4'-azobis-(4-cyanovaleric acid), 2,2'-azobis-(2-methylpropionate)dimethyl, 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine], 2,2'-azobis[2-(2-imidazolin-2-yl)propane], 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis(N-butyl-2-methylpropionamide), 1,1'-azobis(cyclohexanecarbonitrile), dimethyl 1,1'-azobis(1-cyclohexanecarboxylate), 2,2'-azobis(2-methylbutyronitrile), 2,2 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2-methylbutyronitrile), 1,1'-azobis(1-acetoxy-1-phenylethane), di-tert-butyl peroxide, tert-butyl hydroperoxide, hydrogen peroxide, potassium peroxodisulfate, benzoyl peroxide, cumene hydroperoxide, dicumyl peroxide, diisobutyl peroxide, di(3,5,5-trimethylhexanoyl)peroxide, disuccinic acid peroxide, diisopropyl peroxydicarbonate, dipropyl peroxydicarbonate, di-2-ethylhexyl peroxydicarbonate, and di-sec-butyl peroxydicarbonate. Among these, azobisisobutyronitrile, 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 4,4'-azobis(4-cyanovaleric acid), and 2,2'-azobis(2-methylpropionic acid) can be mentioned. The radical generator may contain at least one selected from the group consisting of azobisisobutyronitrile, 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 4,4'-azobis(4-cyanovaleric acid), and 2,2'-azobis(2-methylpropionic acid).

[0075] The amount of the radical generator used may be in excess relative to the thiocarbonylthio group-containing block copolymer, and may be 5 moles or more, or 10 moles or more, and 100 moles or less, or 80 moles or less, relative to 1 mole of the thiocarbonylthio group-containing block copolymer.

[0076] The above-mentioned radical generators can efficiently introduce polar groups due to their decomposition temperature, solubility in reaction solvents, and the low susceptibility to side reactions. When an azo-based or peroxide-based radical generator is used as the radical generator, a reaction mixture containing the thiocarbonylthio group-containing block copolymer and the radical generator can be reacted under conditions of, for example, 30 to 120°C and 1 to 48 hours.

[0077] The method for introducing a mercapto group as a polar group into the terminal of the thiocarbonylthio group-containing block copolymer may be a method for converting the terminal group containing a thiocarbonylthio group into a mercapto group (—SH) by hydrolysis.

[0078] The hydrolysis may be alkaline hydrolysis carried out under basic conditions, for example, by reacting the thiocarbonylthio group-containing block copolymer in the presence of a base such as ethylamine, propylamine, butylamine, hexylamine, octylamine, benzylamine, ethylenediamine, hydrazine, piperidine, or ethanolamine.

[0079] The concentration of the base in the reaction solution may be 0.01 M or more, or 0.05 M or more, and may be 1 M or less, or 0.5 M or less.

[0080] When hydrolysis is carried out using a base, a reaction mixture containing the thiocarbonylthio group-containing block copolymer and the base can be reacted, for example, at 5 to 50° C. for 10 to 180 minutes.

[0081] One embodiment of the present invention provides a method for producing a temperature-responsive block copolymer having a polar group introduced at the end of block segment (A) or (B). The method includes a step of reacting a thiocarbonylthio group-containing block copolymer with a radical generator having a polar group to convert the end group containing the thiocarbonylthio group to an end group containing a polar group, thereby obtaining the temperature-responsive block copolymer. This step can be carried out by applying the embodiment described above as the method for introducing a polar group at the end.

[0082] As one embodiment of the present invention, there is provided a method for producing a temperature-responsive block copolymer having a mercapto group introduced at the end of block segment (A) or (B). The method includes a step of hydrolyzing a thiocarbonylthio group-containing block copolymer to convert the thiocarbonylthio group-containing end group to a mercapto group, thereby obtaining a temperature-responsive block copolymer. This step can be carried out by applying the embodiment described above as the method for introducing a mercapto group at the end.

[0083] [Coating Agent] The coating agent according to this embodiment contains a temperature-responsive block copolymer and a solvent. The coating agent may further contain additives as needed. By using this coating agent, temperature responsiveness can be imparted to the surface of a cell culture substrate by the simple method of dropping the coating agent onto the surface of the cell culture substrate and drying it.

[0084] Examples of the solvent include water, an organic solvent, or a mixture thereof. Examples of the organic solvent include alcohols such as methanol, ethanol, 1-propanol, 2-propanol, and 1-butanol; acetonitrile, formamide, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, 1,4-dioxane, and methyl ethyl ketone.

[0085] The content of the temperature-responsive block copolymer in the coating agent may be 0.01 to 50 mass%, 0.05 to 20 mass%, 0.1 to 10 mass%, or 0.5 to 5 mass%, based on the total mass of the coating agent.

[0086] [Cell Culture Substrate] The cell culture substrate according to this embodiment comprises a substrate and a coating layer that covers at least a portion of the surface of the substrate. The coating layer contains the above-described temperature-responsive block copolymer. The change in bubble contact angle on the surface of the coating layer measured in water at 21°C and in water at 37°C is 5.0° or more. The explanation given for the temperature-responsive block copolymer can be applied to the change in bubble contact angle on the surface of the coating layer.

[0087] The cell culture substrate may include a region that has cell adhesive properties and temperature-responsive detachment properties, or may include a region that has cell adhesive properties and temperature-responsive detachment properties, and a region that is not cell adhesive.

[0088] The region having cell adhesive properties and temperature-responsive releasability may be a region further having cell proliferation properties. At least a portion of the region having cell adhesive properties and temperature-responsive releasability is provided with a coating layer containing a temperature-responsive block copolymer.

[0089] The material of the substrate is not particularly limited, but may be any of the materials commonly used in cell culture, such as glass and polystyrene, as well as generally shapeable materials, such as polymers such as polycarbonate, polyethylene terephthalate, polyvinylidene fluoride, polytetrafluoroethylene, polyethylene, polypropylene, and polymethyl methacrylate, as well as ceramics and metals. From the perspective of ease of culturing operations, the substrate material preferably comprises at least one material selected from the group consisting of glass, polystyrene, polycarbonate, polyethylene terephthalate, polyethylene, and polypropylene. It is more preferable to comprise at least one material selected from the group consisting of glass, polystyrene, polycarbonate, polyethylene terephthalate, and polyethylene. Because this material is suitable for enhancing flexibility, it is particularly preferable to comprise at least one material selected from the group consisting of polystyrene, polycarbonate, polyethylene terephthalate, and polyethylene. The substrate may be polystyrene, which can be more easily molded.

[0090] The substrate is preferably polycarbonate because it can be easily formed into a thin film and cell detachment is further improved when a temperature-responsive block copolymer is used.

[0091] The shape of the substrate is not particularly limited, and may be a planar shape such as a plate or film, or may be a fiber, porous particle, porous membrane, or hollow fiber. The substrate may also be a container generally used for cell culture (cell culture dish such as a Petri dish, flask, plate, bag, etc.). From the viewpoint of ease of culture operation, the substrate is preferably a planar shape such as a plate or film, or a flat porous membrane. Because it is suitable for large-scale culture, the substrate is preferably a plate, film, flask, or bag.

[0092] At least a portion of the surface of the substrate may be coated with a hydrophilic polymer. That is, at least a portion of the surface of the substrate may be provided with a layer containing a hydrophilic polymer. At least a region of the surface of the substrate where cells do not adhere may contain the hydrophilic polymer.

[0093] The hydrophilic polymer may be a hydrophilic polymer containing a phosphorylcholine group or a hydroxyl group. By containing a phosphorylcholine group or a hydroxyl group, the area coated with the hydrophilic polymer can be made into an area to which cells do not adhere. In addition, since such a hydrophilic polymer does not need to be completely decomposed and removed, the area can be made into an area with cell adhesive properties by a modification process. Examples of commercially available hydrophilic polymers include Lipidure® CM5206 (manufactured by NOF Corporation), Lipidure® CM2001 (manufactured by NOF Corporation), and BIOSURFINE®-AWP (manufactured by Toyo Gosei Co., Ltd.).

[0094] The hydrophilic polymer preferably contains a compound represented by the following general formula (1), a compound represented by the following general formula (2), or a compound represented by the following general formula (3).

[0095] [In general formula (1), R １ and R ２ each independently represents a hydrogen atom or a methyl group, R ３represents a hydrogen atom or an arbitrary organic group, and m and n each independently represent a positive integer.

[0096] [In general formula (2), R ４ , R ５ and R ６ each independently represents a hydrogen atom or a methyl group, R ７ represents a hydrogen atom or an arbitrary organic group, and x, y, and z each independently represent a positive integer.

[0097] [In general formula (3), R ８ R and ９ each independently represents a hydrogen atom or a methyl group, R １０ represents a hydrogen atom or an arbitrary organic group, and a and b each independently represent a positive integer.

[0098] When the hydrophilic polymer contains the compound represented by the general formula (1), the compound represented by the general formula (2), or the compound represented by the general formula (3), cells can be easily attached and proliferated, and it is suitable for forming cell aggregates of uniform shape in the recesses. ３ , R ７ , and R １０ are suitable for immobilizing a hydrophilic polymer on a substrate, and therefore are preferably hydrophobic groups or functional groups reactive to active energy rays (e.g., UV, electron beams, etc.). As the hydrophobic group, linear or cyclic alkyl groups such as methyl, ethyl, propyl, butyl, and cyclohexyl groups can be preferably used. Furthermore, as the functional group reactive to active energy rays (e.g., UV, electron beams, etc.), azide groups, acrylate groups, methacrylate groups, vinyl groups, and epoxy groups can be used, and an azide group can be preferably used.

[0099] The thickness of the layer containing a hydrophilic polymer may be, for example, 10 nm or more, 20 nm or more, 30 nm or more, or 40 nm or more, and may be 50 nm or more, 60 nm or more, or 70 nm or more because this is suitable for suppressing adhesion of cells to areas other than the area having cell adhesiveness and temperature-responsive detachability. The thickness of the layer containing a hydrophilic polymer may be 1500 nm or less, and may be 1000 nm or less, 500 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, 90 nm or less, or 80 nm or less because this is suitable for increasing the cell viability of the cell aggregate by concentrating cells to the area having cell adhesiveness and temperature-responsive detachability through cell migration.

[0100] The "layer thickness" of the layer containing a hydrophilic polymer refers to the out-of-plane length from the interface between the substrate and the layer containing a hydrophilic polymer to the interface of the layer containing a hydrophilic polymer on the opposite side of the substrate (excluding recesses). When the layer thickness exceeds 10 nm, the cross-sectional image can be measured using a transmission electron microscope using an ultrathin section of the cell culture substrate prepared with a microtome, and the distance can be measured at 10 randomly selected points and averaged to calculate the thickness. When the layer thickness is 10 nm or less, the thickness can be measured using an ellipsometer.

[0101] The thickness of the substrate is preferably 0.01 mm to 0.5 mm, 0.05 mm to 0.4 mm, 0.1 mm to 0.3 mm, or 0.15 mm to 0.25 mm. When the thickness of the substrate is within this range, the fluorescent image of the cells becomes clearer when observed at high magnification.

[0102] Commercially available products can be used as the substrate. Examples of the substrate include commercially available culture substrates (for example, manufactured by Sumitomo Bakelite Co., Ltd., trade name: PrimeSurface (registered trademark), and manufactured by AGC Technoglass Co., Ltd., trade name EZ-BindShut (registered trademark)). As the substrate, commercially available substrates coated with a hydrophilic polymer, such as PrimeSurface (registered trademark) (manufactured by Sumitomo Bakelite Co., Ltd.), EZ-BindShut (registered trademark) (manufactured by AGC Technoglass Co., Ltd.), and EZ-BindShut II (registered trademark) (manufactured by AGC Technoglass Co., Ltd.), can be suitably used.

[0103] At least a portion of the surface of the substrate may be subjected to a surface modification treatment. The region of the substrate where the coating layer is to be provided (the region having cell adhesiveness and temperature-responsive release properties) may be subjected to a surface modification treatment. Examples of surface modification treatments include plasma treatment, corona treatment, and UV treatment. Surface modification treatments such as plasma treatment, corona treatment, and UV treatment can be carried out under conditions commonly used when treating the surface of a cell culture substrate to have cell adhesiveness and cell proliferation properties.

[0104] The thickness of the coating layer may be 1000 nm or less, 500 nm or less, 200 nm or less, 100 nm or less, 80 nm or less, 60 nm or less, 50 nm or less, 40 nm or less, or 35 nm or less, since cell adhesiveness is further improved. The thickness of the coating layer may be 5 nm or more, more than 5 nm, 10 nm or more, 15 nm or more, 20 nm or more, or 25 nm or more, since cell detachment properties are further improved. The thickness of the coating layer may be 5 to 1000 nm, 10 to 500 nm, 10 to 200 nm, 10 to 100 nm, 10 to 50 nm, 15 to 200 nm, 15 to 100 nm, 15 to 50 nm, 20 to 200 nm, 20 to 100 nm, 20 to 50 nm, 25 to 200 nm, 25 to 100 nm, or 25 to 50 nm, since cell detachment properties are further improved.

[0105] The thickness of the coating layer is measured using a scanning probe microscope (AFM) (for example, SPM-9600 manufactured by Shimadzu Corporation). A BL-AC40TS-C2 cantilever is used, and the thickness of the coating layer is measured by scratching the surface with tweezers and measuring the depth of the scratch.

[0106] The coating layer may have a phase-separated structure or a surface structure derived from phase separation at the culture temperature. The culture temperature may be, for example, 30 to 42°C, 32 to 40°C, 36 to 38°C, or 37°C. The method for forming the surface structure by phase separation is not particularly limited, and examples thereof include a method in which phase separation occurs when a block copolymer is applied to a substrate, and a method in which a block copolymer that phase separates at the culture temperature is applied.

[0107] The cell culture substrate may be sterilized. There are no particular limitations on the sterilization method, but high-pressure steam sterilization, UV sterilization, gamma-ray sterilization, ethylene oxide gas sterilization, etc. can be used. From the viewpoint of suppressing denaturation of the block copolymer, high-pressure steam sterilization, UV sterilization, and ethylene oxide gas sterilization are preferred. From the viewpoint of suppressing deformation of the substrate, UV sterilization or ethylene oxide gas sterilization are preferred. From the viewpoint of excellent mass productivity, ethylene oxide gas sterilization is preferred.

[0108] The cell culture substrate can be obtained by a method including a step of forming a coating layer containing the above-mentioned temperature-responsive block copolymer on a substrate. By including the step of forming a coating layer on a substrate, the cell detachability of the cell culture substrate is improved. The method for forming the coating layer is not particularly limited, and various commonly known methods can be used, such as painting, brush coating, dip coating, spin coating, bar coding, flow coating, spray coating, roll coating, air knife coating, blade coating, gravure coating, microgravure coating, and slot die coating.

[0109] A cell culture substrate comprising a cell-adhesive and temperature-responsive release region and a cell-nonadhesive region can be produced by a method comprising the steps of: surface-modifying a portion of the surface of a substrate coated with a hydrophilic polymer (the portion to be the cell-adhesive and temperature-responsive release region); and forming a coating layer containing a temperature-responsive block copolymer on the surface-modified substrate. Examples of methods for surface-modifying a portion of the substrate surface include placing a mask (e.g., a metal mask) with holes of a predetermined shape (e.g., circular) on the substrate, and then surface-modifying the substrate with the mask placed on it. This method allows the surface-modified region (the mask hole region) to be patterned as a cell-adhesive region, while the unmodified region (the mask region) can be patterned as a cell-nonadhesive region.

[0110] [Cell Production Method] The cell production method includes a culture step of culturing cells on the above-described cell culture substrate at a temperature equal to or higher than the temperature at which the degree of hydrophilicity and hydrophobicity of the temperature-responsive block copolymer changes (hereinafter referred to as the "response temperature"), and a detachment step of cooling the cell culture substrate to below the response temperature and then detaching the cells from the cell culture substrate. The cell production method may further include a seeding step of seeding cells on the cell culture substrate before the culture step.

[0111] In the seeding step, cells are seeded onto a cell culture substrate. After seeding, the cells adhere to the cell substrate. Cell seeding is performed by contacting the cell suspension with the cell culture substrate, for example, by applying a medium in which cells are dispersed (hereinafter also referred to as a "cell suspension") onto the cell culture substrate or by injecting the medium into the culture substrate.

[0112] Examples of cells include, but are not limited to, various established cell lines such as Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, human embryonic kidney-derived HEK293 cells, and human cervical cancer-derived HeLa cells, as well as epithelial cells and endothelial cells that constitute various tissues and organs in the body, contractile skeletal muscle cells, smooth muscle cells, and cardiac muscle cells, neuronal cells, glial cells, and fibroblasts that constitute the nervous system, hepatic parenchymal cells involved in the metabolism of the body, non-parenchymal hepatic cells, and adipocytes. Examples of cells that can differentiate include stem cells present in various tissues such as mesenchymal stem cells, bone marrow cells, and Muse cells, as well as stem cells with pluripotency (pluripotent stem cells) such as ES cells and iPS cells, and cells induced to differentiate therefrom. From the viewpoint of cell proliferation and detachment on a cell culture substrate, stem cells or pluripotent stem cells are preferred, mesenchymal stem cells or pluripotent stem cells are more preferred, pluripotent stem cells are even more preferred, and iPS cells are most preferred.

[0113] In the culturing step, cells are cultured on the cell culture substrate at a temperature equal to or higher than the response temperature. The cells are cultured in a medium. The temperature of the medium during cell culture is equal to or higher than the response temperature, and is preferably 30 to 42°C, 32 to 40°C, or 36 to 38°C.

[0114] The medium can be selected appropriately depending on the type of cells, etc. The medium may contain additives depending on the type of cells. When the cells are stem cells, the medium may contain factors for maintaining the undifferentiated state of the stem cells. Examples of factors for maintaining the undifferentiated state of stem cells include insulin, transferrin, selenium, ascorbic acid, sodium bicarbonate, basic fibroblast growth factor, transforming growth factor β (TGFβ), CCL2, activin, and 2-mercaptomethanol.

[0115] Examples of media include DMEM (manufactured by Sigma-Aldrich Co. LLC), Ham's F12 (manufactured by Sigma-Aldrich Co. LLC), D-MEM / Ham's F12 (manufactured by Sigma-Aldrich Co. LLC), Primate ES Cell Medium (manufactured by REPROCELL Co., Ltd.), StemFit AK02N (manufactured by Ajinomoto Co., Inc.), StemFit AK03 (manufactured by Ajinomoto Co., Inc.), mTeSR1 (manufactured by STEMCELL TECHNOLOGIES), and TeSR-E8 (manufactured by STEMCELL TECHNOLOGIES). TECHNOLOGIES), ReproNaive (manufactured by REPROCELL Co., Ltd.), ReproXF (manufactured by REPROCELL Co., Ltd.), ReproFF (manufactured by REPROCELL Co., Ltd.), ReproFF2 (manufactured by REPROCELL Co., Ltd.), NutriStem (manufactured by Biological Industries, Inc.), iSTEM (manufactured by Takara Bio Inc.), GS2-M (manufactured by Takara Bio Inc.), hPSC Growth Medium DXF (manufactured by PromoCell Co., Ltd.), and the like.

[0116] The cell density at the start of culture is not particularly limited, but is preferably 1.0 × 10 ２ ~1.0 x 10 ６ pieces / cm ２ , 1.0×10 ３ ~5.0 x 10 ５ pieces / cm ２ , or 1.0 × 10 ４ ~1.0 x 10 ５ pieces / cm ２ It may be.

[0117] The medium may contain, for example, a biologically derived substance. The biologically derived substance may be a natural product, may be artificially synthesized using genetic recombination technology or the like, may be a fragment obtained by cleavage with a restriction enzyme or the like, or may be a synthetic protein or synthetic peptide based on such a biologically derived substance. A specific example of a biologically derived substance is iMatrix-511 (manufactured by Nippi Corporation). The concentration of the biologically derived substance in the medium may be, for example, 1.0 to 10 μL / mL, or 2.0 to 5 μL / mL, based on the total volume of the medium.

[0118] The medium may further contain a Rho-associated kinase inhibitor, such as (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide.2HCl.H ２ Examples of Rho-binding kinase inhibitors that can be used include 1-(5-isoquinolinesulfonyl) homopiperazine hydrochloride (Y-27632, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 1-(5-isoquinolinesulfonyl) homopiperazine hydrochloride (HA1077, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). The concentration of the Rho-binding kinase inhibitor may be within a range that is effective for maintaining the survival of human stem cells and does not affect the undifferentiated state of human stem cells, and may be, for example, 1 μM to 50 μM, 3 μM to 20 μM, 5 μM to 15 μM, or 8 μM to 12 μM based on the total volume of the medium.

[0119] The culture period can be set appropriately depending on the type of cells, etc. When culturing stem cells, it is preferable to culture the cells using the following method. The first medium change is performed 22 to 26 hours after the start of cell culture. The second medium change is performed 48 to 72 hours after the start of the first culture. After the second medium change, the medium change is performed every 24 to 48 hours. The cells grow during culture and form cell clusters called colonies. Culture may be continued until the colonies reach a size of approximately 1 mm.

[0120] In the detachment step, the cell culture substrate is cooled to below the response temperature, and then the cells are detached from the cell culture substrate. Cooling the cell culture substrate to below the response temperature changes the degree of hydrophilicity and hydrophobicity of the coating layer on the cell culture substrate, making it easier to detach the cells from the cell culture substrate. The cell culture substrate can be cooled, for example, by removing the medium after the culture step and then adding a liquid below the response temperature. The temperature of the liquid below the response temperature may be, for example, 0 to 20°C, 1 to 15°C, 2 to 10°C, or 3 to 5°C. The time for which the cell culture substrate is contacted with the liquid below the response temperature may be, for example, 120 minutes or less, 100 minutes or less, 60 minutes or less, or 30 minutes or less, or may be 5 minutes or more, 10 minutes or more, or 15 minutes or more.

[0121] Cell detachment can be achieved by applying an external stimulus to the cell culture substrate. The external stimulus may be, for example, pipetting, tapping, or agitation using a shaker. Pipetting refers to the repeated aspirating and dispensing of a liquid contained in the cell culture medium using an instrument such as a pipette. Tapping refers to the application of vibration to the culture environment by, for example, striking the cell culture substrate. Agitation using a shaker refers to the application of an external stimulus using a shaker, and the agitation method is not particularly limited. Agitation using a shaker may be, for example, an operation of applying vibration (rotational vibration) by circularly moving the cell culture substrate. The shaker may be a device capable of rotating the cell culture substrate in a horizontal circle, and a commercially available microplate shaker or the like can be used.

[0122] The present invention will be described in detail below with reference to embodiments for carrying out the present invention. However, these are merely examples for explaining the present invention and are not intended to limit the present invention to the following content. Furthermore, the present invention can be practiced with appropriate modifications within the scope of the gist of the present invention. Unless otherwise specified, commercially available reagents were used.

[0123] <Polymer Composition> The composition of the polymer was determined by proton nuclear magnetic resonance spectroscopy ( １H-NMR) spectral analysis, or carbon nuclear magnetic resonance spectroscopy ( １３ C-NMR spectral analysis.

[0124] <Molecular Weight and Molecular Weight Distribution of Polymer> The weight average molecular weight (Mw), number average molecular weight (Mn), and molecular weight distribution (Mw / Mn) were measured by gel permeation chromatography (GPC). The GPC apparatus used was an HLC-8320GPC manufactured by Tosoh Corporation, and two TSKgel Super AWM-H columns manufactured by Tosoh Corporation were used. The column temperature was set to 40°C, and the eluent was N,N-dimethylformamide containing 10 mM lithium bromide. The measurement sample was prepared at 1.0 mg / mL and measured. A molecular weight calibration curve was prepared using polymethyl methacrylate (manufactured by Polymer Laboratories Ltd.) of known molecular weight.

[0125] <Polymerization Degree of Block Segment (A)> The polymerization degree of the block segment (A) was calculated based on the following formula: Polymerization Degree of Block Segment (A) = (Total Molar Amount of Monomers Dissolved for Polymerization Reaction) x (Conversion Rate of Monomers to Polymer) / (Molar Amount of Chain Transfer Agent)

[0126] <Coating Layer Thickness> The thickness of the coating layer containing a block copolymer provided on the substrate was measured using an AFM device (SPM-9600 manufactured by Shimadzu Corporation). A BL-AC40TS-C2 cantilever was used, and the thickness of the coating layer was measured by scratching the surface with tweezers and measuring the depth of the scratch.

[0127] Example 1 [Synthesis of Block Copolymer] 0.10 g (0.25 mmol) of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 2.99 g (21 mmol) of n-butyl methacrylate, and 8 mg (0.05 mmol) of azobisisobutyronitrile were placed in a test tube and dissolved in 18 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, the mixture was reacted at 70°C for 17 hours to obtain an n-butyl methacrylate polymer solution. The conversion of n-butyl methacrylate at this stage was 80%.

[0128] A 100 mL eggplant-shaped flask was charged with 21 mL (0.25 mmol) of the n-butyl methacrylate polymer solution, 7.92 g (70 mmol) of N-isopropylacrylamide, and 8 mg (0.03 mmol) of azobisisobutyronitrile, and dissolved in 2 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, the mixture was reacted at 70°C for 3 hours. The conversion of N-isopropylacrylamide at this stage was 85%. After completion of the reaction, the reaction solvent was diluted with 70 mL of acetone and poured into 3 L of hexane. The precipitated solid was collected and dried under reduced pressure to prepare block copolymer A. Block copolymer A is a block copolymer containing a block segment (A) containing N-isopropylacrylamide as a monomer unit and a block segment (B) containing n-butyl methacrylate as a monomer unit.

[0129] 1 g of block copolymer A and 0.32 g (2.0 mmol) of azobisisobutyronitrile were placed in a test tube and dissolved in 20 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, the mixture was reacted at 80°C for 3 hours. After completion of the reaction, the reaction solution was poured into 1 L of hexane, and the precipitated solid was collected and dried under reduced pressure. It was then redissolved in acetone and poured into 1 L of pure water. The precipitated gel-like product was collected and freeze-dried. This yielded the block copolymer of Example 1.

[0130] The block copolymer of Example 1 is a copolymer in which a cyano group is introduced at the end of the block segment (A). The degree of polymerization of the block segment (A) in the block copolymer of Example 1 was 238. The Mn×10 －３ The molecular weight was 38.4 and the Mw / Mn was 1.15.

[0131] [Measurement of Bubble Contact Angle] The block copolymer of Example 1 was diluted with ethanol to prepare a 0.8% by mass solution. 100 μL of the prepared 0.8% by mass solution was dropped onto a polycarbonate film (manufactured by Teijin Limited, product name: Panlite 180) and spin-coated at 2000 rpm for 60 seconds to form a film of the block copolymer of Example 1. The polycarbonate film coated with the film of the block copolymer of Example 1 was immersed in water at 21° C. or 37° C. for 10 minutes, and the contact angle (°) of 1 μL of a bubble in water was measured using a contact angle meter DM300 manufactured by Kyowa Interface Science Co., Ltd. In measuring the bubble contact angle, the film thickness of the block copolymer was 40 nm.

[0132] The bubble contact angle measured using the block copolymer of Example 1 was 145.1° at 21°C and 156.2° at 37°C, and the change in bubble contact angle was 11.1°.

[0133] [Preparation of Cell Culture Substrate] A 35 mm diameter dish (manufactured by Sumitomo Bakelite Co., Ltd., product name: PrimeSurface®) coated with a hydrophilic polymer was covered with a metal mask (manufactured by Mitani Micronics Co., Ltd.) having a plurality of 0.5 mm diameter circular holes. Plasma treatment (20 Pa gas pressure, 20 mA conduction current, 60 seconds of irradiation) was performed from above the metal mask using a plasma irradiation device (manufactured by Vacuum Device Co., Ltd., product name: Plasma Ion Bombarder PIB-20). This produced a cell culture substrate with cell adhesive and cell proliferation regions. 50 μL of a 0.6% by mass ethanol solution of the block copolymer of Example 1 was dropped onto this cell culture substrate, and the substrate was spin-coated at 2000 rpm for 60 seconds to form a coating layer containing the block copolymer of Example 1 on the substrate, thereby producing the cell culture substrate of Example 1. The coating layer thickness in the cell culture substrate of Example 1 was 30 nm.

[0134] [Cell culture and cell detachment evaluation] The culture medium StemFit AK02N (Ajinomoto Co., Inc.) was applied to two substrates coated with the block copolymer of Example 1 at a rate of 0.2 mL / cm. ２ In addition, human iPS cells 201B7 strain were added at 15,000 cells / cm ２, iMatrix-511 solution (manufactured by Nippi Corporation) was added at a concentration of 2.5 μL / mL. ２ The cells were cultured in an environment with a concentration of 5%. Furthermore, Y-27632 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (concentration: 10 μM) was added to the medium until 24 hours after cell seeding. After 24 and 96 hours, the state of the cells was observed using a phase-contrast microscope, and the medium was replaced with fresh medium. It was confirmed that cell clusters had formed on the pattern on the bottom surface of the culture substrate 144 hours after cell seeding.

[0135] After removing the medium from one of the two culture substrates, the substrate was washed with phosphate-buffered saline (PBS(-)), and methanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added and allowed to stand for 30 minutes to immobilize the cell clumps. Thereafter, the methanol was removed and the substrate was washed again with PBS(-), and Giemsa staining solution was added and stained at room temperature for 30 minutes. After removing the staining solution and washing with PBS(-), the substrate was dried and the entire bottom surface of the substrate was photographed with a fluorescence microscope (manufactured by Keyence Corporation, product name: BZ-X800), and the number of stained cell clumps was counted using an image analysis application (manufactured by Media Cybernetics, Image-Pro (registered trademark)).

[0136] After removing the medium from the other culture substrate, 4°C PBS(-) was added and left to stand for 20 minutes, and the side of the substrate was struck to apply vibration to detach the cell clumps. After removing the PBS along with the detached cell clumps, Giemsa staining was performed using the method described above, and the number of cell clumps that did not detach was counted.

[0137] The percentage of detached cell clusters among the cell clusters formed on the culture substrate was calculated from the number of each cell cluster measured, and was found to be 79.4%.

[0138] Example 2 [Synthesis of Block Copolymer] In the same manner as in the method described in [Synthesis of Block Copolymer] of Example 1, a block copolymer A including a block segment (A) containing N-isopropylacrylamide as a monomer unit and a block segment (B) containing n-butyl methacrylate as a monomer unit was synthesized.

[0139] 1 g of block copolymer A and 0.43 g (2.0 mmol) of 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide] were placed in a test tube and dissolved in 20 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, the mixture was reacted at 90°C for 14 hours. After completion of the reaction, the reaction solution was poured into 1 L of hexane, and the precipitated solid was collected and dried under reduced pressure. It was then redissolved in acetone and poured into 1 L of pure water. The precipitated gel-like product was collected and freeze-dried. This yielded the block copolymer of Example 2.

[0140] The block copolymer of Example 2 is a copolymer in which a hydroxyl group is introduced at the end of the block segment (A). The degree of polymerization of the block segment (A) in the block copolymer of Example 2 was 238. Mn×10 －３ The molecular weight was 38.0 and the Mw / Mn was 1.18.

[0141] [Measurement of Bubble Contact Angle] A film coated with the block copolymer was prepared and the bubble contact angle was measured in the same manner as in [Measurement of Bubble Contact Angle] in Example 1, except that the block copolymer of Example 2 was used. The bubble contact angles measured using the block copolymer of Example 2 were 155.0° at 21° C. and 122.7° at 37° C., and the change in bubble contact angle was 32.3°.

[0142] [Preparation of Cell Culture Substrate] The cell culture substrate of Example 2 was prepared in the same manner as in Example 1 [Preparation of Cell Culture Substrate], except that the block copolymer of Example 2 was used.

[0143] [Cell culture and cell detachment evaluation] Cell culture and cell detachment evaluation were performed in the same manner as in Example 1 [Cell culture and cell detachment evaluation], except for using the cell culture substrate of Example 2. The proportion of detached cell masses among the cell masses formed on the prepared cell culture substrate was 85.0%.

[0144] Example 3 [Synthesis of Block Copolymer] In the same manner as in the method described in [Synthesis of Block Copolymer] of Example 1, a block copolymer A including a block segment (A) containing N-isopropylacrylamide as a monomer unit and a block segment (B) containing n-butyl methacrylate as a monomer unit was synthesized.

[0145] 1 g of block copolymer A and 0.42 g (2.0 mmol) of 4,4'-azobis-(4-cyanovaleric acid) were placed in a test tube and dissolved in 20 mL of 1,4-dioxane. After degassing by bubbling nitrogen for 30 minutes, the mixture was reacted at 90°C for 14 hours. After completion of the reaction, the reaction solution was poured into 1 L of hexane, and the precipitated solid was collected and dried under reduced pressure. It was then redissolved in acetone and poured into 1 L of pure water. The precipitated gel-like product was collected and freeze-dried. This yielded the block copolymer of Example 3.

[0146] The block copolymer of Example 3 is a copolymer in which a carboxyl group is introduced at the end of the block segment (A). The degree of polymerization of the block segment (A) in the block copolymer of Example 3 was 238. Mn×10 －３ The molecular weight was 35.3 and the Mw / Mn was 1.18.

[0147] [Measurement of Bubble Contact Angle] A film coated with the block copolymer was prepared and the bubble contact angle was measured in the same manner as in [Measurement of Bubble Contact Angle] in Example 1, except that the block copolymer of Example 3 was used. The bubble contact angles measured using the block copolymer of Example 3 were 159.3° at 21° C. and 135.6° at 37° C., and the change in bubble contact angle was 23.7°.

[0148] [Preparation of Cell Culture Substrate] The cell culture substrate of Example 3 was prepared in the same manner as in Example 1 [Preparation of Cell Culture Substrate], except that the block copolymer of Example 3 was used.

[0149] [Cell culture and cell detachment evaluation] Cell culture and cell detachment evaluation were carried out in the same manner as in Example 1 [Cell culture and cell detachment evaluation], except for using the cell culture substrate of Example 3. The proportion of detached cell masses among the cell masses formed on the prepared culture substrate was 88.4%.

[0150] Example 4 [Synthesis of Block Copolymer] In the same manner as in the method described in [Synthesis of Block Copolymer] of Example 1, a block copolymer A including a block segment (A) containing N-isopropylacrylamide as a monomer unit and a block segment (B) containing n-butyl methacrylate as a monomer unit was synthesized.

[0151] 1 g of block copolymer A and 0.35 g (2.0 mmol) of 2,2'-azobis-(2-methylpropionic acid) were placed in a test tube and dissolved in 20 mL of 1,4-dioxane. After degassing by bubbling nitrogen for 30 minutes, the mixture was reacted at 80°C for 3 hours. After completion of the reaction, the reaction solution was poured into 1 L of hexane, and the precipitated solid was collected and dried under reduced pressure. It was then redissolved in acetone and poured into 1 L of pure water. The precipitated gel-like product was collected and freeze-dried. This yielded the block copolymer of Example 4.

[0152] The block copolymer of Example 4 had an ester group (—C(═O)OCH ３ The degree of polymerization of the block segment (A) in the block copolymer of Example 4 was 238. The Mn×10 －３ The molecular weight was 37.5 and the Mw / Mn was 1.16.

[0153] [Measurement of Bubble Contact Angle] A film coated with the block copolymer was prepared and the bubble contact angle was measured in the same manner as in [Measurement of Bubble Contact Angle] in Example 1, except that the block copolymer of Example 4 was used. The bubble contact angles measured using the block copolymer of Example 4 were 160.1° at 21° C. and 133.5° at 37° C., and the change in bubble contact angle was 26.6°.

[0154] [Preparation of Cell Culture Substrate] The cell culture substrate of Example 4 was prepared in the same manner as in Example 1 [Preparation of Cell Culture Substrate], except that the block copolymer of Example 4 was used.

[0155] [Cell culture and cell detachment evaluation] Cell culture and cell detachment evaluation were performed in the same manner as in Example 1 [Cell culture and cell detachment evaluation], except for using the cell culture substrate of Example 4. The percentage of detached cell masses among the cell masses formed on the prepared cell culture substrate was 89.6%.

[0156] Example 5 [Synthesis of Block Copolymer] In the same manner as in the method described in [Synthesis of Block Copolymer] of Example 1, a block copolymer A including a block segment (A) containing N-isopropylacrylamide as a monomer unit and a block segment (B) containing n-butyl methacrylate as a monomer unit was synthesized.

[0157] 1 g of block copolymer A and 0.52 g (4.0 mmol) of octylamine were added to a test tube and dissolved in 20 mL of 1,4-dioxane, followed by reaction at 25°C for 30 minutes. After completion of the reaction, the reaction solution was poured into 1 L of hexane, and the precipitated solid was collected and dried under reduced pressure. It was then redissolved in acetone and poured into 1 L of pure water, and the precipitated gel-like product was collected and freeze-dried. This yielded the block copolymer of Example 5.

[0158] The block copolymer of Example 5 is a copolymer in which a mercapto group is introduced at the end of the block segment (A). The degree of polymerization of the block segment (A) in the block copolymer of Example 5 was 238. Mn×10 －３ The molecular weight was 36.0 and the Mw / Mn was 1.25.

[0159] [Measurement of Bubble Contact Angle] A film coated with the block copolymer was prepared and the bubble contact angle was measured in the same manner as in [Measurement of Bubble Contact Angle] in Example 1, except that the block copolymer of Example 5 was used. The bubble contact angles measured using the block copolymer of Example 5 were 151.2° at 21°C and 127.1° at 37°C, and the change in bubble contact angle was 24.1°.

[0160] [Preparation of Cell Culture Substrate] The cell culture substrate of Example 5 was prepared in the same manner as in Example 1 [Preparation of Cell Culture Substrate], except that the block copolymer of Example 5 was used.

[0161] [Cell culture and cell detachment evaluation] Cell culture and cell detachment evaluation were performed in the same manner as in Example 1 [Cell culture and cell detachment evaluation], except for using the cell culture substrate of Example 5. The proportion of detached cell masses among the cell masses formed on the prepared cell culture substrate was 80.3%.

[0162] Comparative Example 1 [Synthesis of Block Copolymer] 0.10 g (0.25 mmol) of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 2.99 g (21 mmol) of n-butyl methacrylate, and 8 mg (0.05 mmol) of azobisisobutyronitrile were placed in a test tube and dissolved in 18 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, the mixture was reacted at 70°C for 17 hours to obtain an n-butyl methacrylate polymer solution. The conversion of n-butyl methacrylate at this stage was 80%.

[0163] A 100 mL eggplant-shaped flask was charged with 21 mL (0.25 mmol) of n-butyl methacrylate polymer solution, 7.92 g (70 mmol) of N-isopropylacrylamide, and 8 mg (0.03 mmol) of azobisisobutyronitrile, and dissolved in 2 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, the mixture was reacted at 70°C for 3 hours. The conversion of N-isopropylacrylamide at this stage was 85%. After completion of the reaction, the reaction solvent was diluted with 70 mL of acetone and poured into 3 L of hexane. The precipitated solid was collected and dried under reduced pressure. 1 g of the resulting polymer was redissolved in 15 mL of acetone and poured into 1 L of purified water. The precipitated gel-like product was collected and freeze-dried. This yielded the block copolymer of Comparative Example 1.

[0164] The block copolymer of Comparative Example 1 was an unmodified block copolymer with a RAFT agent-derived structure remaining at the end. The degree of polymerization of the block segment (A) in the block copolymer of Comparative Example 1 was 238. The Mn×10 －３The molecular weight was 35.6 and the Mw / Mn was 1.17.

[0165] [Measurement of bubble contact angle] A film coated with the block copolymer was prepared and the bubble contact angle was measured in the same manner as in Example 1 [Measurement of bubble contact angle], except that the block copolymer of Comparative Example 1 was used. The bubble contact angles measured using the block copolymer of Comparative Example 1 were 155.5° at 20°C and 151.2° at 37°C, and the change in bubble contact angle was 4.3°.

[0166] [Preparation of Cell Culture Substrate] The cell culture substrate of Comparative Example 1 was prepared in the same manner as in Example 1 [Preparation of Cell Culture Substrate], except that the block copolymer of Comparative Example 1 was used.

[0167] [Cell culture and cell detachment evaluation] Cell culture and cell detachment evaluation were performed in the same manner as in Example 1 [Cell culture and cell detachment evaluation], except for using the cell culture substrate of Comparative Example 1. The proportion of detached cell masses among the cell masses formed on the prepared cell culture substrate was 44.2%.

[0168] Comparative Example 2 [Preparation of Cell Culture Substrate] A cell culture substrate of Comparative Example 2 was prepared in the same manner as in Example 1 [Preparation of Substrate Coated with Terminally Modified Block Copolymer], except for the step of coating the substrate with the block copolymer.

[0169] [Cell culture and cell detachment evaluation] Cell culture and cell detachment evaluation were carried out in the same manner as in Example 1 [Cell culture and cell detachment evaluation], except for using the cell culture substrate of Comparative Example 2. The proportion of detached cell masses among the cell masses formed on the prepared culture substrate was 3.1%.

[0170] Comparative Example 3 [Measurement of Bubble Contact Angle] The bubble contact angle of a polycarbonate film not coated with a block copolymer was measured in the same manner as [Measurement of Bubble Contact Angle] in Example 1. The measured bubble contact angles were 131.7° at 21°C and 129.9° at 37°C, and the change in bubble contact angle was 1.8°.

[0171] Example 6 [Preparation of a cell culture substrate with a film-formed bottom] 0.1 mL of an 80 wt% ethanol aqueous solution containing a hydrophilic polymer (manufactured by Toyo Gosei Co., Ltd., product name: BIOSURFINE (registered trademark)-AWP) at a solids concentration of 0.6 wt% was dropped onto the polycarbonate film and spin-coated (2000 rpm, 60 seconds), and then left to stand under a high-pressure mercury lamp for 1 hour to cure the hydrophilic polymer by UV irradiation to form a layer containing the hydrophilic polymer (layer thickness 75 nm). This hydrophilic polymer-coated film was covered with the metal mask having a plurality of circular holes with a diameter of 0.5 mm, and plasma treatment was performed from above the metal mask using a plasma irradiation device (20 Pa gas pressure, 20 mA conduction current, 60 seconds of irradiation) to produce a film having cell adhesive and cell proliferation regions. Furthermore, 0.1 mL of an ethanol solution containing the block copolymer of Example 1 at a solids concentration of 0.8 wt % was dropped onto this treated film and spin-coated (2000 rpm, 60 seconds) to form a coating layer (layer thickness 40 nm) containing the block copolymer of Example 1, thereby obtaining a cell culture substrate with a two-layer structure consisting of a layer containing a hydrophilic polymer and a coating layer containing the block copolymer of Example 1. This film was attached to the bottom of a bottomless 6-well plate to produce a cell culture substrate with a bottom formed of a polycarbonate film.

[0172] [Evaluation of cell culture and cell detachment properties on a cell culture substrate with a film-formed bottom surface] Except for using the cell culture substrate described above, cell culture and cell detachment properties were evaluated in the same manner as in [Evaluation of cell culture and cell detachment properties] in Example 1. The percentage of detached cell masses among the cell masses formed on the prepared cell culture substrate was 99.3%.

[0173] [Changing the cell detachment method] A cell culture substrate was prepared using the same method as in Example 6 [Preparation of a cell culture substrate with a film-based bottom surface], except that the metal mask with multiple 0.2 mm diameter circular holes was used. Cell detachment was evaluated by stirring at 300 rpm for 20 seconds using a microplate shaker (manufactured by Isis Co., Ltd.). The percentage of detached cell masses among the cell masses formed on the prepared cell culture substrate was 90.6%.

[0174] [Evaluation of the Relationship between the Thickness of the Coating Layer Containing a Block Copolymer and Cell Detachability] After preparing a film having cell adhesive and cell proliferation regions using the method described in "Preparation of Cell Culture Substrates with a Film-Based Bottom" in Example 6, 0.2 wt%, 0.4 wt%, 0.6 wt%, 0.8 wt%, or 1.0 wt% ethanol solutions of the block copolymer of Example 1 were prepared, and 0.1 mL of each solution was added and spin-coated (2000 rpm, 60 seconds) to obtain cell culture substrates with a coating layer containing the block copolymer of 10 nm, 20 nm, 30 nm, 40 nm, or 50 nm. These cell culture substrates were attached to the bottom of a bottomless 6-well plate to prepare cell culture substrates with a polycarbonate film-based bottom containing a coating layer of different thicknesses.

[0175] Except for using this cell culture substrate, cell culture and cell detachment evaluation were performed in the same manner as in Example 1 [Evaluation of cell culture and cell detachment]. The percentages of detached cell masses among the cell masses formed on the prepared cell culture substrates were 93.2%, 99.3%, 99.4%, 99.9%, and 99.9%, respectively.

[0176] Example 7 [Preparation of cell culture substrate with a bottom surface formed of a film] The cell culture substrate of Example 7 was prepared in the same manner as in Example 6 [Preparation of cell culture substrate with a bottom surface formed of a film], except that the block copolymer of Example 2 was used.

[0177] [Evaluation of cell culture and cell detachment properties on a cell culture substrate with a film-formed bottom surface] Cell culture and cell detachment properties were evaluated in the same manner as in Example 1 [Evaluation of cell culture and cell detachment properties], except that the cell culture substrate of Example 7 was used. The percentage of detached cell masses among the cell masses formed on the prepared cell culture substrate was 99.9%.

[0178] Example 8 [Preparation of cell culture substrate with a bottom surface formed of a film] The cell culture substrate of Example 8 was prepared in the same manner as in Example 6 [Preparation of cell culture substrate with a bottom surface formed of a film], except that the block copolymer of Example 3 was used.

[0179] [Evaluation of cell culture and cell detachment properties on a cell culture substrate with a film-formed bottom surface] Cell culture and cell detachment properties were evaluated in the same manner as in Example 1 [Evaluation of cell culture and cell detachment properties], except that the cell culture substrate of Example 8 was used. The proportion of detached cell masses among the cell masses formed on the prepared cell culture substrate was 99.0%.

[0180] Example 9 [Preparation of cell culture substrate with a bottom surface formed of a film] The cell culture substrate of Example 9 was prepared in the same manner as in Example 6 [Preparation of cell culture substrate with a bottom surface formed of a film], except that the block copolymer of Example 4 was used.

[0181] [Evaluation of cell culture and cell detachability on a cell culture substrate with a film-formed bottom surface] Cell culture and cell detachability were evaluated in the same manner as in Example 1 [Evaluation of cell culture and cell detachability], except for using the cell culture substrate of Example 9. The proportion of detached cell masses among the cell masses formed on the prepared cell culture substrate was 100.0%.

[0182] Example 10 [Preparation of cell culture substrate with a bottom surface formed of a film] The cell culture substrate of Example 10 was prepared in the same manner as in Example 6 [Preparation of cell culture substrate with a bottom surface formed of a film], except that the block copolymer of Example 5 was used.

[0183] [Evaluation of cell culture and cell detachability on a cell culture substrate with a film-formed bottom surface] Cell culture and cell detachability were evaluated in the same manner as in Example 1 [Evaluation of cell culture and cell detachability], except that the cell culture substrate of Example 10 was used. The proportion of detached cell masses among the cell masses formed on the prepared cell culture substrate was 100.0%.

[0184] Example 11 [Synthesis of Block Copolymer] 0.10 g (0.25 mmol) of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 2.99 g (21 mmol) of n-butyl methacrylate, and 8 mg (0.05 mmol) of azobisisobutyronitrile were placed in a test tube and dissolved in 18 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, the mixture was reacted at 70°C for 17 hours to obtain an n-butyl methacrylate polymer solution. The conversion of n-butyl methacrylate at this stage was 78%.

[0185] To a 100 mL eggplant-shaped flask, 21 mL (0.25 mmol) of the n-butyl methacrylate polymer solution, 11.32 g (100 mmol) of N-isopropylacrylamide, and 8 mg (0.05 mmol) of azobisisobutyronitrile were added and dissolved in 10 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, the mixture was reacted at 70°C for 3 hours. The conversion of N-isopropylacrylamide at this stage was 96%. After completion of the reaction, the reaction solvent was diluted with 70 mL of acetone and poured into 3 L of hexane. The precipitated solid was collected and dried under reduced pressure to prepare block copolymer B. Like block copolymer A, block copolymer B is a block copolymer containing a block segment (A) containing N-isopropylacrylamide as a monomer unit and a block segment (B) containing n-butyl methacrylate as a monomer unit.

[0186] The entire amount of the obtained block copolymer B and 0.82 g (5.0 mmol) of azobisisobutyronitrile were added to a 200 mL eggplant-shaped flask and dissolved in 112 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, the mixture was reacted at 80°C for 3 hours. After completion of the reaction, the reaction solution was poured into 3 L of hexane, and the precipitated solid was collected and dried under reduced pressure. It was then redissolved in 150 mL of acetone and poured into 5 L of pure water. The precipitated gel-like product was collected and freeze-dried. This yielded the block copolymer of Example 11.

[0187] The block copolymer of Example 11 is a copolymer in which a cyano group is introduced at the end of the block segment (A). The degree of polymerization of the block segment (A) in the block copolymer of Example 11 was 387. The Mn×10 －３ The molecular weight was 49.0 and the Mw / Mn was 1.20.

[0188] [Measurement of Bubble Contact Angle] A film coated with the block copolymer was prepared and the bubble contact angle was measured in the same manner as in [Measurement of Bubble Contact Angle] in Example 1, except that the block copolymer of Example 11 was used. The bubble contact angles measured using the block copolymer of Example 11 were 160.8° at 21° C. and 135.1° at 37° C., and the change in bubble contact angle was 25.6°.

[0189] [Preparation of Cell Culture Substrate] The cell culture substrate of Example 11 was prepared in the same manner as in Example 1 [Preparation of Cell Culture Substrate], except that the block copolymer of Example 11 was used.

[0190] [Cell culture and cell detachment evaluation] Cell culture and cell detachment evaluation were carried out in the same manner as in Example 1 [Cell culture and cell detachment evaluation], except for using the cell culture substrate of Example 11. The proportion of detached cell masses among the cell masses formed on the prepared culture substrate was 98.2%.

[0191] Example 12 [Preparation of cell culture substrate having a bottom surface formed of a film] The cell culture substrate of Example 12 was prepared in the same manner as in Example 6 [Preparation of cell culture substrate having a bottom surface formed of a film], except that the block copolymer of Example 11 was used.

[0192] [Evaluation of cell culture and cell detachability on a cell culture substrate with a film-formed bottom surface] Cell culture and cell detachability were evaluated in the same manner as in Example 1 [Evaluation of cell culture and cell detachability], except for using the cell culture substrate of Example 12. The proportion of detached cell masses among the cell masses formed on the prepared cell culture substrate was 99.9%.

[0193] [Evaluation of the relationship between the thickness of the coating layer containing a block copolymer and cell detachment properties] Cell culture and cell detachment properties were evaluated in the same manner as in [Evaluation of the relationship between the thickness of the coating layer containing a block copolymer and cell detachment properties] in Example 6, except for using the block copolymer of Example 11. The percentages of detached cell masses among the cell masses formed on the cell culture substrates having a block copolymer layer of Example 11 with a thickness of 10 nm, 20 nm, 30 nm, 40 nm, or 50 nm were 98.2%, 99.5%, 100.0%, 100.0%, or 99.9%, respectively.

[0194] Comparative Example 4 [Preparation of cell culture substrate having a bottom surface formed of a film] The cell culture substrate of Comparative Example 4 was prepared in the same manner as in Example 6 [Preparation of cell culture substrate having a bottom surface formed of a film], except that the block copolymer of Comparative Example 1 was used.

[0195] [Evaluation of cell culture and cell detachability on cell culture substrates with a film-formed bottom surface] Cell culture and cell detachability were evaluated in the same manner as in Example 1 [Evaluation of cell culture and cell detachability], except that the cell culture substrate of Comparative Example 4 was used. The proportion of detached cell masses among the cell masses formed on the prepared cell culture substrate was 70.9%.

[0196] Comparative Example 5 [Measurement of Bubble Contact Angle] 0.1 mL of an 80 wt% ethanol aqueous solution containing a hydrophilic polymer at a solids concentration of 0.6 wt% was dropped onto the polycarbonate film and spin-coated (2000 rpm, 60 seconds), and then the film was left to stand under a high-pressure mercury lamp for 1 hour to cure the hydrophilic polymer by UV irradiation, forming a hydrophilic polymer layer (layer thickness 75 nm). The polycarbonate film coated with the hydrophilic polymer film was immersed in water at 21°C or 37°C for 10 minutes, and the contact angle (°) of a 1 μL bubble in the water was measured using a contact angle meter DM300 manufactured by Kyowa Interface Science Co., Ltd.

[0197] The bubble contact angle measured using the hydrophilic polymer was 145.0° at 21°C and 141.9° at 37°C, and the change in bubble contact angle was 3.1°.

[0198] [Preparation of cell culture substrate with a film-formed bottom] 0.1 mL of an 80 wt% ethanol aqueous solution containing a hydrophilic polymer (manufactured by Toyo Gosei Co., Ltd., product name: BIOSURFINE (registered trademark)-AWP) at a solids concentration of 0.6 wt% was dropped onto the polycarbonate film and spin-coated (2000 rpm, 60 seconds), and then left to stand under a high-pressure mercury lamp for 1 hour to harden the hydrophilic polymer by UV irradiation to form a hydrophilic polymer layer (layer thickness 75 nm). This hydrophilic polymer-coated film was covered with the metal mask having a plurality of circular holes with a diameter of 0.5 mm, and plasma treatment was performed from above the metal mask using a plasma irradiation device (20 Pa gas pressure, 20 mA conduction current, irradiation time 60 seconds) to prepare a film having cell adhesive and cell proliferation regions. This film was attached to the bottom of a bottomless 6-well plate to prepare a cell culture substrate of Comparative Example 5, the bottom of which was formed from a polycarbonate film.

[0199] [Evaluation of cell culture and cell detachability on cell culture substrates with a film-formed bottom surface] Cell culture and cell detachability were evaluated in the same manner as in Example 1 [Evaluation of cell culture and cell detachability], except for using the cell culture substrate of Comparative Example 5. The proportion of detached cell masses among the cell masses formed on the prepared cell culture substrate was 20.3%.

[0200] Comparative Example 6 [Synthesis of Block Copolymer] 0.10 g (0.25 mmol) of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 5.69 g (40 mmol) of n-butyl methacrylate, and 8 mg (0.05 mmol) of azobisisobutyronitrile were placed in a test tube and dissolved in 18 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, the mixture was reacted at 70°C for 17 hours to obtain an n-butyl methacrylate polymer solution. The conversion of n-butyl methacrylate at this stage was 73%.

[0201] To a 100 mL eggplant-shaped flask, 32 mL (0.25 mmol) of the n-butyl methacrylate polymer solution, 13.58 g (120 mmol) of N-isopropylacrylamide, and 8 mg (0.05 mmol) of azobisisobutyronitrile were added and dissolved in 20 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, the mixture was reacted at 70°C for 5 hours. The conversion of N-isopropylacrylamide at this stage was 85%. After completion of the reaction, the reaction solvent was diluted with 70 mL of acetone and poured into 3 L of hexane. The precipitated solid was collected and dried under reduced pressure. It was then redissolved in 150 mL of acetone and poured into 5 L of pure water. The precipitated gel-like product was collected and freeze-dried. This yielded the block copolymer of Comparative Example 6.

[0202] The block copolymer of Comparative Example 6 was an unmodified block copolymer with a RAFT agent-derived structure remaining at the end. The degree of polymerization of the block segment (A) in the block copolymer of Comparative Example 6 was 408. The Mn×10 －３ The molecular weight was 52.3 and the Mw / Mn was 1.31.

[0203] [Measurement of Bubble Contact Angle] A film coated with the block copolymer was prepared and the bubble contact angle was measured in the same manner as in [Measurement of Bubble Contact Angle] in Example 1, except that the block copolymer of Comparative Example 6 was used. The bubble contact angles measured using the block copolymer of Comparative Example 6 were 152.0° at 21° C. and 150.3° at 37° C., and the change in bubble contact angle was 1.7°.

[0204] [Preparation of Cell Culture Substrate] A cell culture substrate of Comparative Example 6 was prepared in the same manner as in Example 1 [Preparation of Cell Culture Substrate], except that the block copolymer of Comparative Example 6 was used.

[0205] [Evaluation of cell culture and cell detachment] Cell culture and cell detachment were evaluated in the same manner as in Example 1 [Evaluation of cell culture and cell detachment], except for using the cell culture substrate of Comparative Example 6. The proportion of detached cell masses among the cell masses formed on the prepared culture substrate was 57.4%.

[0206] Comparative Example 7 [Preparation of cell culture substrate with a bottom surface formed of a film] The cell culture substrate of Comparative Example 7 was prepared in the same manner as in Example 6 [Preparation of cell culture substrate with a bottom surface formed of a film], except that the block copolymer of Comparative Example 6 was used.

[0207] [Evaluation of cell culture and cell detachability on cell culture substrates with a film-formed bottom surface] Cell culture and cell detachability evaluation were performed in the same manner as in Example 1 [Evaluation of cell culture and cell detachability], except for using the cell culture substrate of Comparative Example 7. The proportion of detached cell masses among the cell masses formed on the prepared cell culture substrate was 58.9%.

[0208] <Results> Table 1 shows the measurement results of the change in bubble contact angle and the peeling rate measured using the block copolymers or cell culture substrates of Examples 1 to 5 and 11 and Comparative Examples 1 to 3 and 6. Table 2 shows the measurement results of the peeling rate measured using the cell culture substrates of Examples 6 to 10 and 12 and Comparative Examples 4 to 5 and 7. In the cell culture substrates of the Examples, the coating layers had a phase-separated structure of the block copolymer at the culture temperature (37°C).

[0209] Detachment rate 1 in Table 1 indicates the detachment rate calculated by forming cell clumps in a polystyrene dish and counting the number of detached cell clumps. Detachment rate 2 in Tables 2 to 4 indicates the detachment rate calculated by forming cell clumps in a plate having a polycarbonate film on the bottom surface of the substrate and counting the number of detached cell clumps.

[0210]

[0211] As shown in Tables 1 and 2, it was shown that the cell detachment rate was improved when the change in bubble contact angle was 5.0° or more.

[0212] As shown in Tables 3 and 4, it was confirmed that even when the film thickness was changed, the cell detachment rate improved when the change in the bubble contact angle was 5.0° or more.

Claims

1. A temperature-responsive block copolymer comprising a block segment (A) having a lower critical solution temperature (LCST) in water and a water-insoluble block segment (B), wherein when a film is formed from the temperature-responsive block copolymer, the change in bubble contact angle on the surface of the film measured in water at 21°C and in water at 37°C is 5.0° or more.

2. The temperature-responsive block copolymer according to claim 1, wherein the change in the bubble contact angle is 10° or more and 40° or less.

3. The temperature-responsive block copolymer according to claim 1 or 2, wherein at least one functional group selected from the group consisting of a hydroxyl group, a mercapto group, an amino group, a substituted amino group, a cyano group, an ester group, an amide group, a carbonyl group, and a carboxyl group is introduced to at least one end of the block segments (A) and (B).

4. The temperature-responsive block copolymer according to claim 1 or 2, wherein at least one functional group selected from the group consisting of a hydroxyl group, a mercapto group, an amino group, a substituted amino group, a cyano group, an ester group, an amide group, a carbonyl group, and a carboxyl group is introduced to the end of the block segment (A).

5. The temperature-responsive block copolymer according to claim 1 or 2, wherein the block segment (B) contains a monomer unit represented by the following general formula (b1): [In the formula, R １ represents a hydrogen atom or a methyl group, Q represents an ester group, R ２ represents a hydrocarbon group having 1 to 30 carbon atoms.

6. The temperature-responsive block copolymer according to claim 1 or 2, wherein the degree of polymerization of the block segment (A) is 100 or more and less than 400.

7. A cell culture substrate comprising a substrate and a coating layer covering at least a portion of the surface of the substrate, the coating layer comprising a temperature-responsive block copolymer, the temperature-responsive block copolymer comprising a block segment (A) having a lower critical solution temperature (LCST) in water and a water-insoluble block segment (B), and wherein the change in bubble contact angle on the surface of the coating layer measured in water at 21°C and in water at 37°C is 5.0° or more.

8. The cell culture substrate according to claim 7, wherein the coating layer has a thickness of 5 to 1,000 nm.

9. The cell culture substrate according to claim 7 or 8, wherein the substrate is any one of a plate, a film, a flask, and a bag.

10. The cell culture substrate according to claim 7 or 8, comprising an area capable of cell adhesion and temperature-responsive detachment, and an area not capable of cell adhesion.

11. The cell culture substrate according to claim 10, wherein the non-cell-adhesive region comprises a hydrophilic polymer.

12. The cell culture substrate according to claim 7 or 8, wherein the coating layer has a thickness of 10 to 50 nm.

13. The cell culture substrate according to claim 7 or 8, wherein the substrate is polycarbonate.

14. A method for producing a temperature-responsive block copolymer comprising a block segment (A) having a lower critical solution temperature (LCST) in water and a water-insoluble block segment (B), in which a polar group has been introduced at an end of the block segment (A) or (B), comprising: and an end group containing a thiocarbonylthio group represented by formula (I): [in formula (I), the wavy line represents a bond on the block segment (A) or (B) side, and * represents a bond on the terminal side], with a radical generator having a polar group to convert the end group containing the thiocarbonylthio group into an end group containing the polar group, thereby obtaining the temperature-responsive block copolymer.

15. The method according to claim 14, wherein the radical generator having a polar group comprises at least one member selected from the group consisting of azobisisobutyronitrile, 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 4,4'-azobis-(4-cyanovaleric acid), and 2,2'-azobis-(2-methylpropionic acid).

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