WO2020004508A1 - Temperature-responsive cell culture substrate - Google Patents

Abstract

A temperature-responsive cell culture substrate is provided which has better cell detachability and which suppresses elution of a temperature-responsive polymer during use. This temperature-responsive cell culture substrate has a temperature responsive layer (A), and the temperature-responsive layer (A) contains a temperature-responsive polymer. Of all the temperature-responsive polymers included in the temperature-responsive layer (A) that have a 4.0 or higher a common logarithm (log M) of the molecular weight (M), the total of temperature-responsive polymers that have a 5.5 or higher log M is greater than or equal to 60%.

Description

Temperature-responsive cell culture substrate

The present disclosure relates to a temperature-responsive cell culture substrate.

基材 A substrate having a temperature-responsive surface is used as a cell culture substrate. These substrates are mainly used for the culture of adherent cells. Adherent cells proliferate by attaching to the surface of the carrier. By using a substrate having a surface that changes from hydrophobic to hydrophilic or vice versa depending on the temperature response, it becomes possible to control the adhesion of the adherent cells to the surface by changing the temperature. Such a cell culture substrate is referred to as a “temperature-responsive cell culture substrate”.

Conventionally, adherent cells cultured on a substrate are chemically converted into proteins between the substrate and the cells using a protease such as trypsin or a metal chelating agent such as ethylenediaminetetraacetic acid (EDTA). However, there has been no choice but to peel off from the surface of the base material due to breakage (Patent Document 1).

If a temperature-responsive cell culture substrate is used, all of these operations become unnecessary, and the cultured cells can be separated by simply changing the temperature. In addition, the cells detached from the temperature-responsive cell culture substrate and the extracellular matrix adhering to the cells have an advantage that the degree of damage is low (Patent Document 1).
Specifically, for example, it becomes possible to collect a sheet-like cell group (cell sheet).

種 々 Various materials have been developed as temperature-responsive cell culture substrates. For example, a culture substrate in which poly-N-isopropylacrylamide (PNIPAM) having a lower critical solution temperature [Lower Critical Solution Temperature (LCST)] of 32 ° C. is immobilized on the surface has been developed (Patent Document 1). By dehydrating the polymer chains on the surface of the substrate that has been exposed to water at a temperature of 32 ° C. or higher, the surface of the substrate has a relatively hydrophobic property as compared to the case of being exposed to water at a temperature of 32 ° C. or lower. Show. In this state, the adherent cells can be adhered to the substrate surface and cultured. On the other hand, when the surface of the substrate comes into contact with water at a temperature of 32 ° C. or lower, a temperature response occurs, and the polymer chains on the surface hydrate with water molecules. As a result, the surface of the base material becomes more hydrophilic than before the temperature response, and the adhered cells can be separated.

Furthermore, temperature-responsive cell culture substrates in which a block polymer having a temperature-responsive polymer as a part thereof are immobilized on the surface have been reported (Patent Documents 2 and 3).

Japanese Patent Application Laid-Open No. H2-211865 International Publication No. WO2014 / 133168 International Publication No. WO2012 / 029882

An object of the present disclosure is to provide a temperature-responsive cell culture substrate having excellent cell detachability.

Item 1.
A temperature-responsive cell culture substrate having a temperature-responsive layer (A),
The temperature-responsive layer (A) contains a temperature-responsive polymer,
The temperature-responsive polymer having a logM of 5.5 or more among the total of the temperature-responsive polymers having a common logarithm (logM) of a molecular weight M of 4.0 or more contained in the temperature-responsive layer (A). Is 60% or more,
Temperature-responsive cell culture substrate.
Item 2.
The temperature-responsive polymer having a logM of 6.0 or more among the total of the temperature-responsive polymers having a common logarithm (logM) of a molecular weight M of 4.0 or more contained in the temperature-responsive layer (A). Is greater than or equal to 15%,
Item 10. The temperature-responsive cell culture substrate according to Item 1.
Item 3.
Item 3. The temperature-responsive cell culture substrate according to Item 1 or 2, wherein the temperature-responsive polymer includes a block polymer in which a temperature-responsive block is bonded to a terminal of a dendritic polymer.
Item 4.
Item 4. The temperature-responsive cell culture substrate according to Item 3, wherein the dendritic polymer is a dendritic polymer having a styrene skeleton or a siloxane skeleton.
Item 5.
At least one of the temperature-responsive polymers,
Temperature-responsive polymer obtainable by polymerizing a monomer containing at least one selected from the group consisting of (meth) acrylamide, N- (or N, N-di) -substituted (meth) acrylamide and vinyl ether, or polyvinyl alcohol Item 5. The temperature-responsive cell culture substrate according to any one of Items 1 to 4, which comprises at least a part of partially acetylated product.
Item 6.
The N- (or N, N-di) -substituted (meth) acrylamide is poly-N-isopropyl (meth) acrylamide, poly-N, N-diethyl (meth) acrylamide, and poly-N, N-dimethyl (meth) acrylamide. Item 6. The temperature-responsive cell culture substrate according to Item 5, which is at least one selected from the group consisting of acrylamide.
Item 7.
Item 7. The method according to any one of Items 1 to 6, further comprising a base material layer (B), wherein the temperature-responsive layer (A) is arranged on at least one surface of the base material layer (B). Temperature-responsive cell culture substrate.
Item 8.
Item 8. The temperature-responsive cell culture substrate according to Item 7, wherein the substrate layer (B) contains polystyrene.
Item 9.
A composition comprising a temperature-responsive polymer, wherein, among the total of the temperature-responsive polymers having a common logarithm (logM) of a molecular weight M of 4.0 or more, the temperature-responsive polymer having a logM of 5.5 or more. Is greater than or equal to 60%.
Item 10.
Item 10. The composition according to Item 9, which is used for producing the temperature-responsive cell culture substrate according to any one of Items 1 to 8.

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

In the temperature-responsive cell culture substrate of the present disclosure, among the components contained in the temperature-responsive layer (A) and having a common logarithm (logM) of molecular weight M of 4.0 or more, logM of 5.5 or more is used. It is the schematic diagram which showed how to calculate | require the ratio [B / (A + B) * 100 (%)] of a certain component.

The present inventors have found that there is a point in the conventional temperature-responsive cell culture substrate that should be improved in cell detachability during temperature response. The present inventors have intensively studied and are a temperature-responsive cell culture substrate having a temperature-responsive layer containing a temperature-responsive polymer, and adjusting the molecular weight distribution of components contained in the temperature-responsive layer. Has found that the above problem can be solved. The present disclosure includes the following embodiments.

1. Temperature-responsive cell culture substrate The temperature-responsive cell culture substrate of the present disclosure is:
A temperature-responsive cell culture substrate having a temperature-responsive layer (A),
The temperature-responsive layer (A) contains a temperature-responsive polymer,
The temperature-responsive polymer having a logM of 5.5 or more among the total of the temperature-responsive polymers having a common logarithm (logM) of a molecular weight M of 4.0 or more contained in the temperature-responsive layer (A). Is 60% or more,
It is a temperature-responsive cell culture substrate.
1.1 Temperature-responsive layer (A)

The temperature-responsive layer (A) contains at least a temperature-responsive polymer, and its surface shows temperature-responsiveness. In the temperature-responsive cell culture substrate of the present disclosure, the surface exhibiting this temperature response is used as a surface for performing cell culture.

In the present disclosure, the term “temperature-responsive polymer” includes a polymer whose entirety is a temperature-responsive region, and further includes a polymer having a temperature-responsive region and a region that does not exhibit a temperature-responsive property. included. The latter may be referred to as "partially temperature-responsive polymer" as necessary.

に お い て In the above description, the “temperature responsive region” may be composed of only one kind of polymer, or may be composed of a block polymer having two or more kinds of polymer blocks.

Specific examples of the partially temperature-responsive polymer include the following.
P [NIPAM / B (M) A] -bp (Styrene)
P [NIPAM / F (M) A] -bp (Styrene)
NIPAM: N-isopropylacrylamide B (M) A: butyl (meth) acrylate F (M) A: fluoroalkyl (meth) acrylate In the above, P [NIPAM / B (M) A] etc. It means a copolymer of NIPAM and B (M) A, and -b- means a block polymer having polymer blocks at both ends of the symbol. In the above description, P [NIPAM / B (M) A] and P [NIPAM / F (M) A] both correspond to the temperature-responsive region, and P (Styrene) (polystyrene) corresponds to the non-temperature-responsive region. Equivalent to.

The temperature responsive layer (A) contains at least one temperature responsive polymer.

In an embodiment of the present disclosure, which is characterized in that cell detachability during temperature response is excellent and / or elution of the temperature-responsive polymer is suppressed, the temperature-responsive layer (A) comprises In the form of a thermally responsive polymer.

The temperature at which the common logarithm (logM) of the molecular weight M is 5.5 or more in the total of the temperature-responsive polymers having the common logarithm (logM) of the molecular weight M of 4.0 or more contained in the temperature-responsive layer (A). The sum of the responsive polymers is 60% or more. The temperature-responsive cell culture substrate according to the present disclosure has excellent cell detachability at the time of temperature response because the temperature-responsive layer (A) has the above characteristics. These effects are obtained when the above ratio is in the range of 60 to 100%. It is preferably from 62 to 100%, more preferably from 65 to 100%, from the viewpoint of more excellent cell detachability.

In view of having more excellent cell detachability at the time of temperature response, the molecular weight M of the total of the temperature-responsive polymers having a common logarithm (logM) of the molecular weight M contained in the temperature-responsive layer (A) of 4.0 or more is 4.0 or more. The total of the temperature-responsive polymers having a common logarithm (logM) of 6.0 or more is preferably 15% to 100%, and more preferably 17% to 100%.

上 記 The molecular weight distribution of the temperature-responsive polymer in the temperature-responsive cell culture substrate of the present disclosure is specifically determined as follows. That is, the temperature-responsive layer (A) is immersed in pure water cooled to 4 ° C., shaken for 24 hours, and the temperature-responsive polymer extracted into the pure water is added to the temperature-responsive layer (A). Calculated as temperature responsive polymer included. The molecular weight distribution of these polymers is measured by GPC measurement (column: Styragel HR-4E + HR-5E, eluent: 50 mM LiBr in DMF, standard: polystyrene).

Of the total temperature-responsive polymers having logM of 4.0 or more, the total of temperature-responsive polymers having logM of x or more is determined as follows. A molecular weight distribution curve (FIG. 1) is prepared in which the logarithm of the molecular weight (LogM) is plotted on the horizontal axis and the differential distribution value (dW / dLogM) is plotted on the vertical axis. The differential distribution value refers to a weight fraction per LogM. That is, the interpretation of the numerical value of the molecular weight distribution diagram can be performed in the range of M to M + ΔM on the horizontal axis and the area surrounded by the distribution curve. For the total of the temperature-responsive polymers, the area A of the molecular weight distribution less than LogM = x and the area B of the molecular weight distribution curve more than LogM = x are calculated. Using these area values, the content (%) of the polymer having LogM> x is calculated by the equation (1).
B / (A + B) × 100 (%) (1)

The temperature-responsive layer (A) preferably does not contain a temperature-responsive polymer having a common logarithm (logM) of molecular weight M of 3.5 or less. It is more preferable that the temperature-responsive layer (A) does not contain a temperature-responsive polymer having a common logarithm (logM) of molecular weight M of 4.0 or less.

The temperature-responsive layer (A) preferably does not contain a temperature-responsive polymer having a common logarithm (logM) of molecular weight M of 8.0 or more.

方法 The method for obtaining the temperature-responsive polymer having the above-mentioned molecular weight distribution is not particularly limited, and can be widely used. As an example, a method of dissolving a temperature-responsive polymer in a good solvent such as acetone, toluene, and tetrahydrofuran (THF) and performing a reprecipitation operation of precipitating in a poor solvent such as hexane and water to perform fractionation may be mentioned. . When using water, it is preferable to use water at 40 to 60 ° C. Further, a method of fractionation by GPC (Gel Permeation Chromatography) and the like can also be mentioned.

In order to improve the cell detachability at the time of temperature response, the proportion of the total of the temperature-responsive polymer among the components contained in the temperature-responsive layer (A) is preferably 70% by mass or more. Preferably, it is 80% by mass or more, more preferably 90% by mass or more, even more preferably 95% by mass or more.

In the temperature-responsive cell culture substrate of the present disclosure, the temperature-responsive polymer has a surface area of 0.5 μg / cm ² or more, preferably 1.0 μg / cm ² or more, more preferably 1 μg / cm ² or more in terms of a temperature-responsive region. It is preferably immobilized on the surface of the substrate at a concentration of 0.5 μg / cm ² or more. In the temperature-responsive cell culture substrate of the present disclosure, when the amount of the temperature-responsive polymer immobilized is equal to or more than the above, the cultured cells on the polymer are not easily peeled even when the temperature is changed.

In the temperature-responsive cell culture substrate of the present disclosure, the total of the temperature-responsive polymers is 10 μg / cm ² or less, preferably 5 μg / cm ² or less, more preferably 4 μg / cm ² or less, in terms of the temperature-responsive region. When immobilized on the surface of the substrate, the cells can easily adhere to the surface before the temperature response, and the cells can easily be sufficiently adhered.

In summary, the temperature-responsive cell culture substrate of the present disclosure is preferably such that the total of the temperature-responsive polymers is 0.5 to 10 μg / cm ² , in terms of the temperature-responsive region, immobilized on the surface, It is more preferably immobilized on the surface at 1 to 5 μg / cm ² , more preferably 1.5 to 4 μg / cm ² .

に お い て In the present disclosure, the amount of the temperature-responsive polymer immobilized on the substrate surface can be calculated from the amount of the temperature-responsive polymer applied to the substrate surface. However, if necessary, the amount of the temperature-responsive polymer immobilized can be measured according to a conventional method. Examples of such a measuring method include a Fourier transform infrared spectroscopic total reflection attenuating method (FT-IR-ATR method), an elemental analysis method, an XPS method, and the like. Any measurement method may be selected as long as the measurement results do not vary, but in the case where the measurement results vary, a measurement result by the FT-IR-ATR method is employed in the present disclosure.

測定 The measurement by the FT-IR-ATR method is specifically performed as follows. A case in which a polystyrene cell culture dish is used as the base material and PNIPAM is used as the temperature-responsive polymer will be described as an example, but the same applies to the case where another base material and / or polymer is used by applying the following example. Can be measured.

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

A known amount of PNIPAM (1 to 10 μg / cm ² ) is applied to a polystyrene substrate, and a calibration curve is prepared in advance from the absorption intensity ratio obtained by the formula (5). The unknown PNIPAM amount can be determined. (Reference: Langmuir 2004, 20, 5506-5511).

1.2 Polymer Constituting Temperature-Responsive Region The polymer constituting the temperature-responsive region is not particularly limited, and can be widely selected. Specific examples of the polymer constituting the temperature-responsive region include a polymer having a lower critical solution temperature (LCST) or a polymer having an upper critical solution temperature (Upper Critical Solution empture (UCST)). There may be. The block polymer may include one type of temperature-responsive block, or may include a plurality of types of temperature-responsive blocks.

ポ リ マ ー As the polymer constituting the temperature responsive region, for example, the polymer described in Japanese Patent Publication No. 06-104061 can be mentioned. Specifically, for example, a polymer having a structural unit based on at least one of the following monomers may be mentioned. Examples of the monomer include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives and vinyl ether derivatives.

ポ リ マ ー Examples of the polymer constituting the temperature-responsive region include polyvinyl alcohol partially acetylated products and nitrogen-containing cyclic polymers.

Examples of the polymer constituting the temperature-responsive region include an alkyl-substituted cellulose derivative, a polyalkylene oxide block copolymer, and a polyalkylene oxide block copolymer.

(4) Usually, it is preferable that the detachment of the cultured cells is performed in a range of 5 ° C. to 50 ° C., and a polymer having an LCST or UCST within this range is preferable as a polymer constituting the temperature-responsive region. A polymer obtained by polymerizing a poly (N- (or N, N-di) alkyl-substituted (meth) acrylamide derivative having such a temperature response (poly (N- (or N, N-di) alkyl-substituted) Specific examples of (meth) acrylamide)) include poly-Nn-propylacrylamide (lower critical solution temperature: 21 ° C.), poly-Nn-propyl methacrylamide (27 ° C.), poly-N-isopropylacrylamide (32 ° C), poly-N-isopropylmethacrylamide (43 ° C), poly-N-cyclopropylacrylamide (45 ° C), poly-N-ethoxyethylacrylamide (35 ° C), poly-N- Ethoxyethyl methacrylamide (about 45 ° C), poly-N-tetrahydrofurfurylacrylamide (about 28 ° C), poly- -Tetrahydrofurfuryl methacrylamide (about 35 ° C), poly-N, N-ethylmethylacrylamide (56 ° C), poly-N, N-diethylacrylamide (32 ° C), poly (N-ethylacrylamide), Examples thereof include poly (N-isopropylmethacrylamide), poly (N-cyclopropylacrylamide), and poly (N-cyclopropylmethacrylamide).

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

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

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

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

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

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

モ ノ マ ー Further, examples of the monomer having a sulfonic acid include (meth) acrylamidoalkylsulfonic acid and the like.

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

As the fluorinated monomer, for example, an acrylate ester having a fluoroalkyl group directly or via a divalent organic group to a carboxyl group and having an ester bond or an amide bond and having a substituent at the α-position (hereinafter referred to as an acrylate ester) , "Fluoroalkyl group-containing acrylic acid ester"), or "fluoroalkyl group-containing acrylamide".

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

[In the formula, X is a hydrogen atom, a linear or branched alkyl group having 1 to 21 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a CFX ¹ X ² group (provided that X ¹ and X ² is a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom), a cyano group, a linear or branched fluoroalkyl group having 1 to 21 carbon atoms, a substituted or unsubstituted benzyl group, or A substituted or unsubstituted phenyl group;
Y is -O- or -NH-;
Z is an aliphatic group having 1 to 10 carbon atoms, an aromatic group or a cycloaliphatic group having 6 to 10 carbon atoms,
—CH ₂ CH ₂ N (R ¹ ) SO ₂ — group (where R ¹ is an alkyl group having 1 to 4 carbon atoms).
), -CH ₂ CH (OZ ¹ ) CH ₂ -group (where Z ¹ is a hydrogen atom or an acetyl group),-(CH ₂ ) _m -SO _2- (CH ₂ ) _n -group,-( CH ₂ ) _m —S— (CH ₂ ) _n — group (where m is 1 to 10 and n is 0 to 10) or — (CH ₂ ) _m —COO— group (m is 1 to 10) There is));
Rf is a linear or branched fluoroalkyl group having 1 to 20 carbon atoms which may have a hetero atom. And acrylamide represented by the following formula:

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

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

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

Further, the fluorinated monomer is preferably a non-telomer, and in this regard, the Rf group includes a fluoroalkyl group having 1 to 2 carbon atoms or a C1 to C2 having 2 or more carbon atoms interposed by a hetero atom. Fluoroalkyl groups are preferred. Specific examples include C ₃ F ₇ OCF (CF ₃ ) CF ₂ OCF (CF ₃ ) — and (CF ₃ ) ₂ NC _p F _2p − (p = 1 to 6).

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

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

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

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

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

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

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

フ ル オ ロ The above fluoroalkyl group-containing acrylates and fluoroalkyl group-containing acrylamides can be used alone or in combination of two or more.

As the polymer constituting the temperature-responsive region, a monomer composition containing at least one selected from the group consisting of (meth) acrylamide, N- (or N, N-di) -substituted (meth) acrylamide and vinyl ether is polymerized. Or a partially acetylated polyvinyl alcohol obtained by the reaction.

ブ ロ ッ ク Alternatively, a block polymer having as a segment the polymer constituting the temperature-responsive region may be used. In addition, as long as the intrinsic properties of the polymer are not impaired, a crosslinked polymer constituting the temperature-responsive region may be used. The crosslinking monomer used at this time is not particularly limited, and can be widely selected. For example, N, N'-methylenebis (meth) acrylamide can be mentioned.

1.3 Dendritic Block Copolymer In the present disclosure, the temperature responsive layer (A) is a block polymer in which a polymer constituting the temperature responsive region is bonded to a terminal of the dendritic polymer (in the present specification, “dendritic block copolymer”). Block copolymer "). Thereby, it becomes easy to obtain a temperature-responsive cell culture substrate having more excellent cell detachability and / or suppressing elution of the temperature-responsive polymer during use.

In particular, the core dendritic polymer portion excluding the temperature-responsive region (in this specification, this portion is simply referred to as “dendritic polymer” in order to distinguish it from the entire dendritic block copolymer. Is a dendritic polymer having a styrene skeleton or a siloxane skeleton. According to the study of the present inventors, having such a skeleton makes it easier for the dendritic polymer portion to be regularly arranged on the substrate surface, thereby stably fixing it to the substrate surface. As a result, it is possible to obtain an effect that the cells are not easily released not only in cell culture but also in detachment of cells by temperature response.

Further, a dendritic block copolymer in which a polymer constituting the temperature-responsive region is bonded to a terminal of a dendritic polymer having a styrene skeleton has a water-insoluble styrene skeleton dendritic polymer portion and a temperature-responsive This is the combination of the region and the region. Therefore, it is expected that a fine phase-separated structure is formed on the surface of the substrate by coating the surface of the substrate with the dendritic block copolymer and drying. When the cells adhere to the surface of the substrate, it is preferable that the surface of the substrate has a phase-separated structure because the denaturation of the cells can be suppressed.

As the dendritic polymer, a dendritic polymer having 15 or more terminals is preferable. When the number of terminals is 15 or more, the density per unit volume of the polymer constituting the temperature-responsive region bound to the terminals can be set in a preferable range, which is an improvement in cell detachability during temperature response. Contribute. In this regard, the number of terminals of the dendritic polymer having a styrene skeleton is preferably 15 or more, more preferably 20 or more, and even more preferably 30 or more.

The number of terminals of the dendritic polymer is preferably 100 or less, more preferably 50 or less, from the viewpoint that the time for the reaction for adding the polymer constituting the temperature-responsive region can be shortened.

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

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

In the present disclosure, Mw of the dendritic polymer is measured by GPC under the following conditions.
Note that, instead of the following devices and reagents, equivalents may be used.
Apparatus: Not particularly limited.
Detector: Differential refractive index detector RI
Column: Styragel HR-4E + HR-5E
Solvent: DMF containing 50 mM LiBr
Flow rate: 1.0 ml / min
Column temperature: 28 ° C
Injection volume: 100 μl
Standard sample: polystyrene

で あ れ ば If the Mw of the dendritic polymer is 1,000,000 or less, the introduction rate of the polymer constituting the temperature-responsive region can be kept within a preferable range. In this regard, the Mw of the dendritic polymer is preferably 500,000 or less, more preferably 300,000 or less, and most preferably 100,000 or less.

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

Further, if necessary, in the dendritic block copolymer in a state in which the polymer constituting the temperature-responsive region is bonded, a hydroxyl group, a carboxyl group, an amino group, a carbonyl group, an aldehyde group, a sulfonic acid group, etc. A group having a positive or negative charge may remain. A group having a positive or negative charge such as a hydroxyl group, a carboxyl group, an amino group, a carbonyl group, an aldehyde group, or a sulfonic acid group may be provided in or at the terminal constituting the temperature-responsive region.

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

The dendritic block copolymer is a dendritic polymer having a terminal number of 15 or more and a polymer constituting a temperature-responsive region having an Mw of 3000 or more bound to the terminal of the dendritic polymer by 50 to 99.5% by mass based on the entire dendritic block copolymer. Is preferred. In the dendritic block copolymer, the polymer constituting the temperature-responsive region is sufficiently bonded to the terminal of the dendritic polymer, so that even when the temperature is changed, the cultured cells on the polymer are not easily separated. . In this regard, the dendritic block copolymer of the present disclosure is preferably configured such that the polymer constituting the temperature-responsive region is bonded to the terminal of the dendritic polymer by 70% by mass or more based on the entire dendritic block copolymer. % Is more preferable.

The dendritic block copolymer is preferably one in which the polymer constituting the temperature-responsive region is bonded to the terminal of the dendritic polymer at 99.5% by mass or less based on the entire dendritic block copolymer. In this regard, the dendritic block copolymer is preferably such that the polymer constituting the temperature-responsive region is bonded to the terminal of the dendritic polymer at 98% by mass or less based on the entire dendritic block copolymer, and 97% by mass or less. More preferably.

デ ン The dendritic block copolymer of the present disclosure preferably has a Mw (weight basis) of 550,000 to 10,000,000. When the measured Mw is 50,000 or more, the polymer constituting the temperature-responsive region becomes easy to be introduced into the terminal of the dendritic polymer, and the blended ratio can be kept at an appropriate level, and the cell detachment property decreases. Can be avoided.

に お い て In the dendritic block copolymer of the present disclosure, the method of bonding the polymer constituting the temperature-responsive region to the terminal of the dendritic polymer is not particularly limited, and can be widely selected.

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

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

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

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

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

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

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

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

More specifically, for the purpose of introducing R ² , it is preferable to polymerize using at least a styrene derivative having a functional group as a monomer. Examples of such styrene derivatives include halogenated methyl styrene. Chloromethylstyrene, bromomethylstyrene and the like are used as halogenated methylstyrene. The styrene derivatives may be used alone or as a mixture of two or more.

の 通 り When the styrene skeleton dendritic polymer is produced by the polymerization reaction as described above, the ratio of the styrene derivative having a functional group to the total monomers used is preferably 5% or more. When the proportion of the styrene derivative having a functional group is 5% or more, the efficiency of introducing the polymer chain constituting the temperature-responsive region becomes good, and the introduction of the polymer constituting the temperature-responsive region targeted in the present disclosure is achieved. Rate is easier to achieve. In this regard, the ratio of the styrene derivative having a functional group is more preferably 10% or more, further preferably 15% or more, and most preferably 20% or more.

When the ratio of the styrene derivative having a functional group is 90% or less, the obtained dendritic block copolymer becomes less soluble in water while maintaining the efficiency of introducing the polymer chain constituting the temperature-responsive region in a favorable range. In addition, the possibility that the dendritic block copolymer elutes into the medium or the like is reduced. In this regard, the ratio of the styrene derivative having a functional group is more preferably 80% or less, further preferably 70% or less, and most preferably 60% or less.

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

The siloxane skeleton dendritic polymer can be widely selected. The siloxane skeleton dendritic polymer is, for example, at least one kind selected from the group consisting of bis (dimethylvinylsiloxane) methylsilane, tris (dimethylvinylsiloxane) silane, bis (dimethylallylsiloxane) methylsilane, and tris (dimethylallylsiloxane) silane. Examples include those that can be obtained by polymerizing monomers. Further, it can be obtained by polymerizing at least one monomer selected from the group consisting of bis (dimethylsiloxy) methylvinylsilane, tris (dimethylsiloxy) vinylsilane, bis (dimethylsiloxy) methylallylsilane and tris (dimethylsiloxy) allylsilane. And the like. Such a siloxane skeleton dendritic polymer can be obtained, for example, by a method described in WO2004 / 074177.

1.4. Method of disposing temperature-responsive layer (A) on surface of base material layer (B)

温度 The temperature-responsive cell culture substrate of the present disclosure can be obtained by disposing the temperature-responsive layer (A) on at least one surface of the substrate layer (B). Specifically, for example, the temperature responsive layer (A) is fixed directly or indirectly to the surface of the base material layer (B) with the polymer constituting the temperature responsive region, so that the temperature responsive layer (A) is fixed to the base material layer (B). Can be placed on the surface of.

The temperature-responsive layer (A) may have two or more regions having different UCSTs or LCSTs, and the regions may be arranged so as to form a two-dimensional pattern.

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

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

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

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

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

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

When immobilizing the polymer constituting the temperature-responsive region or the composite containing the same on the surface of the base material layer (B), a solution containing the polymer constituting the temperature-responsive region or the composite containing the same is used as a substrate. It is preferable to apply uniformly on the surface of the layer (B). The method is not particularly limited, and can be selected widely. For example, a method using a dispenser, a method in which the base material layer (B) is allowed to stand on a horizontal table, and the like are exemplified.

After applying the solution containing the polymer constituting the temperature-responsive region or the complex containing the same to the surface of the substrate layer (B), the solvent is removed to obtain the temperature-responsive cell culture substrate of the present disclosure. Can be The method for removing the solvent is not particularly limited, and can be widely selected. For example, a method of slowly evaporating at room temperature and in the air over time, a method of slowly evaporating at room temperature and in a solvent saturated environment over time, a method of evaporating under heating, and an evaporating under reduced pressure Method and the like. In particular, from the viewpoint that a uniform surface of the temperature responsive layer (A) can be obtained, a method of slowly evaporating at room temperature over time in a solvent-saturated environment is preferable. Specifically, it is preferable to place under the vapor of the solvent for 2 hours or more.

(4) From the viewpoint that a uniform surface of the temperature-responsive layer (A) can be obtained, it is preferable to slowly evaporate the solvent over a long period of time in a solvent-saturated environment, and then wash the surface once and then dry it.

2. Base material layer (B)
The material of the base material layer (B) is not particularly limited as long as it has no nitrogen atom. Usually, it is not particularly limited as long as it is used for cell culture. For example, glass, modified glass, various resins and the like can be mentioned. Further, in addition to these, a material that can be generally given a form may be used. Such a material is not particularly limited, and can be widely selected. For example, resins, ceramics, metals and the like can be mentioned.

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

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

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

The surface of the base material layer (B) on the side where the temperature responsive layer (A) is disposed may be smooth or may have a three-dimensional structure such as a hole, a protrusion, or a wall. You may. Examples of the substrate layer (B) having such a three-dimensional structure on its surface include, for example, a commercially available three-dimensional structure cell culture substrate, NanoCulture Plate manufactured by SCIVAX, nano pillar plate manufactured by Hitachi High-Technologies, 3D manufactured by Biomatrix. Perfecta3D or Kuraray ELPLASIA can be used.

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

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

温度 The temperature-responsive cell culture substrate of the present disclosure may have another layer in addition to the temperature-responsive layer (A) and the substrate layer (B), if necessary. Other layers include, for example, a support layer used for the purpose of shape retention.

形状 The shape of the temperature-responsive cell culture substrate of the present disclosure is not particularly limited. It may be a cell culture dish such as a Petri dish or a plate, fiber or particle. The particles may be porous. Further, another container shape generally used for cell culture or the like may be used. Examples of such a container shape include a flask and a bag.

温度 The temperature-responsive cell culture substrate of the present disclosure can be used for cells in general. For example, cells such as animals, insects and plants, and bacteria can be mentioned. Examples of the origin of animal cells include humans, monkeys, dogs, cats, rabbits, rats, nude mice, mice, guinea pigs, pigs, sheep, Chinese hamsters, cows, marmosets, African green monkeys, and the like.

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

各種 Various stem cells can be used as the adherent cells. Examples of adherent stem cells include embryonic stem cells (embryonic stem cells: ES cells), embryonic germ cells (embryonic stem cells), germline stem cells (germline stem cells: GS cells), and induced pluripotent stem cells. (IPS cells; induced pluripotent stem cells) and other pluripotent stem cells, mesenchymal stem cells, hematopoietic stem cells, multipotent stem cells such as neural stem cells, etc., myocardial progenitor cells, vascular endothelial progenitor cells, neural progenitor cells, fat progenitors Stem cells such as cells, skin fibroblasts, skeletal myoblasts, osteoblasts, and monopotent stem cells (progenitor cells) such as odontoblasts.

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

In the temperature-responsive substrate for cell culture of the present disclosure, the cultured cells are enzymatically treated by setting the temperature of the entire or a part of the culture substrate to not less than UCST or not more than LCST of the polymer constituting the temperature-responsive region. It can be peeled off without any problem. The exfoliation due to this temperature change may be performed in a culture solution or in another isotonic solution. In addition, the substrate can be dabbed or shaken for the purpose of detaching and collecting cells faster and more efficiently. If necessary, the medium may be stirred using a pipette or the like. As an advantage of changing the temperature of only a part of the base material, for example, in the differentiation induction of iPS cells, only the differentiated cell colonies can be selectively detached.

The temperature-responsive substrate for cell culture of the present disclosure is preferably such that the polymer constituting the temperature-responsive region is firmly fixed to the surface, and in this case, it can be used for reuse.
In particular, the temperature-responsive substrate for cell culture having the surface on which the dendritic block copolymer of 1.3 is immobilized has a polymer in which the temperature-responsive region is particularly firmly immobilized on the surface. It can be preferably used for applications.

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

4. Temperature-Responsive Polymer-Containing Composition The composition of the present disclosure is a composition containing a temperature-responsive polymer, wherein the composition has a common logarithm (logM) of a molecular weight M of 4.0 or more contained in the composition. The composition wherein the total of the temperature-responsive polymers having a logM of 5.5 or more is 60% or more of the total of the reactive polymers. Such a composition is preferably used for producing the above-mentioned temperature-responsive cell culture substrate.

The composition of the present disclosure, by having the above-mentioned properties, in the temperature-responsive cell culture substrate obtained using the same, excellent cell detachability during temperature response, and these effects, Obtained when the above ratio is in the range of 60 to 100%. It is preferably from 62 to 100%, more preferably from 65 to 100%, from the viewpoint of more excellent cell detachability.

Of the total temperature-responsive polymers having a logarithm of molecular weight M (log M) of 4.0 or more contained in the above composition, a common logarithm of molecular weight M in that the composition has more excellent cell detachability during temperature response. The total of the temperature-responsive polymers having (logM) of 6.0 or more is preferably 15% to 100%, and more preferably 17% to 100%.

組成 The composition of the present disclosure contains at least one temperature-responsive polymer.

In a preferred embodiment of the present disclosure, the composition of the present disclosure is excellent in the effect that cell detachability during temperature response is excellent and / or elution of the temperature-responsive polymer during use is suppressed. The temperature responsive polymer is included in the form of a partially temperature responsive polymer.

The temperature-responsive polymer is as described in 1.2 and 1.3 above.

組成 It is preferable that the composition of the present disclosure does not contain a temperature-responsive polymer having a common logarithm (logM) of a molecular weight M of 3.5 or less. Further, it is more preferable that the composition of the present disclosure does not contain a temperature-responsive polymer having a common logarithm (logM) of molecular weight M of 4.0 or less.

組成 It is preferable that the composition of the present disclosure does not contain a temperature-responsive polymer having a common logarithm (logM) of a molecular weight M of 8.0 or more.

When used to produce a temperature-responsive cell culture substrate, the effect is that cell detachability during temperature response is more excellent and / or elution of the temperature-responsive polymer during use is suppressed. In the components contained in the composition of the present disclosure, the proportion occupied by the temperature-responsive polymer is preferably 70% by mass or more, more preferably 80% by mass or more, and more preferably 90% by mass or more. More preferably, it is still more preferably 95% by mass or more.

When used to produce a temperature-responsive cell culture substrate, cell exfoliation during temperature response is excellent, and / or elution of the temperature-responsive polymer during use is suppressed. In a preferred embodiment of the present disclosure, the ratio of the partially temperature-responsive polymer among the components contained in the composition of the present disclosure is preferably 70% by mass or more, and more preferably 80% by mass or more. Is more preferably 90% by mass or more, even more preferably 95% by mass or more.

Although the embodiments have been described above, it will be understood that various changes in form and details can be made without departing from the spirit and scope of the claims.

Hereinafter, the present disclosure will be specifically described, but the present disclosure is not limited to the following examples.

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

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

[Synthesis of Dendritic Block Copolymer HBP-0]
Into a 10-ml flask equipped with a branch tube under a nitrogen atmosphere, the above-mentioned dendritic polymer 1 (0.028 g), N-isopropylacrylamide (NIPAM, 1 g, 8.85 × 10 ⁻³ mol), dehydrated tetrahydrofuran (THF, 3 ml) And stirred to dissolve.
Further, AIBN (4.0 × 10 ⁻³ g, 4.6 × 10 ⁻⁶ mol) was added, and the mixture was stirred at 70 ° C. for 10 hours. After the reaction, 3 ml of THF was added for dilution, and the resulting mixture was dropped and reprecipitated in hexane to recover an egg-colored solid. It was again dissolved in 5 ml of THF and reprecipitated in hexane to obtain 0.706 g of a white powdery solid HBP-0.

[Fractionation of dendritic block copolymer]
The block polymer HBP-0 obtained in the preceding section was fractionated according to the difference in the elution time of GPC to obtain polymers HBP-1 to 5 having different molecular weight distributions. The molecular weight distribution of these polymers was measured by GPC measurement (column: Styragel HR-4E + HR-5E, eluent: 50 mM LiBr in DMF, standard: polystyrene).

Table 1 shows the weight average molecular weight (Mw) and polydispersity (Mw / Mn) of the polymer HBP-0 before fractionation and the polymers HBP-1 to 5 having different molecular weight distributions after fractionation.

In addition, in each molecular weight distribution curve in which the logarithm of the molecular weight (LogM) is plotted on the horizontal axis and the differential distribution value (dW / dLogM) is plotted on the vertical axis, the area A of the molecular weight distribution where LogM is less than 5.5, The area B of the molecular weight distribution curve of LogM = 5.5 or more was calculated. Using these area values, the content (%) of the polymer having a LogM> 5.5 was calculated by the equation (1).
B / (A + B) × 100 (%) (1)

高分子 The polymer content of LogM> 6 was similarly obtained, and is shown in Table 2 together with the polymer content of LogM> 5.5.

<Example 1>
[Preparation of coating solution]
10 mg of each of the polymer HBP-1 synthesized and fractionated in Production Example 1 was dissolved in a mixed solvent (4 ml) of THF / MeOH = 1/4 (v / v). This is called mother liquor. A mixed solvent of THF / MeOH = 1/4 (v / v) such that 50 μl of the mother liquor is applied to a cell culture dish having a culture area of 9.6 cm ^{2 so that} the amount of PNIPAM applied is 3.5 μg / cm ^2. For further dilution.

[Immobilization of polymer on polystyrene substrate]
The coating solution (50 μl) obtained by the above method was applied to a polyculture cell culture dish (Falcon 3001, manufactured by Corning, culture area: 9.6 cm ² ) and capped. The mixture was allowed to stand and dried for 2.5 hours to obtain a temperature-responsive cell culture substrate.

<Examples 2 to 5>
Temperature-responsive cell culture substrates were obtained in the same manner as in Example 1 except that the polymers used for preparing the coating solution were HBP-2 to HBP-5 (Table 2).

<Comparative Example 1>
A temperature-responsive cell culture substrate was obtained in the same manner as in Example 1, except that the polymer used for preparing the coating solution was HBP-0 before fractionation.

<Comparative Example 2>
An untreated φ3.5 cm polystyrene cell culture dish (Falcon 3001) was prepared.

<Comparative Example 3>
A commercially available φ3.5 cm temperature-responsive cell culture container (UpCell manufactured by Cell Seed, culture area: 9.6 cm ² ) having a homopolymer of PNIPAM on the surface was prepared.

[Evaluation of cell adhesion]
(1) Culture conditions Culture medium: 10% FBS (fetal bovine serum) and 1% penicillin (Penicillin-Streptomycin) -containing DMEM (Dulbecco's modified Eagle's medium) -high glase solution

(2) Preparation method of cell suspension for evaluation In advance, 3T3 mouse fibroblasts are cultured in a cell culture vessel, and the number of cells is counted after collecting the cells. The cell count becomes 5 × 10 ⁴ cells / mL with respect to the medium. Cell suspension was prepared as described above.

Comparative Example 1 using a substrate coated with HBP-0 before fractionation, Comparative Example 2 using an untreated dish (Falcon 3001), and Comparative Example using a commercially available temperature-responsive cell culture vessel having a homopolymer of PNIPAM on the surface. It was set to 3. Into the dish prepared in the comparative example and the dish prepared in the example, 2 ml of the cell suspension was injected, and the cells were seeded. Thereafter, the cells were cultured in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for 4 days.

(3) Measurement of Cell Detachment Time After the completion of the culture, the state of adherence of the cultured cells and whether the cultured cells were growing in the dish until they became full (confluent) were observed using an inverted microscope. Then, it was allowed to stand still at room temperature, and vibration was applied more slowly after 10 minutes. The observation was performed for 50 minutes after taking out from the incubator. The time until the cell sheet spontaneously peeled was measured.

Table 2 shows the results of the evaluations of Examples 1 to 5 and Comparative Examples 1 to 3, respectively. In each example, evaluation was performed at n = 3, and the average was determined. Since the untreated PS cell culture dish of Comparative Example 2 was not treated with the temperature-responsive polymer, the cells did not detach. In addition, when the commercially available temperature-responsive cell culture container of Comparative Example 3 was used, peeling was performed 20 minutes after removal from the incubator. In Comparative Example 1 using HBP-0, peeling was performed 21 minutes after removal from the incubator. On the other hand, the cell culture dishes prepared in Examples 1 to 5 in which the content of the temperature-responsive polymer having a logM of 5.5 or more was 60% or more showed high detachability. In particular, Examples 1 to 4 in which the content of the temperature-responsive polymer having a logM of 6.0 or more was 15% or more had particularly high releasability. This revealed that there was a correlation between the cell detachability and the molecular weight distribution of the temperature-responsive polymer.

[Polymer dissolution test]
No. 1 fractionated in Production Example 1 With respect to HBP-1 and -2, a coating solution was prepared in the same manner as in Example 1 to prepare a coated sample. After the coating and drying, the coating was left standing in water at room temperature for 1 hour and then dried. As a comparative example, a commercially available temperature-responsive cell culture vessel was similarly left standing in water at room temperature for 1 hour and then dried. For these samples, the PNIPAM abundance before and after the polymer elution test was measured by the IR-ATR method, and the presence or absence of HBP elution was confirmed.

IR-ATR measurement results are shown in Table 3. In contrast to Comparative Examples in which dissolution was observed, in both Examples 1 and 2, desorption was not observed before and after the polymer dissolution test, and it was confirmed that there was almost no dissolution.

 

Claims (10)

1. A temperature-responsive cell culture substrate having a temperature-responsive layer (A),
   The temperature-responsive layer (A) contains a temperature-responsive polymer,
   The temperature-responsive polymer having a logM of 5.5 or more among the total of the temperature-responsive polymers having a common logarithm (logM) of a molecular weight M of 4.0 or more contained in the temperature-responsive layer (A). Is 60% or more,
   Temperature-responsive cell culture substrate.
2. The temperature-responsive polymer having a logM of 6.0 or more among the total of the temperature-responsive polymers having a common logarithm (logM) of a molecular weight M of 4.0 or more contained in the temperature-responsive layer (A). Is greater than or equal to 15%,
   The temperature-responsive cell culture substrate according to claim 1.
3. The temperature-responsive cell culture substrate according to claim 1 or 2, wherein the temperature-responsive polymer includes a block polymer in which a temperature-responsive block is bonded to a terminal of a dendritic polymer.
4. The temperature-responsive cell culture substrate according to claim 3, wherein the dendritic polymer is a dendritic polymer having a styrene skeleton or a siloxane skeleton.
5. At least one of the temperature-responsive polymers,
   Temperature-responsive polymer obtainable by polymerizing a monomer containing at least one selected from the group consisting of (meth) acrylamide, N- (or N, N-di) -substituted (meth) acrylamide and vinyl ether, or polyvinyl alcohol The temperature-responsive cell culture substrate according to any one of claims 1 to 4, wherein at least a part of the substrate is partially acetylated.
6. The N- (or N, N-di) -substituted (meth) acrylamide is poly-N-isopropyl (meth) acrylamide, poly-N, N-diethyl (meth) acrylamide, and poly-N, N-dimethyl (meth) acrylamide. The temperature-responsive cell culture substrate according to claim 5, which is at least one selected from the group consisting of) acrylamide.
7. The device according to any one of claims 1 to 6, further comprising a substrate layer (B), wherein the temperature-responsive layer (A) is disposed on at least one surface of the substrate layer (B). The described temperature-responsive cell culture substrate.
8. The temperature-responsive cell culture substrate according to claim 7, wherein the substrate layer (B) contains polystyrene.
9. A composition comprising a temperature-responsive polymer, wherein, among the total of the temperature-responsive polymers having a common logarithm (logM) of a molecular weight M of 4.0 or more, the temperature-responsive polymer having a logM of 5.5 or more. Is greater than or equal to 60%.
10. The composition according to claim 9, which is used for producing the temperature-responsive cell culture substrate according to any one of claims 1 to 8.
    
    

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