JP7271870B2 - Substrate for culture of pluripotent stem cells and method for producing pluripotent stem cells - Google Patents

Description

The present invention provides a culture substrate that can exfoliate pluripotent stem cells by temperature change by having a layer of a temperature-responsive polymer on the substrate, and a culture substrate that is excellent in mass production and does not damage cells. The present invention relates to a method for producing pluripotent stem cells capable of producing pluripotent stem cells.

Pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) are cells that have the ability to differentiate into various tissues in the body (pluripotency), and are used in the field of regenerative medicine. As a source, it has received a lot of attention. To apply pluripotent stem cells to regenerative medicine, it is essential to first proliferate the required number of pluripotent stem cells in an undifferentiated state and collect them as single cells or cell aggregates (excluding cell sheets). be.

In order to maintain the undifferentiated state of pluripotent stem cells, there is a method of culturing feeder cells prepared by gamma-ray irradiation or administration of antibiotics to mouse fetal fibroblasts as a scaffold. However, the method of culturing pluripotent stem cells using feeder cells complicates the culturing process, and endogenous virus infection may occur between different species of animals. Particularly in medical applications, there is a demand for a culture method that avoids the use of feeder cells between different kinds of animals as much as possible.

In order to solve the above problems, an iPS cell culture substrate coated with an extracellular matrix such as laminin (Patent Document 1) is known. With such a base material, pluripotent stem cells can be cultured while maintaining an undifferentiated state without using feeder cells, ie, in a feeder-free environment. However, a proteolytic enzyme is used to detach the pluripotent stem cells proliferated on such a substrate from the substrate. Proteases are responsible for degrading proteins on the surface of multi-cells and severing the bonds between pluripotent stem cells and substrates and between pluripotent stem cells. On the other hand, proteases are known to adversely affect cell viability, and a method for separating cells from substrates without using proteases is important as a method that does not damage cells.

As a method for exfoliating cells without using a proteolytic enzyme, a method using a temperature-responsive substrate is known. For example, in Patent Document 2, a cell culture support coated with a temperature-responsive polymer having an upper or lower critical dissolution temperature in water of 0 to 80° C. is used, and the cells are exfoliated by temperature change to recover the cell sheet. A method for doing so is disclosed. However, the method described in Patent Document 2 has a problem that it cannot be applied to pluripotent stem cells. Furthermore, in Patent Document 2, the main purpose is to collect a cell sheet, and it is not intended to obtain a single cell or a clump of cells.

JP 2015-178526 A JP 2009-131275 A

An object of the present invention is to allow pluripotent stem cells to be cultured in a feeder-free environment, and by changing the temperature of the culture substrate, the pluripotent stem cells can be detached from the culture substrate without using proteolytic enzymes. and to provide a culture substrate from which pluripotent stem cells can be collected as single cells or cell aggregates.

Another object of the present invention is to provide a method for producing pluripotent stem cells that is excellent in mass productivity and capable of producing pluripotent stem cells as single cells or cell aggregates without damaging the cells.

In view of the above points, the present inventors have made intensive studies and found that a culture substrate having a layer of a temperature-responsive polymer and a method for producing specific pluripotent stem cells can solve the above problems. He found the headline and completed the present invention.

That is, according to the present invention, the base material surface has a layer of a temperature-responsive polymer having a response temperature to water in the range of 0° C. to 50° C., and the layer is composed of matrigel, laminin, fibronectin, vitronectin, and collagen. Provided is a substrate for culturing pluripotent stem cells characterized by immobilizing at least one biological substance selected from the group consisting of:

The present invention also relates to a method for producing pluripotent stem cells, which produces undifferentiated pluripotent stem cells through the following steps [1] to [3].
[1] A step of seeding pluripotent stem cells onto the culture substrate.
[2] A step of culturing the pluripotent stem cells seeded on the culture substrate in a liquid at a temperature equal to or higher than the response temperature of the temperature-responsive polymer.
[3] A step of cooling the culture substrate to a temperature lower than the response temperature of the temperature-responsive polymer and exfoliating the pluripotent stem cells cultured in the liquid from the substrate.

According to the present invention, pluripotent stem cells can be cultured in a feeder-free environment, and by changing the temperature of the culture substrate, the pluripotent stem cells can be detached from the substrate without using a proteolytic enzyme, A culture substrate from which pluripotent stem cells can be collected as single cells or cell aggregates can be provided. Moreover, it is possible to provide a method for producing pluripotent stem cells that is excellent in mass productivity and capable of producing pluripotent stem cells as single cells or cell aggregates without damaging the cells.

Schematic diagram showing components of the culture substrate of the present invention An example of LCST measurement results by measuring the transmittance of an aqueous solution An example of measurement results of response temperature by water contact angle measurement Underwater AFM image of the culture substrate surface of Example 8

EMBODIMENT OF THE INVENTION Hereinafter, the form (only henceforth "this Embodiment") for implementing this invention is demonstrated in detail. The following embodiments are examples for explaining the present invention, and are not intended to limit the present invention to the following contents. The present invention can be appropriately modified and implemented within the scope of its gist.

As used herein, the term "temperature-responsive polymer" refers to a polymer whose degree of hydrophilicity/hydrophobicity changes with temperature changes. In addition, when the temperature of the polymer is increased from a low temperature to a high temperature in the presence of water molecules, the degree of hydrophilicity/hydrophobicity of the polymer changes to become more hydrophobic at a certain temperature. There is a temperature-responsive polymer that changes from water-insoluble to water-insoluble, and this boundary temperature is called "Lower Critical Solution Temperature (LCST)". In general, when a temperature-responsive polymer dissolves in water at a temperature below the LCST, it becomes insoluble in water at a temperature above the LCST. When the temperature-responsive polymer is water-insoluble, it does not have an LCST, but has a response temperature at which the degree of hydrophilicity/hydrophobicity changes with temperature changes.

In the present specification, the term "biologically-derived substance" refers to a substance that exists in the body of an organism, but it may be a natural product or one that is artificially synthesized by genetic recombination technology or the like. Alternatively, it may be a chemically synthesized substance based on the biological substance. Although there is no particular limitation on substances derived from living organisms, for example, nucleic acids, proteins, and polysaccharides, which are basic materials that constitute living organisms, nucleotides, nucleosides, amino acids, and various sugars, which are constituents thereof, or lipids, vitamins, and hormones. be.

Also, as used herein, the term "pluripotent stem cell adhesiveness" means that pluripotent stem cells adhere to a culture substrate at the temperature at which the pluripotent stem cells are cultured.

Further, in the present specification, the block segment "has water insolubility" means that at least part of the homopolymer consisting of only the monomer units constituting the block segment is insoluble in water.

In addition, as used herein, the term “pluripotent stem cell proliferativeness” indicates the ease with which pluripotent stem cells proliferate at the culture temperature. Adheres to wood and shows that it can grow. Furthermore, high proliferativeness means proliferating into more pluripotent stem cells when compared during the same culture period.

In the present specification, "pluripotent stem cell detachability" refers to the ease with which pluripotent stem cells proliferated on a culture substrate can be detached from the culture substrate, and "having detachability" , indicates that pluripotent stem cells proliferated on a culture substrate can be detached from the culture substrate by an external stimulus. Furthermore, the term "high detachability" indicates that the pluripotent stem cells are detached from the culture substrate by a weaker external stimulus such as short-term cooling or weak stress. In addition, "having cooling peelability" indicates that the culture substrate has peelability, and cooling the culture substrate enhances the peelability as compared to the case where the culture substrate is not cooled. Here, the term "external stimulus" as used herein refers to mechanical stimuli such as ultrasonic waves, vibrations and convection, electromagnetic stimuli such as light, electricity and magnetism, and thermodynamic stimuli such as heating and cooling. , Excludes biological reactions such as enzymatic reactions.

The culture substrate of the present invention has a layer of a temperature-responsive polymer having a water response temperature in the range of 0°C to 50°C on the surface of the substrate. By having a layer of a temperature-responsive polymer, the culture substrate of the present invention has cooling exfoliation properties for pluripotent stem cells. When there is no layer of temperature-responsive polymer, the pluripotent stem cells do not have cooling detachability. Here, in the present invention, the term “culture substrate” refers to the entire article for culturing pluripotent stem cells (for example, the portion indicated by reference numeral 10 in FIG. 1), which is composed of the substrate and the temperature-responsive polymer. Including layers. In the present invention, the term "substrate" refers to a base substrate coated with a layer of temperature-responsive polymer (for example, the portion indicated by reference numeral 1 in FIG. 1).

In the present invention, the temperature-responsive polymer has a response temperature in the range of 0°C to 50°C. Since the response temperature is in the range of 0° C. to 50° C., the culture substrate of the present invention has pluripotent stem cell adhesion and proliferative properties near body temperature (37° C.) and does not damage the cells. It has cooling peelability in If it does not have a response temperature, it does not have cooling peelability. The response temperature is in the range of 10°C to 40°C because it is suitable for imparting adhesion and proliferative properties to pluripotent stem cells near body temperature and imparting cooling detachability in a temperature range that does not damage cells. , preferably in the range of 15°C to 35°C, particularly preferably in the range of 15°C to 30°C, most preferably in the range of 15°C to 25°C. At a temperature equal to or higher than the response temperature, the temperature-responsive polymer is hydrophobic, so proteins are easily adsorbed, and adherent culture of cells is possible using the adsorbed protein as a scaffold. On the other hand, when the temperature is lowered, cell detachment is promoted by changing to hydrophilicity. If the response temperature is less than 0°C, it becomes difficult to impart cooling exfoliation in a temperature range that does not damage cells, and if it exceeds 50°C, pluripotent stem cells do not have adhesiveness and proliferative properties near body temperature. , making cell culture difficult. In addition, when using a medium at room temperature during medium exchange in the culture operation, the response temperature is in the range of 0 ° C. to 30 ° C. because it is suitable for suppressing detachment of cells during medium exchange. more preferably in the range of 5°C to 25°C, particularly preferably in the range of 5°C to 20°C, most preferably in the range of 10°C to 20°C.

In the present invention, the type of the temperature-responsive polymer is not particularly limited. A polymer or the like immobilized on a substrate by coalescing, applying a monomer to the substrate, and performing electron beam polymerization or radical polymerization on the substrate can be preferably used.

In the present invention, the temperature-responsive polymer is preferably a block copolymer containing at least the following block segments (A) and (B).
(A) A temperature-responsive polymer block segment having an LCST in the range of 0°C to 80°C.
(B) A block segment having water insolubility or a block segment having pluripotent stem cell adhesion.

By containing the block segment (A) in the block copolymer, the culture substrate of the present invention is easily imparted with pluripotent stem cell adhesion and proliferative properties near body temperature, and the temperature range does not damage the cells. It is easy to impart cold peelability to.

Here, in the present invention, the expression "LCST of block segment (A)" indicates the LCST of a homopolymer consisting only of repeating units constituting block segment (A). The LCST of the block segment (A) is the temperature at which the homopolymer becomes insoluble in water. It can be obtained by measuring the transmittance of light having a wavelength of 500 nm. Although the transmittance is generally constant from a low temperature until it reaches the LCST, the transmittance drops sharply due to cloudiness near the LCST, and after that, a curve in which the transmittance becomes generally constant again is obtained (for example, FIG. 2 ). In this curve, the LCST can be determined by determining the temperature at which the transmittance is the average value of the transmittance at temperatures below the LCST and the transmittance at temperatures above the LCST (midpoint method). The temperature range for measurement includes a temperature range of 5°C or higher where the transmittance is generally constant at temperatures below the LCST, and a temperature range of 5°C or higher where the transmittance is constant at temperatures above the LCST.

In addition, when the block copolymer of the present invention has a water-insoluble block segment, it is insoluble in water, but the response temperature is measured by determining the temperature at which the degree of hydrophilicity/hydrophobicity changes depending on the temperature change. be able to. For example, the degree of hydrophilicity/hydrophobicity of the block copolymer is measured by immersing the base material coated with the block copolymer in water and measuring the contact angle of air bubbles in water. Next, the temperature of the block copolymer is changed by changing the temperature of the water, and after waiting until the temperature stabilizes, the contact angle of the bubbles is measured again to obtain the contact angle at various temperatures. . Below the response temperature, the bubble contact angle is large (the water contact angle is small) and generally constant. A curve that is approximately constant above the response temperature is obtained (eg, FIG. 3). In this curve, the response temperature can be determined by determining the temperature at which the contact angle is the average value of the contact angles at temperatures below the response temperature and at temperatures above the response temperature (midpoint method). The temperature range to be measured includes a temperature range of 10°C or higher where the contact angle is generally constant at temperatures below the response temperature, and a temperature range of 10°C or higher where the contact angle is generally constant at temperatures above the response temperature. is.

A representative method for controlling the response temperature of a temperature-responsive polymer is to adjust the properties of the monomers to be copolymerized and the copolymerization rate thereof. In the temperature-responsive polymer copolymerized with a hydrophilic monomer, the response temperature shifts to higher temperatures as the composition increases. On the other hand, the response temperature of a temperature-responsive polymer copolymerized with a hydrophobic monomer shifts to the low temperature side as the composition increases. From such a point of view, the suitable LCST of the block segment (A) varies depending on the properties of the monomer of the block segment (B) to be copolymerized. Therefore, the LCST of the block segment (A) is preferably 10 to 75°C, more preferably 10 to 60°C, and particularly preferably 20 to 50°C.

The monomer unit constituting the block segment (A) is not particularly limited, but examples include (meth)acrylamide compounds such as acrylamide and methacrylamide; N,N-diethylacrylamide, N-ethylacrylamide, Nn - propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N - N-alkyl-substituted (meth)acrylamide derivatives such as tetrahydrofurfuryl acrylamide and N-tetrahydrofurfuryl methacrylamide; N,N-dimethyl(meth)acrylamide, N,N-ethylmethylacrylamide, N,N-diethylacrylamide and the like N,N-dialkyl-substituted (meth)acrylamide derivatives of; 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, 4-(1-oxo-2- (meth)acrylamide derivatives having a cyclic group such as methyl-2-propenyl)-morpholine; vinyl ethers such as methyl vinyl ether; proline derivatives such as N-proline methyl ester acrylamide; Since it is suitable for 0 to 50 ° C., N,N-diethylacrylamide, Nn-propylacrylamide, N-isopropylacrylamide, Nn-propylmethacrylamide, N-ethoxyethylacrylamide, N-tetrahydro Furfuryl acrylamide and N-tetrahydrofurfuryl methacrylamide are preferred, Nn-propyl acrylamide and N-isopropyl acrylamide are more preferred, and N-isopropyl acrylamide is particularly preferred. In addition, when using a medium at room temperature when exchanging the medium in the culture operation, it is preferable to lower the response temperature of the block copolymer to a temperature lower than room temperature. Methyl ester acrylamide is preferred.

In the present invention, it is preferable that the block copolymer contains the water-insoluble block segment (B) because it is easy to suppress the temperature-responsive polymer from being mixed into the culture solution.

The block segment (B) in the present invention is also suitable for enhancing the adhesion of pluripotent stem cells to the culture substrate. Adhesion is preferred. Those having pluripotent stem cell adhesiveness are not particularly limited, but for example, those having an ionic group, a hydrophilic group, a hydrophobic group, etc., and after coating them on the substrate surface, Examples include those subjected to surface treatment such as gamma ray irradiation, plasma treatment, and corona treatment.

The block segment (B) in the present invention is also suitable for setting the response temperature of the block copolymer in the range of 0 to 50 ° C., so that it is a block that contributes to the control of the response temperature of the block copolymer. is preferred. Since the block copolymer has the block segment (B), the response temperature of the block segment (A) is shifted to the high temperature side or the low temperature side, and the response temperature of the block copolymer is controlled within the range of 0 to 50°C. be able to. Since the block copolymer has the block segment (B), it is possible to control the response temperature of the block copolymer, and various monomers can be used as the monomer units constituting the block segment (B). be. In order to control the response temperature of the block copolymer, for example, a hydrophilic monomer, a hydrophobic monomer, or both may be used in the block segment (B).

In addition, since the block segment (B) in the present invention is suitable for enhancing detachment of pluripotent stem cells, it is expected that the block contributes to shortening the cooling time required for detachment of pluripotent stem cells. preferable. Since the block copolymer has the block segment (B), when the temperature of the culture substrate is lower than the response temperature of the block copolymer, water easily enters the block copolymer, and the cells and the block copolymer are separated. The adhesive strength of pluripotent stem cells can be weakened easily, and the time required for detachment of pluripotent stem cells can be shortened. Therefore, when the block copolymer has the block segment (B), the time required for detachment of the pluripotent stem cells can be shortened. In order to shorten the cooling time required for detachment of pluripotent stem cells, it is preferable to use, for example, a hydrophilic monomer for the block segment (B).

Furthermore, the block segment (B) in the present invention is preferably a block that contributes to adhesion to the substrate, since it is suitable for firmly fixing the block copolymer to the substrate. Since the block segment (B) is a block that contributes to adhesion to the substrate, the block copolymer can be stably coated on the substrate even when the temperature of the culture substrate is lower than the response temperature of the block copolymer. can continue. For this reason, since the block segment (B) is a block that contributes to adhesion to the substrate, immobilization of the block copolymer to the substrate is not limited to chemical coating, but physical coating. You can also let In order to increase the adhesion of the block copolymer to the substrate, it is preferable to use, for example, a hydrophobic monomer or a monomer having a reactive functional group for the block segment (B).

In the present invention, the block segment (B) is a block weight containing at least one type of repeating unit among repeating units represented by the following general formula (1), since it is suitable for increasing water insolubility. It is preferably coalesced.

[In the formula, R ¹ is a hydrogen atom or a methyl group, Q is a divalent bond selected from an ester bond, an amide bond, a urethane bond or an ether bond, and R ² is the following general formula (2), ( 3), (4), (5), (6), (7) or (8), a hydrocarbon group having 1 to 30 carbon atoms, or a hydrogen atom.

(In the formula, R ³ is a divalent hydrocarbon group having 1 to 10 carbon atoms, and R ⁴ and R ⁵ are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms.)

(wherein R ⁶ is a divalent hydrocarbon group having 1 to 10 carbon atoms, R ⁷ is a divalent hydrocarbon group having 1 to 4 carbon atoms, and R ⁸ and R ⁹ are each independently , a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms, and X is a sulfonate anion group, a carboxylate anion group or a phosphate anion group.)

(Wherein, R ¹⁰ is a hydrogen atom or an alkyl group having 1 to 30 carbon atoms, i is an integer of 1 to 300, and j is an integer of 0 to 60.)

(In the formula, R ¹¹ is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms.)

(Wherein, R ¹² is a divalent hydrocarbon group having 1 to 12 carbon atoms or a (poly)oxyethylene group, R ¹³ is a divalent hydrocarbon group having 1 to 4 carbon atoms, R ¹⁴ , R ¹⁵ and R ¹⁶ are each independently a hydrogen atom or a hydrocarbon group having 1 to 2 carbon atoms.)

(In the formula, A is an ether bond or a methylene group, R ¹⁷ represents a divalent hydrocarbon group having 3 to 5 carbon atoms, R ¹⁸ represents a fluorine atom, and n represents an integer of 0 to 4. )

(In the formula, R ¹⁹ represents a divalent hydrocarbon group having 1 to 5 carbon atoms, but may be absent. R ²⁰ , R ²¹ , R ²² , R ²³ and R ²⁴ are each independently hydrogen represents an atom, a hydroxyl group, a carboxyl group, an amino group, or a hydrocarbon group having 1 to 4 carbon atoms.)]
In general formula (1) in the present invention, ^R1 is a hydrogen atom or a methyl group. Q is a divalent bond selected from an ester bond, an amide bond, a urethane bond or an ether bond, preferably an ester bond or an amide bond, and particularly preferably an ester bond. In general formula (1), R ² is a substituent represented by general formula (2), (3), (4), (5), (6), (7) or (8) and has 1 to 30 hydrocarbon groups or hydrogen atoms are shown.

In general formula (1), R ² is used to increase water insolubility, to increase adhesiveness and proliferation of pluripotent stem cells, to shift the response temperature of the block copolymer to a higher temperature side, or to increase pluripotent stem cell It is preferable to use the substituent represented by the general formula (2) because it is suitable for enhancing the releasability of the film. In general formula (2), R ³ is a divalent hydrocarbon group having 1 to 10 carbon atoms, which is suitable for increasing the exfoliation of pluripotent stem cells, and therefore preferably has 1 to 6 carbon atoms. A divalent hydrocarbon group, especially an alkylene group is used. Examples of such alkylene group include methylene group, ethylene group, propylene group, butylene group, pentamethylene group, hexamethylene group and the like, preferably ethylene group. Each of R ⁴ and R ⁵ is independently ^a hydrogen atom or ^a hydrocarbon group having 1 to 4 carbon atoms, and is suitable for enhancing exfoliation of pluripotent stem cells. A methyl group or an ethyl group is preferred, and a methyl group is particularly preferred.

Examples of substituents represented by general formula (2) in the present invention include aminomethyl group, N,N-dimethylaminomethyl group, N,N-diethylaminomethyl group, aminoethyl group and N,N-dimethylaminoethyl group. , N,N-diethylaminoethyl group, 3-aminopropyl group, 3-(N,N-dimethylamino)-propyl group, 3-(N,N-diethylamino)-propyl group, etc., but pluripotent N,N-dimethylaminomethyl group, N,N-dimethylaminoethyl group, and N,N-diethylaminoethyl group are preferable because they are suitable for enhancing exfoliation of stem cells.

The monomer unit represented by general formula (2) for R ² in general formula (1) is not particularly limited, but examples include 2-dimethylaminoethyl acrylate, 2-dimethylaminoethyl methacrylate, 2-diethylamino Ethyl acrylate, 2-diethylaminoethyl methacrylate, N-[3-(dimethylamino)propyl]acrylamide and the like can be mentioned.

In general formula (1), R ² is used to increase water insolubility, to increase adhesiveness and proliferation of pluripotent stem cells, to shift the response temperature of the block copolymer to a higher temperature side, or to increase pluripotent stem cell A substituent represented by the general formula (3) can be used because it is suitable for enhancing the releasability of the film. In the general formula (3), R ⁶ is a divalent hydrocarbon group having 1 to 10 carbon atoms, which is suitable for increasing the exfoliation of pluripotent stem cells, and thus is preferably a methylene group or an ethylene group. , a propylene group, a butylene group, a pentamethylene group, a hexamethylene group, and a divalent alkylene group having 1 to 6 carbon atoms, more preferably an ethylene group. R ⁷ is a divalent hydrocarbon group having 1 to 4 carbon atoms, preferably an alkylene group such as a methylene group, ethylene group, propylene group, butylene group, etc., in order to enhance the exfoliation of pluripotent stem cells. and more preferably an ethylene group. R ⁸ and R ⁹ are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms, and are suitable for enhancing exfoliation of pluripotent stem cells, so R ⁸ and R ⁹ are preferably They are a hydrogen atom or a methyl group at the same time, more preferably a methyl group at the same time. X is a sulfonate anion group, a carboxylate anion group or a phosphate anion group.

Examples of the substituent represented by the general formula (3) in the present invention include dimethyl (ethyl) (carboxylatomethyl) aminium group, dimethyl (ethyl) (2-carboxylatoethyl) aminium group, dimethyl (ethyl) (3- carboxylatopropyl) aminium group, dimethyl(propyl)(3-sulfonatopropyl) aminium group, dimethyl(propyl)(4-sulfonatobutyl) aminium group, dimethyl(ethyl)(2-sulfonatoethyl) aminium group, dimethyl(ethyl ) (3-sulfonatopropyl) aminium group, dimethyl (ethyl) (2-phosphonatoethyl) aminium group, dimethyl (ethyl) (3-phosphonatopropyl) aminium group and the like can be exemplified. preferably dimethyl(ethyl)(carboxylatomethyl)aminium group, dimethyl(ethyl)(2-carboxylatoethyl)aminium group, dimethyl(propyl)(3-sulfonatopropyl)aminium group , dimethyl(propyl)(4-sulfonatobutyl)aminium group and dimethyl(ethyl)(2-sulfonatoethyl)aminium group.

In general formula (1), the monomer unit represented by general formula (3) for R ² is not particularly limited, but for example, N-(3-sulfopropyl)-N-methacryloyloxyethyl- N,N-dimethylammonium betaine, N-methacryloyloxyethyl-N,N-dimethylammonium-α-N-methylcarboxybetaine and the like can be mentioned.

In general formula (1), R ² increases water insolubility, increases pluripotent stem cell adhesiveness and proliferation, shifts the response temperature of the block copolymer to a higher temperature side or a lower temperature side, or exfoliates It is preferable to use the substituent represented by the general formula (4) because it is suitable for enhancing the property. In general formula (4), R ¹⁰ is a hydrogen atom or an alkyl group having 1 to 30 carbon atoms, methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, isobutyl group, tert. -Butyl group, n-hexyl group, isohexyl group and the like can be exemplified, but methyl group, ethyl group, n-propyl group and isopropyl group are preferable because they are suitable for increasing the exfoliation of pluripotent stem cells. be. However, in general formula (4), i is an integer of 1-300 and j is an integer of 0-60.

Examples of substituents represented by general formula (4) in the present invention include polyethylene glycol group, 2-hydroxyethyl group, 2-hydroxyethyl group, hydroxymethyl group, 2-methoxyethyl group, furfuryl group and tetrahydrofurfuryl group. etc., but polyethylene glycol group, 2-methoxyethyl group or tetrahydrofurfuryl group is preferable because it is suitable for enhancing the exfoliation of pluripotent stem cells.

In general formula (1), the monomer unit represented by general formula (4) for R ² is not particularly limited, but examples include hydroxyethyl acrylate, hydroxyethyl methacrylate, and N-(2-hydroxyethyl). acrylamide, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, polypropylene glycol monomethacrylate, methoxypolyethylene glycol monoacrylate, methoxypolyethylene glycol monomethacrylate, 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 Acrylates, furfuryl methacrylate, tetrahydrofurfuryl acrylate, tetrahydrofurfuryl methacrylate and the like can be mentioned.

In general formula (1), R ² is used to increase water insolubility, to increase pluripotent stem cell adhesiveness and proliferation, to shift the response temperature of the block copolymer to a higher temperature side or to a lower temperature side, or to increase water insolubility. Substituents represented by general formula (5) can be used because they are suitable for enhancing the exfoliation of potential stem cells. In general formula (5), R ¹¹ is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms.

In general formula (1), the monomer unit represented by general formula (5) for R ² is not particularly limited, but examples include methoxymethyl acrylate, methoxymethyl methacrylate, 2-ethoxymethyl acrylate, 2- Ethoxymethyl methacrylate, 3-butoxymethyl acrylate, 3-butoxymethyl methacrylate, 3-butoxymethyl acrylamide and the like can be mentioned.

In general formula (1), R ² is used to increase water insolubility, to increase adhesiveness and proliferation of pluripotent stem cells, to shift the response temperature of the block copolymer to a higher temperature side, or to increase pluripotent stem cell A substituent represented by the general formula (6) can be used because it is suitable for enhancing the releasability of the film. In general formula (6), R ¹² is a divalent hydrocarbon group having 1 to 12 carbon atoms or a (poly)oxyethylene group, which is suitable for shortening the cooling time required for detachment of pluripotent stem cells. Therefore, it is preferably a divalent hydrocarbon group having 1 to 6 carbon atoms, particularly an alkylene group. Examples of such an alkylene group include a methylene group, ethylene group, propylene group, butylene group, pentamethylene group, hexamethylene group and the like, preferably ethylene group. R ¹³ is a divalent hydrocarbon group having 1 to 4 carbon atoms, which is suitable for shortening the cooling time required for exfoliation of pluripotent stem cells, and therefore is preferably an alkylene group having 1 to 4 carbon atoms. Groups such as methylene group, ethylene group, propylene group, butylene group and the like are exemplified, and ethylene group is particularly preferred.

R ¹⁴ , R ¹⁵ and R ¹⁶ are each independently a hydrogen atom or a hydrocarbon group having 1 to 2 carbon atoms, such as a methyl group or an ethyl group, which are suitable for enhancing exfoliation of pluripotent stem cells. Therefore, it is particularly preferred that R ¹⁴ , R ¹⁵ and R ¹⁶ are simultaneously a hydrogen atom or a methyl group, especially a methyl group.

The substituent represented by the general formula (6) in the present invention includes a 2-ethylphosphorylcholine group, a 3-propylphosphorylcholine group, a 4-butylphosphorylcholine group, a 6-hexylphosphorylcholine group, a 10-decylphosphorylcholine group, ω-( A poly)oxyethylene phosphorylcholine group and the like can be exemplified, but a 2-ethylphosphorylcholine group is preferably used because it is suitable for enhancing the exfoliation of pluripotent stem cells.

In the general formula (1), the monomer unit represented by the general formula (6) for R ² is not particularly limited. (Meth)acryloyloxypropylphosphorylcholine, 4-(meth)acryloyloxybutylphosphorylcholine, 6-(meth)acryloyloxyhexylphosphorylcholine, 10-(meth)acryloyloxydecylphosphorylcholine, ω-(meth)acryloyl(poly)oxyethylenephosphorylcholine , 2-acrylamidoethylphosphorylcholine, 3-acrylamidopropylphosphorylcholine, 4-acrylamidobutylphosphorylcholine, 6-acrylamidohexylphosphorylcholine, 10-acrylamidodecylphosphorylcholine, ω-(meth)acrylamido(poly)oxyethylene phosphorylcholine and the like.

In general formula (1), R ² is used to increase water insolubility, to increase adhesion and proliferation of pluripotent stem cells, to shift the response temperature of the block copolymer to the low temperature side, or to block copolymers on the base material. A substituent having a phenylazide group represented by the general formula (7) can be used because it is suitable for coating the polymer by chemical bonding. In general formula (7), A is an ether bond or an ester bond. R ¹⁷ represents a divalent hydrocarbon group having 1 to 5 carbon atoms, such as a propylene group, a butylene group, a pentamethylene group and a phenylene group. R ¹⁸ represents a fluorine atom and n represents an integer of 0-4.

In general formula (1), the monomer unit represented by general formula (7) for R ² is not particularly limited, but examples include 4-azidophenyl acrylate, 4-azidophenyl methacrylate, 2-(( 4-azidobenzoyl)oxy)ethyl acrylate, 2-((4-azidobenzoyl)oxy)ethyl methacrylate and the like.

In the general formula (1), R ² enhances water insolubility, enhances adhesion and proliferation of pluripotent stem cells, shifts the response temperature of the block copolymer to the high temperature side or the low temperature side, Since it is suitable for coating the block copolymer by physical interaction or for coating the block copolymer on the substrate by chemical bonding, it is represented by the general formula (8) , it is preferable to use a substituent having an aromatic ring. In general formula (8), R ¹⁹ represents a divalent hydrocarbon group having 1 to 5 carbon atoms, but may be absent. R ²⁰ , R ²¹ , R ²² , R ²³ and R ²⁴ each independently represent a hydrogen atom, a hydroxyl group, a carboxyl group, an amino group or a hydrocarbon group having 1 to 4 carbon atoms. In general formula (1), the monomer unit represented by general formula (8) for R ² is not particularly limited, but examples include 2-hydroxyphenyl acrylate, 2-hydroxyphenyl methacrylate, 3-hydroxyphenyl Acrylates, 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 the like.

In general formula (1), R ² is used to increase water insolubility, to increase adhesion and proliferation of pluripotent stem cells, to shift the response temperature of the block copolymer to the low temperature side, or to block copolymers on the base material. It is preferable to use a hydrocarbon group having 1 to 30 carbon atoms because it is suitable for coating the polymer by physical interaction. It is a hydrocarbon group having 4 to 15 carbon atoms. The monomer unit in which R ² is a hydrocarbon group having 1 to 30 carbon atoms is not particularly limited, but examples 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-tetra Decyl acrylate, n-tetradecyl methacrylate and the like can be mentioned.

The structure of the block copolymer containing the block segments (A) and (B) in the present invention is not particularly limited, but a diblock copolymer containing at least the block segments (A) and (B) It is preferably coalesced. Moreover, the block segment (A) and the block segment (B) may be directly bound or may be bound via a spacer. When there are two or more kinds of repeating units constituting the block segment (B), their arrangement may be a block arrangement, a random arrangement, or an alternating arrangement.

The structural unit ratio of the block segment (A) in the block copolymer containing the block segments (A) and (B) in the present invention is preferably 40 to 40 from the viewpoint of enhancing the cooling exfoliation property of pluripotent stem cells. 99 wt %, more preferably 60 to 99 wt %, particularly preferably 80 to 99 wt %, most preferably more than 90 wt %. On the other hand, from the viewpoint of increasing the water insolubility of the block copolymer, the structural unit ratio of the block segment (B) is preferably 1 to 50 wt%, preferably 1 to 25 wt%, more preferably 3 to 15 wt%. %, most preferably 5-15 wt %.

The molecular weight of the temperature-responsive polymer in the present invention is not particularly limited, but since it is suitable for increasing the strength of the block copolymer, the number average molecular weight is preferably 1000 to 1,000,000, preferably 2000 to 500,000. is more preferable, 5000 to 300,000 is particularly preferable, and 10,000 to 200,000 is most preferable.

The temperature-responsive polymer in the present invention may contain a chain transfer agent, a polymerization initiator, a polymerization inhibitor, etc., if necessary. The chain transfer agent is not particularly limited, and commonly used ones can be preferably used. Noic acid, 2-cyanopropan-2-yl N-methyl-N-(pyridin-4-yl) carbamodithioate, methyl methyl 2-propionate (4-pyridinyl) carbamodithioate can be mentioned. . In addition, the polymerization initiator is not particularly limited, and commonly used ones can be preferably used. -butyl peroxide, tert-butyl hydroperoxide, hydrogen peroxide, potassium peroxodisulfate, benzoyl peroxide, triethylborane, diethylzinc and the like. Furthermore, the polymerization inhibitor is not particularly limited, and commonly used ones can be preferably used, but hydroquinone, p-methoxyphenol, triphenylferdazyl, 2,2,6,6-tetramethylpiperidinyl Nil-1-oxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl and the like can be mentioned.

The method for synthesizing the temperature-responsive polymer in the present invention is not particularly limited. 161-225 (2010) can be used.

In the present invention, the temperature-responsive polymer layer preferably has a layer thickness of 5 to 1,000 nm, more preferably 5 to 200 nm, because it is suitable for enhancing adhesion, proliferation, and detachment of pluripotent stem cells. It is more preferably 20 to 100 nm, most preferably 35 to 80 nm.

In the present invention, the type of the temperature-responsive polymer is not particularly limited. A copolymer or a mixture of two types of block copolymers is preferred, and one type of block copolymer is more preferred. Here, in the present invention, regarding the "type of temperature-responsive polymer", when all the blocks constituting the block copolymer are the same, they are regarded as the same type of block copolymer. Here, the block being the same means that when the block is mainly composed of one type of monomer unit, the monomer unit contained most in wt% ratio is the same, and , indicates the case where the block is composed of two or more types of monomer units, and the two monomer units with the highest wt % ratio are the same. The polydispersity of each block (weight-average molecular weight M _w /number-average molecular weight M _n ) is preferably 1 to 20, because it is more suitable for homogenizing the state of cultured pluripotent stem cells. It is more preferably 1-10, particularly preferably 1-5, and most preferably 1-2.

In the present invention, the layer of the temperature-responsive polymer has a biological substance immobilized on the layer, and the biological substance is selected from the group consisting of Matrigel, laminin, fibronectin, vitronectin and collagen. is at least one type.

The culture substrate of the present invention can culture pluripotent stem cells by having a biological substance immobilized on a layer made of a temperature-responsive polymer. When the temperature-responsive polymer layer does not contain a biological substance, the adhesion of pluripotent stem cells to the culture substrate is weak, and pluripotent stem cells cannot be cultured.

In addition, it is preferable that the biological substance is immobilized by a non-covalent bond rather than a covalent bond, because the denaturation of the biological substance can be suppressed and the proliferation of pluripotent stem cells can be enhanced. Here, "non-covalent bond" in the present invention means electrostatic interaction, hydrophobic interaction, hydrogen bond, π-π interaction, dipole-dipole interaction, London dispersion force, other van der Waals Indicates a binding force other than covalent bonding derived from intermolecular forces, such as interaction. Immobilization of the biological substance to the block copolymer may be by a single binding force or by a combination of multiple binding forces.

In the present invention, the method for immobilizing the biological substance is not particularly limited. For example, the biological substance is immobilized by applying a solution of the biological substance to a base material having a layer of a temperature-responsive polymer for a predetermined period of time. A method of immobilizing pluripotent stem cells by adding a biological substance to a culture solution for culturing pluripotent stem cells can be suitably used.

In the present invention, the biological substance is at least one selected from the group consisting of matrigel, laminin, vitronectin, fibronectin and collagen. By using any one of matrigel, laminin, fibronectin, vitronectin, collagen, or a combination thereof as the biological substance, the culture substrate of the present invention has pluripotent stem cell adhesion and proliferative properties. If the biological material is not matrigel, laminin, vitronectin, fibronectin, or collagen, it does not have pluripotent stem cell adhesion and proliferation. A combination of at least 4 types of laminin is more preferable because it is suitable for conferring adhesion and proliferation of pluripotent stem cells, and any one of laminin and matrigel, laminin and fibronectin, or laminin and collagen are particularly preferred, and laminin alone is most preferred.

These biological substances may be natural products or artificially synthesized by genetic recombination technology or the like. Alternatively, it may be a synthetic peptide.

In the present invention, as the Matrigel, for example, Matrigel (manufactured by Corning Incorporated) and Geltrex (manufactured by Thermo Fisher Scientific) can be suitably used as commercially available products due to their easy availability.

Although the type of laminin is not particularly limited, for example, laminin 511, laminin 521, or laminin 511-511, which has been reported to exhibit high activity against α6β1 integrin expressed on the surface of human iPS cells. An E8 fragment can be used. The laminin may be a natural product, one artificially synthesized by gene recombination technology or the like, or a synthetic protein or synthetic peptide based on the laminin. For example, iMatrix-511 (manufactured by Nippi Co., Ltd.) can be preferably used as a commercial product because of its easy availability.

The vitronectin may be a natural product, may be artificially synthesized by genetic recombination technology or the like, or may be a synthetic protein or synthetic peptide based on the vitronectin. Commercially available products such as vitronectin, derived from human plasma (manufactured by Wako Pure Chemical Industries, Ltd.), synthemax (manufactured by Corning Incorporated), and Vitronectin (VTN-N) (manufactured by Thermo Fisher Scientific) are preferably used. be able to.

The fibronectin may be a natural product, may be artificially synthesized by genetic recombination technology or the like, or may be a synthetic protein or synthetic peptide based on the fibronectin. Commercially available products such as fibronectin solution, human plasma-derived (manufactured by Wako Pure Chemical Industries, Ltd.) and Retronectin (manufactured by Takara Bio Inc.) can be preferably used because of their easy availability.

Although the type of collagen is not particularly limited, for example, type I collagen or type IV collagen can be used. The collagen may be a natural product, may be artificially synthesized by genetic engineering or the like, or may be a synthetic peptide based on the collagen. Commercially available products such as Collagen I, Human (manufactured by Corning Incorporated) and Collagen IV, Human (manufactured by Corning Incorporated) can be suitably used.

In the present invention, the laminin adsorption rate according to the following test is preferably 10% or more, more preferably 20% or more, because it is suitable for increasing the proliferation and exfoliation of pluripotent stem cells. Particularly preferred is 20% to 80%, most preferred is 30% to 60%.
[Laminin adsorption rate test]
A solution obtained by adding 2 to 2.5 μL of a laminin 511-E8 fragment solution with a concentration of 0.5 mg/mL to 1 mL of phosphate-buffered saline was added in an amount of 0.2 mL/cm per unit area of the culture substrate. ² Laminin adsorption rate determined from the following formula when dropped onto a substrate and allowed to stand at 37°C for 24 hours.

Here, there is no particular limitation on the method for measuring the weight of the laminin 511-E8 fragment adsorbed to the culture substrate and the weight of the laminin 511-E8 fragment contained in the solution dropped onto the culture substrate. A laminin 511-E8 fragment fluorescently labeled with HiLyte Fluor ^™ 647 Labeling Kit-NH ₂ (manufactured by Dojindo Laboratories) is used, and the laminin adsorption rate is determined by fluorescence measurement.

In the present invention, the amount of laminin 511-E8 fragment adsorbed on the culture substrate is preferably 5 to 5000 ng/cm ² because it is suitable for increasing the proliferation and detachment of pluripotent stem cells. More preferably 10 to 1000 ng/cm ² , particularly preferably 15 to 500 ng/cm ² and most preferably 20 to 100 ng/cm ² . As the laminin 511-E8 fragment, commercially available products such as iMatrix-511 (manufactured by Nippi) can be used.

In the present invention, the material of the substrate coated with the layer of the temperature-responsive polymer is not particularly limited, but it is not limited to materials such as glass and polystyrene that are commonly used for cell culture, and can generally be shaped. such as polymeric compounds such as polycarbonate, polyethylene terephthalate, polyvinylidene fluoride, polytetrafluoroethylene, polyethylene, polypropylene, and polymethyl methacrylate, ceramics, and metals. From the viewpoint of facilitating culture operations, the material of the substrate preferably contains at least one of glass, polystyrene, polycarbonate, polyethylene terephthalate, polyvinylidene fluoride, polytetrafluoroethylene, polyethylene, and polypropylene, and glass, polystyrene, and polycarbonate. , polyethylene terephthalate, and polyethylene are more preferable, and polystyrene, polycarbonate, polyethylene terephthalate, and polyethylene are particularly preferable because they are suitable for increasing flexibility.

Also, the substrate preferably has pluripotent stem cell adhesiveness.

The shape of the substrate is not particularly limited, and may be a planar shape such as a plate or film, or may be fibers, porous particles, porous membranes, or hollow fibers. In addition, a container generally used for cell culture or the like (cell culture dish such as Petri dish, flask, plate, etc.) may also be used. From the viewpoint of easiness of culturing operation, it is preferably a planar shape such as a plate or film, or a flat porous membrane.

In the present invention, the base material is a porous base material, and the pore diameter of the porous base material is smaller than that of the pluripotent stem cells to be cultured, since this is suitable for enhancing the exfoliation of the pluripotent stem cells. Preferably. Detachability of pluripotent stem cells can be enhanced by using a porous substrate as the substrate. In addition, since the pore size of the porous substrate is smaller than that of the pluripotent stem cells, the adhesiveness and proliferative properties of the pluripotent stem cells can be enhanced. In addition, the pore size is more preferably 8 μm or less, particularly preferably 3 μm or less, most preferably 1 μm or less, because it is suitable for enhancing adhesion and proliferation of pluripotent stem cells. preferable. Furthermore, the pore diameter is more preferably 0.01 μm or more, particularly preferably 0.05 μm or more, particularly preferably 0.1 μm or more, because it is suitable for enhancing the detachment of potential stem cells. is most preferred. Here, in the present invention, the "pore diameter" of the porous substrate indicates the average value of the diameter of the pores of the porous substrate along the in-plane direction of the porous substrate. It can be calculated by measuring the diameter of pores at 20 or more points in the laser microscope image, scanning electron microscope image, and transmission electron microscope image, and calculating the average value.

In the present invention, the pore density of the pores of the porous substrate is 10 to 10 ¹⁰ /cm ² because it is suitable for enhancing pluripotent stem cell adhesion, proliferation and detachment. is preferred, 10 ³ to 10 ⁹ /cm ² is more preferred, 10 ⁵ to 10 ⁹ /cm ² is particularly preferred, and 10 ⁵ to 10 ⁷ /cm ² is most preferred. In addition, since it is suitable for increasing the transparency of the porous substrate and facilitating observation of cells with a microscope, the pore density of the pores of the porous substrate is 10 ⁷ /cm ² or less. more preferably 10 ⁶ /cm ² or less, particularly preferably 10 ⁵ /cm ² or less, most preferably 10 ⁴ /cm ² or less. Here, in the present invention, the "pore density" of the pores of the porous substrate indicates the number of pores present per substrate area of the porous substrate, and is a laser microscope image of the porous substrate. In a scanning electron microscope image and a transmission electron microscope image, it can be calculated by determining the number of pores in a square region having a side length of 200 times or more the pore diameter of the pores of the porous substrate. . In the present invention, the "substrate area" of the porous substrate indicates the surface area of one main surface of the porous substrate, assuming that the porous substrate has no pores.

In the present invention, the shape of the pores of the porous substrate is not particularly limited. A flat membrane is preferred. In addition, since it is suitable for enabling observation of pluripotent stem cells with a phase-contrast microscope, the pores possessed by the porous substrate preferably have a cylindrical shape, and the pores have an independent cylindrical shape. is more preferable. It is suitable for observing the shape of cells on this surface where the pores are cylindrical in shape.

The method of forming a layer of the temperature-responsive polymer on the substrate surface of the present invention includes (1) a method of coating the substrate with the temperature-responsive polymer through chemical bonding to form a layer, and (2) A method of coating and forming a layer by physical interaction can be performed alone or in combination. That is, (1) methods using chemical bonding include ultraviolet irradiation, electron beam irradiation, gamma ray irradiation, plasma treatment, corona treatment, and the like. Furthermore, when the temperature-responsive polymer and the substrate have suitable reactive functional groups, generally used organic reactions such as radical reactions, anionic reactions, and cationic reactions can be used. (2) As a method based on physical interaction, a matrix having good compatibility with the temperature-responsive polymer and good coatability is used as a medium, and coating, brush coating, dip coating, spin coating, bar coating, Various commonly known methods such as flow coating, spray coating, roll coating, air knife coating, blade coating, gravure coating, microgravure coating and slot die coating can be used.

In the present invention, the type of pluripotent stem cells is not particularly limited. cells or ntES cells) can be used, and induced pluripotent stem cells are particularly preferred. In addition, although the animal from which the pluripotent stem cells are derived is not particularly limited, it can be derived from mammals, for example. Examples of mammals include rodents (mice, rats, etc.) and primates (monkeys, humans, etc.), and may be laboratory animals or companion animals. In the present invention, it is preferably derived from primates, particularly preferably human.

The present invention also relates to a method for producing undifferentiated pluripotent stem cells using the culture substrate. The method is a method for producing pluripotent stem cells, which produces undifferentiated pluripotent stem cells through the following steps [1] to [3].
[1] Seeding pluripotent stem cells on the culture substrate.
[2] A step of culturing the pluripotent stem cells seeded on the culture substrate in a liquid at a temperature equal to or higher than the response temperature of the temperature-responsive polymer.
[3] A step of cooling the culture substrate to a temperature lower than the response temperature of the temperature-responsive polymer and exfoliating the pluripotent stem cells cultured in the liquid from the substrate.

The steps [1] to [3] in the production method of the present invention are described in detail below.

The [1] step in the method for producing pluripotent stem cells of the present invention is a step of seeding pluripotent stem cells onto the culture substrate using the culture substrate. In the present invention, "seeding cells" means that a medium in which cells are dispersed (hereinafter referred to as "cell suspension") is applied on a culture substrate, or injected into a culture substrate, etc. It shows contacting the cell suspension and the culture substrate. By using a substrate having a layer of a temperature-responsive polymer with a response temperature in the range of 0° C. to 50° C., pluripotent stem cells are exfoliated from the culture substrate by temperature change in the step [3] described later. can be done. If the culture substrate does not have a layer of the temperature-responsive polymer, the pluripotent stem cells cannot be detached from the substrate by temperature change in step [3]. In addition, since the culture substrate has a biological substance immobilized on a layer of a temperature-responsive polymer, pluripotent stem cells can be cultured in step [2] described later. Pluripotent stem cells cannot be cultured in step [2] if the culture substrate does not have the biological substance immobilized on the layer of the temperature-responsive polymer.

In steps [1] to [3], culture is performed under conditions effective for maintaining the undifferentiated state of the pluripotent stem cells. Conditions effective for maintaining undifferentiation are not particularly limited. For example, it is performed in the presence of a liquid medium. Examples of effective media for maintaining the undifferentiated state of pluripotent stem cells include insulin, transferrin, selenium, ascorbic acid, which are known factors for maintaining the undifferentiated state of pluripotent stem cells. A medium supplemented with one or more of sodium bicarbonate, basic fibroblast growth factor, transforming growth factor β (TGFβ), CCL2, activin, and 2-mercaptomethanol can be preferably used. Insulin, transferrin, selenium, ascorbic acid, sodium bicarbonate, basic fibroblast growth factor, transforming growth factor beta (TGFβ) are particularly suitable for maintaining the undifferentiated nature of pluripotent stem cells. It is more preferred to use a medium containing it, and most preferred to use a medium supplemented with basic fibroblast growth factor.

The type of medium to which the basic fibroblast growth factor is added is not particularly limited. manufactured by), 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. Made), MTESR1 (STEMCELL TECHNOLOGIES), TESR -E8 (STEMCELL TECHNOLOGIES), REPRONAIVE ((made by REPROCELL Corporation)), Reproxf (manufactured by REPROCELL Corporation) ), REPROFF (manufactured by REPROCELL Co., Ltd.), REPROFF2 ((stock) ) REPROCELL), NutriStem (manufactured by Biological Industries), iSTEM (manufactured by Takara Bio Inc.), GS2-M (manufactured by Takara Bio Inc.), hPSC Growth Medium DXF (manufactured by PromoCell Inc.), etc. can be mentioned. Primate ES Cell Medium (manufactured by REPROCELL Co., Ltd.), StemFit AK02N (manufactured by Ajinomoto Co., Inc.) or StemFit AK03 (manufactured by Ajinomoto Co., Inc.) are suitable for maintaining the undifferentiated state of pluripotent stem cells. is preferred, StemFit AK02N (manufactured by Ajinomoto Co.) or StemFit AK03 (manufactured by Ajinomoto Co.) is more preferred, and StemFit AK02N (manufactured by Ajinomoto Co.) is particularly preferred.

In the above step [1], the method for seeding pluripotent stem cells is not particularly limited, but for example, it can be carried out by injecting a cell suspension into the substrate. The cell density during seeding is not particularly limited, but is preferably 1.0×10 ² to 1.0×10 ⁶ cells/cm ² so that the cells can be maintained and proliferated. 5.0×10 ² to 5.0×10 ⁵ cells/cm ² are more preferred, 1.0×10 ³ to 2.0×10 ⁵ cells/cm ² are particularly preferred, and 1.2×10 ³ to 1 0×10 ⁵ cells/cm ² is most preferred.

As the medium used in the step [1], a Rho-binding kinase inhibitor is further added to the medium supplemented with the basic fibroblast growth factor, since it is suitable for maintaining the survival of pluripotent stem cells. It is preferable to use a culture medium prepared with Especially when using human pluripotent stem cells, when the cell density of human pluripotent stem cells is low, if a Rho-binding kinase inhibitor is added, the survival and maintenance of human pluripotent stem cells can be effective. Examples of Rho-binding kinase inhibitors include (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide·2HCl·H2O (manufactured by Wako Pure Chemical Industries, Ltd.). Y-27632), 1-(5-Isoquinolinesulfonyl) homopiperazine Hydrochloride (HA1077 manufactured by Wako Pure Chemical Industries, Ltd.) can be used. The concentration of the Rho-binding kinase inhibitor added to the medium is within a range that is effective for maintaining the survival of human pluripotent stem cells and does not affect the undifferentiated state of human pluripotent stem cells, It is preferably 1 μM to 50 μM, more preferably 3 μM to 20 μM, even more preferably 5 μM to 15 μM, most preferably 8 μM to 12 μM.

Shortly after starting the step [1], the pluripotent stem cells begin to adhere to the culture substrate.

In step [2] in the method for producing pluripotent stem cells of the present invention, the seeded pluripotent stem cells are cultured at a temperature equal to or higher than the response temperature of the temperature-responsive polymer. Pluripotent stem cells can be grown when the culture temperature is equal to or higher than the response temperature of the temperature-responsive polymer. Pluripotent stem cells cannot be grown when the culture temperature is lower than the response temperature of the temperature-responsive polymer. The culture temperature is preferably 30°C to 42°C, more preferably 32°C to 40°C, particularly preferably 36°C to 38°C, and most preferably 37°C, because it is suitable for cell growth ability, physiological activity, and function maintenance. is.

It is preferable to perform the first medium exchange 22 to 26 hours after starting the step [2]. After 48 to 72 hours, the medium is replaced for the second time, and thereafter, preferably, the medium is replaced every 24 to 48 hours. During this time, the pluripotent stem cells proliferate and form cell clusters called colonies. Cultivation is continued until the size of the colonies reaches about 1 mm, and then the process proceeds to step [3].

In step [3] in the method for producing pluripotent stem cells of the present invention, the culture substrate in which the pluripotent stem cells have been cultured is cooled to a temperature lower than the response temperature of the temperature-responsive polymer, and the cultured pluripotent stem cells are The stem cells are detached from the culture substrate. In order to exfoliate the pluripotent stem cells in a short time and reduce damage due to cooling, the cooling temperature is preferably 0 to 30°C, more preferably 3 to 25°C, and still more preferably 5 to 30°C. 20°C. In order to reduce damage to cells, the cooling time is preferably 120 minutes or less, more preferably 90 minutes or less, particularly preferably 75 minutes or less, and most preferably 60 minutes or less.

The cooling method of the culture substrate in the step [3] is not particularly limited, but for example, a method of cooling the culture substrate by placing it in a refrigerator, a method of placing the culture substrate on a cool plate and cooling it, A method of exchanging with a cooled medium or buffer solution and allowing it to stand for a predetermined period of time can be used.

In the above step [3], in order to detach the pluripotent stem cells in a short time and further reduce damage due to cooling, a step of generating convection in the liquid containing the cultured pluripotent stem cells during cooling. may contain The method for generating convection is not particularly limited. Examples include a method of applying physical vibration to the substrate and a method of generating natural convection such as Marangoni convection by applying a temperature difference.

When culturing a large amount of cells, the culture substrate of the present invention is suitable for simplifying the step of detaching the cells and increasing the productivity of the cells. Therefore, it is preferable that the cells be detached with a weak external stimulus. , cooling and pipetting, cooling and tapping are more preferred, and cooling and tapping are particularly preferred. It is most preferable to peel only by cooling. Here, the term "pipetting" as used herein refers to an operation that causes convection in the culture environment by repeatedly aspirating and discharging the culture medium using an instrument such as a Pipetman. Further, "tapping" refers to an operation that vibrates the culture environment by, for example, tapping the culture vessel.

Hereinafter, the present invention will be described in detail with reference to embodiments for carrying out the present invention. Moreover, it can be modified appropriately within the scope of the gist of the present invention. Commercially available reagents were used unless otherwise specified.
<Polymer composition>
Proton nuclear magnetic resonance spectroscopy ( ¹ H-NMR) spectrum analysis using a nuclear magnetic resonance spectrometer (manufactured by JEOL Ltd., trade name JNM-GSX400), or nuclear magnetic resonance spectroscopy (manufactured by Bruker, trade name AVANCEIIIHD500). It was obtained from carbon nuclear magnetic resonance spectroscopy ( ¹³ C-NMR) spectroscopy using .
<Molecular weight and molecular weight distribution of the polymer>
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 uses HLC-8320GPC manufactured by Tosoh Corporation, the column uses two TSKgel Super AWM-H manufactured by Tosoh Corporation, the column temperature is set to 40 ° C., and the eluent contains 10 mM sodium trifluoroacetate. Measured using 1,1,1,3,3,3-hexafluoro-2-isopropanol or N,N-dimethylformamide containing 10 mM lithium bromide. A measurement sample was prepared and measured at 1.0 mg/mL. Polymethyl methacrylate (manufactured by Polymer Laboratories Ltd.) with a known molecular weight was used for the molecular weight calibration curve.
<Surface structure>
Observation of the surface structure of the base material coated with the temperature-responsive polymer at the incubation temperature was performed using an underwater AFM device (SPM-9600 manufactured by Shimadzu Corporation). The cantilever was measured using BL-AC40TS-C2 (manufactured by Olympus Corporation).

Example 1
[Synthesis of block copolymer]
In a test tube, 1.50 g (5.1 mmol) of 2-methacryloyloxyethylphosphorylcholine, 43 mg (106 μmol) of 4-cyano-4-[(dodecylsulfonylthiocarbonyl)sulfonyl]pentanoic acid as a RAFT agent, and azobisiso 1.7 mg (10 μmol) of butyronitrile was added and dissolved in 10.2 mL of a mixed solution of 1,4-dioxane/ethanol=1:1, degassed by nitrogen bubbling for 30 minutes, and reacted at 65° C. for 18 hours. After the reaction, the reaction solution was poured into 200 mL of a mixed solution of acetone:methanol=20:1, and the precipitated yellow solid was filtered and dried under reduced pressure for one day to obtain a polymer of 2-methacryloyloxyethylphosphorylcholine.

1.0 g of the above 2-methacryloyloxyethylphosphorylcholine polymer, 2.40 g (16.9 mmol) of n-butyl methacrylate, and 2.5 mg (15 μmol) of azobisisobutyronitrile were added to a test tube, and 1,4-dioxane/ It was dissolved in 17 mL of ethanol=1:1 mixed solution. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 65° C. for 24 hours. After the reaction, the reaction solution was poured into 300 mL of hexane, and the precipitated pale yellow solid was filtered and dried under reduced pressure for one day. -butyl methacrylate diblock polymer).

0.5 g of the 2-methacryloyloxyethylphosphorylcholine/n-butyl methacrylate diblock polymer, 0.62 g (5.5 mmol) of N-isopropylacrylamide, and 0.2 mg (1 μmol) of azobisisobutyronitrile were added to the test tube. , 1,4-dioxane/ethanol=1:1 mixed solution 5.5 mL. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 65° C. for 24 hours. After the reaction, the reaction solution was poured into 200 mL of hexane, and the precipitated white solid was filtered and dried under reduced pressure for one day to obtain a triblock copolymer.

Table 1 shows the composition, Mn and Mw/Mn of the obtained triblock copolymer.
[Preparation of base material coated with block copolymer]
0.01 g of the block copolymer was dissolved in 4.99 g of ethanol to prepare a 0.2 wt % ethanol solution of the block copolymer. Furthermore, 1 mL of 0.2 wt % ethanol solution and 3 mL of ethanol were mixed to prepare a 0.05 wt % ethanol solution. 0.42 mL of this solution was added to each well of a 6-well plate for cell culture (manufactured by Corning Incorporated, material: polystyrene) and dried under reduced pressure for 2 hours. Furthermore, it was immersed in pure water for 24 hours and washed to prepare a base material coated with a block copolymer.
[LCST measurement of block segment (A)]
4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid 0.40 g (0.1 mmol), N-isopropylacrylamide 2.26 g (20 mmol) and azobis(isobutyronitrile) 33 mg (0.1 mmol). 2 mmol) was dissolved in 20 mL of dioxane. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 70° C. for 24 hours. After completion of the reaction, the reaction solvent was distilled off under reduced pressure using a rotary evaporator to concentrate the reaction solution. The concentrate was poured into 250 mL of hexane, and the precipitated white solid was collected and dried under reduced pressure to obtain an N-isopropylacrylamide polymer. A 0.6 wt % aqueous solution was prepared by dissolving the N-isopropylacrylamide polymer in pure water. This solution is placed in a quartz cell with an optical path length of 1 cm, and the transmittance of light at a wavelength of 500 nm is measured with a spectrophotometer (UH-5300, manufactured by Hitachi High-Technologies Co., Ltd.) while increasing the temperature at a rate of 1 ° C./min. bottom. When the LCST was obtained by the midpoint method, the LCST was 32°C.
[Measurement of response temperature of block copolymer]
The bubble contact angle (θ) (°) of the substrate coated with the block copolymer was measured in water at 37°C, 20°C and 10°C. (180-θ) (°) was calculated. As for θ, the contact angle of 3 μL of air bubbles in water was measured using a contact angle meter DM300 manufactured by Kyowa Interface Science Co., Ltd.

Table 2 shows the water contact angles at 37°C, 20°C and 10°C. The contact angle of water at 20°C is lower than the contact angle of water at 37°C, and the base material coated with the block copolymer exhibits temperature responsiveness, and the response temperature is in the range of 20 to 37°C. I found out. Further, the water contact angle was similarly measured at intervals of 5°C in the range of 20°C to 45°C, and the response temperature was determined by the midpoint method.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Human iPS cells 201B7 strain were seeded at a density of 1300 cells/cm ² on the substrate coated with the block copolymer, and cultured at 37° C. in a CO ₂ concentration of 5%. 0.2 mL/cm ² of StemFit AK02N (manufactured by Ajinomoto Co., Inc.) was added to the medium. In addition, until 24 hours after cell seeding, Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) and iMatrix-511 solution (manufactured by Nippi Co., Ltd.) (2.5 μL / mL) were added to the medium. was cultured using a medium supplemented with

24 hours, 96 hours and 144 hours after seeding the cells, the state of the cells was observed under a phase-contrast microscope. After changing the medium 144 hours after seeding the cells, (1) when the substrate was cooled to 4 ° C., (2) when the substrate was cooled to 4 ° C. and tapped 20 times, (3) the substrate was cooled to 4° C. and pipetting was performed 10 times, and detachment of cells was confirmed. Under the conditions of (3), almost all the cells were detached and collected as aggregates.

Example 2
[Synthesis of block copolymer]
In a test tube, 1.50 g (5.1 mmol) of 2-methacryloyloxyethylphosphorylcholine, 25.3 mg (63 μmol) of 4-cyano-4-[(dodecylsulfonylthiocarbonyl)sulfonyl]pentanoic acid as an RAFT agent, and an initiator. 1 mg (3 μmol) of azobisisobutyronitrile was added and dissolved in 10.2 mL of a mixed solution of 1,4-dioxane/ethanol=1:1. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 65° C. for 18 hours. After the reaction, the reaction solution was poured into 200 mL of a mixed solution of acetone:methanol=20:1, and the precipitated yellow solid was filtered and dried under reduced pressure for one day to obtain a polymer of 2-methacryloyloxyethylphosphorylcholine.

1.50 g of the above 2-methacryloyloxyethylphosphorylcholine polymer, 1.71 g (12.0 mmol) of n-butyl methacrylate, and 2 mg (13 μmol) of azobisisobutyronitrile were added to a test tube, and 1,4-dioxane/ethanol = It was dissolved in 12 mL of a 1:1 mixed solution. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 65° C. for 24 hours. After the reaction, the reaction solution was poured into 300 mL of hexane, and the precipitated pale yellow solid was filtered and dried under reduced pressure for one day to obtain a 2-methacryloyloxyethylphosphorylcholine/n-butyl methacrylate diblock polymer.

A test tube was charged with 0.75 g of the 2-methacryloyloxyethylphosphorylcholine/n-butyl methacrylate diblock copolymer, 0.93 g (8.2 mmol) of N-isopropylacrylamide, and 0.3 mg (2 μmol) of azobisisobutyronitrile. In addition, it was dissolved in 8.2 mL of a mixed solution of 1,4-dioxane/ethanol=1:1. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 65° C. for 24 hours. After the reaction, the reaction solution was poured into 200 mL of hexane, and the precipitated white solid was filtered and dried under reduced pressure for one day to obtain a triblock copolymer.

Table 1 shows the composition, Mn and Mw/Mn of the obtained triblock copolymer.
[Preparation of base material coated with block copolymer]
It was produced in the same manner as in Example 1 [Preparation of base material coated with block copolymer] except that the block copolymer was used.
[Measurement of response temperature of block copolymer]
Measurement was performed in the same manner as in Example 1 [Wettability of block copolymer-coated substrate] except that the block copolymer-coated substrate was used.

Table 2 shows the water contact angles at 37°C, 20°C and 10°C. The contact angle of water at 20°C is lower than the contact angle of water at 37°C, and the base material coated with the block copolymer exhibits temperature responsiveness, and the response temperature is in the range of 20 to 37°C. I found out.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Cultivation was performed according to Example 1 [Pluripotent stem cell culture evaluation and exfoliation evaluation] except that the base material coated with the block copolymer was used.

24 hours, 96 hours and 144 hours after seeding the cells, the state of the cells was observed under a phase-contrast microscope. After changing the medium 144 hours after seeding the cells, (1) when the substrate was cooled to 4 ° C., (2) when the substrate was cooled to 4 ° C. and tapped 20 times, (3) the substrate was cooled to 4° C. and pipetting was performed 10 times, and detachment of cells was confirmed. Under the conditions of (3), almost all the cells were detached and collected as aggregates.

Example 3
[Synthesis of block copolymer]
3.00 g (10.2 mmol) of 2-methacryloyloxyethylphosphorylcholine, 3.37 g (23.7 mmol) of n-butyl methacrylate, and 4-cyano-4-[(dodecylsulfonylthiocarbonyl)sulfonyl] as a RAFT agent were placed in a test tube. 46 mg (113 μmol) of pentanoic acid and 4 mg (23 μmol) of azobisisobutyronitrile were added and dissolved in 30 mL of a mixed solution of 1,4-dioxane/ethanol=1:1. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 65° C. for 21 hours. After the reaction, the reaction solution was poured into 500 mL of a mixed solution of acetone:methanol=20:1, and the precipitated pale yellow solid was filtered and dried under reduced pressure for one day to obtain a 2-methacryloyloxyethylphosphorylcholine/n-butyl methacrylate random copolymer. got

1.50 g of the above 2-methacryloyloxyethylphosphorylcholine/n-butyl methacrylate random copolymer, 1.85 g (16.4 mmol) of N-isopropylacrylamide, and 0.5 mg (3 μmol) of azobisisobutyronitrile were added to the test tube. , 1,4-dioxane/ethanol=1:1 mixed solution 12.6 mL. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 65° C. for 24 hours. After the reaction, the reaction solution was poured into 500 mL of hexane, and the precipitated white solid was filtered and dried under reduced pressure for one day to obtain a diblock copolymer.

Table 1 shows the composition, Mn and Mw/Mn of the resulting diblock copolymer.
[Preparation of base material coated with block copolymer]
It was produced in the same manner as in Example 1 [Preparation of base material coated with block copolymer] except that the block copolymer was used.
[Measurement of response temperature of block copolymer]
Measurement was performed in the same manner as in Example 1 [Wettability of block copolymer-coated substrate] except that the block copolymer-coated substrate was used.

Table 2 shows the water contact angles at 37°C, 20°C and 10°C. The contact angle of water at 20°C is lower than the contact angle of water at 37°C, and the base material coated with the block copolymer exhibits temperature responsiveness, and the response temperature is in the range of 20 to 37°C. I found out.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Cultivation was performed according to Example 1 [Pluripotent stem cell culture evaluation and exfoliation evaluation] except that the base material coated with the block copolymer was used.

24 hours, 96 hours and 144 hours after seeding the cells, the state of the cells was observed under a phase-contrast microscope. After changing the medium 144 hours after seeding the cells, (1) when the substrate was cooled to 4 ° C., (2) when the substrate was cooled to 4 ° C. and tapped 20 times, (3) the substrate was cooled to 4° C. and pipetting was performed 10 times, and detachment of cells was confirmed. Under the conditions of (3), almost all the cells were detached and collected as aggregates.

Example 4
[Synthesis of block copolymer]
0.57 g (1.8 mmol) of propargyl ester of 4-cyanopentanoic acid dithiobenzoate, 12.80 g (90 mmol) of n-butyl methacrylate, and 60 mg of azobis(isobutyronitrile) were placed in a test tube equipped with a three-way stopcock. 0.36 mmol) was added and dissolved in 90 mL of a mixed solution of 1,4-dioxane/ethanol=1:1. The test tube was immersed in liquid nitrogen to freeze, degassed under reduced pressure with a vacuum pump, and returned to room temperature. This operation was repeated three times, and after removing dissolved oxygen in the test tube, the mixture was reacted at 65° C. for 24 hours. After completion of the reaction, the reaction solvent was distilled off under reduced pressure using a rotary evaporator to concentrate the reaction solution. The concentrated liquid was poured into 300 mL of methanol, and the precipitated red oily substance was collected and dried under reduced pressure to obtain an n-butyl methacrylate polymer having a terminal alkyne.

In a test tube equipped with a three-way cock, 5.95 g (0.7 mmol) of the n-butyl methacrylate polymer having a terminal alkyne, 15.84 g (140 mmol) of N-isopropylacrylamide, 11.5 mg of azobisisobutyronitrile ( 0.07 mmol) was added and dissolved in 140 mL of 1,4-dioxane. The test tube was immersed in liquid nitrogen to freeze, degassed under reduced pressure with a vacuum pump, and returned to room temperature. This operation was repeated three times, and after removing dissolved oxygen in the test tube, reaction was carried out at 65° C. for 43 hours. After completion of the reaction, the reaction solvent was distilled off under reduced pressure using a rotary evaporator, and the reaction solution was concentrated. The concentrated liquid was poured into 1000 mL of hexane, and the precipitated pale red solid was collected and dried under reduced pressure to obtain an N-isopropylacrylamide/n-butyl methacrylate diblock copolymer having a terminal alkyne.

Separately, 0.20 g (0.57 mmol) of 3-azidopropyl ester of 4-cyanopentanoic acid dithiobenzoate and 12.01 g (40 mmol) of polyethylene glycol methacrylate (manufactured by Aldrich, Mn=300) were placed in a test tube equipped with a three-way stopcock. , AIBN 18.8 mg (0.11 mmol) was added, and 1,4-dioxane 28 mL was added to dissolve. The test tube was immersed in liquid nitrogen to freeze, degassed under reduced pressure with a vacuum pump, and returned to room temperature. This operation was repeated three times to remove dissolved oxygen in the test tube. The test tube was heated to 65°C and polymerized at 65°C for 2.5 hours. After completion of the reaction, the reaction solvent was distilled off under reduced pressure using a rotary evaporator, and the reaction solution was concentrated. The concentrate was poured into 500 ml of hexane, and a red oily matter adhering to the bottom was recovered. After washing twice with 300 mL of hexane, the red oil obtained was vacuum dried to obtain a polyethylene glycol methacrylate polymer block having a terminal azide.

Subsequently, 0.50 g of the N-isopropylacrylamide/n-butyl methacrylate diblock copolymer having a terminal alkyne and 0.94 g of the polyethylene glycol methacrylate polymer having a terminal azide were added to a test tube equipped with a three-way cock, Nitrogen substitution was performed. 9 mL of nitrogen-bubbled DMF was added and dissolved. A separately prepared solution of 38 mg of copper (I) bromide, 84 mg of 2,2'-bipyridyl, and 1 mL of DMF was added to the test tube under a nitrogen stream and allowed to react at room temperature for 48 hours. After completion of the reaction, the three-way cock was removed and exposed to air to deactivate the copper catalyst. The reaction solution was passed through a column packed with activated alumina to remove the copper catalyst, and the solution was concentrated using a rotary evaporator. The concentrate was slowly poured into 50 mL of pure water, and the precipitated solid content was collected by centrifugation (3000 rpm×3 minutes). 2 ml of methanol was dissolved in the obtained solid content, and slowly poured into 50 ml of pure water again, and the precipitated solid content was recovered by centrifugation (3000 rpm×3 minutes). By vacuum drying, a triblock copolymer consisting of polyethylene glycol methacrylate polymer, n-butyl methacrylate polymer and N-isopropylacrylamide polymer blocks was obtained.

Table 1 shows the composition, Mn and Mw/Mn of the obtained block copolymer.
[Preparation of base material coated with block copolymer]
It was produced in the same manner as in Example 1 [Preparation of base material coated with block copolymer] except that the block copolymer was used.
[Measurement of response temperature of block copolymer]
Measurement was performed in the same manner as in Example 1 [Wettability of block copolymer-coated substrate] except that the block copolymer-coated substrate was used.

Table 2 shows the water contact angles at 37°C, 20°C and 10°C. The contact angle of water at 20°C is lower than the contact angle of water at 37°C, and the base material coated with the block copolymer exhibits temperature responsiveness, and the response temperature is in the range of 20 to 37°C. I found out. Further, the water contact angle was similarly measured at intervals of 5°C in the range of 20°C to 45°C, and the response temperature was determined by the midpoint method.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Cultivation was performed according to Example 1 [Pluripotent stem cell culture evaluation and exfoliation evaluation] except that the base material coated with the block copolymer was used.

24 hours, 96 hours and 144 hours after seeding the cells, the state of the cells was observed under a phase-contrast microscope. After medium change 144 hours after cell seeding, 0.25 mM ethylenediaminetetraacetic acid solution was added, (1) when the substrate was cooled to 4° C., (2) when the substrate was cooled to 4° C. and repeated 20 times. Detachment of cells was confirmed in each of the case of tapping and (3) the case of cooling the substrate to 4° C. and pipetting 10 times. Under the condition (3), almost all cells were detached and recovered as single cells.

Example 5
[Synthesis of block copolymer]
Synthesis was performed in the same manner as in Example 4 [Synthesis of block copolymer] except that 5.20 g (40 mmol) of 2-methoxyethyl acrylate was used instead of 12.01 g of polyethylene glycol methacrylate, and polymerization was performed at 65°C for 9 hours. was performed to synthesize a diblock copolymer.

Table 1 shows the composition, Mn and Mw/Mn of the resulting diblock copolymer.
[Preparation of base material coated with block copolymer]
It was produced in the same manner as in Example 1 [Preparation of base material coated with block copolymer] except that the block copolymer was used.
[Measurement of response temperature of block copolymer]
Measurement was performed in the same manner as in Example 1 [Wettability of block copolymer-coated substrate] except that the block copolymer-coated substrate was used.

Table 2 shows the water contact angles at 37°C, 20°C and 10°C. The contact angle of water at 20°C is lower than the contact angle of water at 37°C, and the base material coated with the block copolymer exhibits temperature responsiveness, and the response temperature is in the range of 20 to 37°C. I found out. Further, the water contact angle was similarly measured at intervals of 5°C in the range of 20°C to 45°C, and the response temperature was determined by the midpoint method.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Cultivation was performed according to Example 1 [Pluripotent stem cell culture evaluation and exfoliation evaluation] except that the base material coated with the block copolymer was used.

24 hours, 96 hours and 144 hours after seeding the cells, the state of the cells was observed under a phase-contrast microscope. After medium change 144 hours after cell seeding, 0.25 mM ethylenediaminetetraacetic acid solution was added, (1) when the substrate was cooled to 4° C., (2) when the substrate was cooled to 4° C. and repeated 20 times. Detachment of cells was confirmed in each of the case of tapping and (3) the case of cooling the substrate to 4° C. and pipetting 10 times. Under the condition (3), almost all cells were detached and recovered as single cells.

Example 6
[Synthesis of block copolymer]
In a test tube, 36 mg (90 μmol) of 4-cyano-4-[(dodecylsulfonylthiocarbonyl)sulfonyl]pentanoic acid, 1.28 g (9 mmol) of n-butyl methacrylate, 3 mg (18 μmol) of azobis(isobutyronitrile) was added and dissolved in 9 mL of a mixed solution of 1,4-dioxane/ethanol=1:1. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 65° C. for 24 hours. After completion of the reaction, the reaction solvent was distilled off under reduced pressure using a rotary evaporator to concentrate the reaction solution. The concentrate was poured into 250 mL of methanol, and the precipitated yellow oily substance was collected and dried under reduced pressure to obtain an n-butyl methacrylate polymer.

0.8 g (1.0 mmol) of the n-butyl methacrylate polymer, 1.0 g (8.8 mmol) of N-isopropylacrylamide, and 1.6 mg (9.9 μmol) of azobisisobutyronitrile were added to a test tube, It was dissolved in 9 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 65° C. for 15 hours. After completion of the reaction, the reaction solvent was distilled off under reduced pressure using a rotary evaporator, and the reaction solution was concentrated. The concentrate was poured into 200 mL of hexane, and the precipitated pale yellow solid was recovered and dried under reduced pressure to obtain a diblock copolymer of N-isopropylacrylamide and n-butyl methacrylate.
[Preparation of base material coated with block copolymer]
It was produced in the same manner as in Example 1 [Preparation of base material coated with block copolymer] except that the block copolymer was used.
[Measurement of response temperature of block copolymer]
Measurement was performed in the same manner as in Example 1 [Wettability of block copolymer-coated substrate] except that the block copolymer-coated substrate was used.

Table 2 shows the water contact angles at 37°C, 20°C and 10°C. The contact angle of water at 20°C is lower than the contact angle of water at 37°C, and the base material coated with the block copolymer exhibits temperature responsiveness, and the response temperature is in the range of 20 to 37°C. I found out. Further, the contact angle of water was similarly measured at intervals of 5°C in the range of 20 to 45°C, and the response temperature was determined by the midpoint method, which was 32°C.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
0.12 mL/cm ² of iMatrix-511 solution (manufactured by Nippi Co., Ltd.) (diluted to about 4.2 μg/mL with PBS(-)) was added to the substrate coated with the block copolymer, and the temperature was maintained at 37°C. , and left for 1 hour in an environment with a CO ₂ concentration of 5%. After that, the iMatrix-511 solution was removed, and human iPS cell line 201B7 was seeded at a density of 1300 cells/cm ² and cultured at 37° C. under a CO ₂ concentration of 5%. 0.2 mL/cm ² of StemFit AK02N (manufactured by Ajinomoto Co., Inc.) was added to the medium. In addition, Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) was added to the medium and cultured until 24 hours after seeding the cells.

Cultivation was performed according to Example 1 [Pluripotent stem cell culture evaluation and exfoliation evaluation] except that the base material coated with the block copolymer was used.

24 hours, 96 hours and 144 hours after seeding the cells, the state of the cells was observed under a phase-contrast microscope. After medium change 144 hours after cell seeding, 0.25 mM ethylenediaminetetraacetic acid solution was added, (1) when the substrate was cooled to 4° C., (2) when the substrate was cooled to 4° C. and repeated 20 times. Detachment of cells was confirmed in each of the case of tapping and (3) the case of cooling the substrate to 4° C. and pipetting 10 times. Under the condition (3), almost all cells were detached and recovered as single cells.

Example 7
[Synthesis of block copolymer]
In a test tube, 0.40 g (0.1 mmol) of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 7.11 g (50 mmol) of n-butyl methacrylate, azobis(isobutyronitrile) 33 mg (0.2 mmol) was added and dissolved in 50 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 70° C. for 24 hours. After completion of the reaction, the reaction solvent was distilled off under reduced pressure using a rotary evaporator to concentrate the reaction solution. The concentrate was poured into 250 mL of methanol, and the precipitated yellow oily substance was collected and dried under reduced pressure to obtain an n-butyl methacrylate polymer.

0.9 g (0.3 mmol) of the n-butyl methacrylate polymer, 2.65 g (23.5 mmol) of N-isopropylacrylamide, and 5 mg (0.03 mmol) of azobisisobutyronitrile were added to a test tube. It was dissolved in 15 mL of 4-dioxane. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 65° C. for 17 hours. After completion of the reaction, the reaction solvent was diluted with acetone and poured into 500 mL of hexane, and the precipitated pale yellow solid was collected and dried under reduced pressure to obtain a diblock copolymer of N-isopropylacrylamide and n-butyl methacrylate.
[Preparation of base material coated with block copolymer]
The block copolymer was dissolved in ethanol to obtain a 0.5 wt % solution. 50 μL of this solution was dropped onto a dish having a diameter of 3.5 cm (manufactured by Corning Incorporated, material: polystyrene) and spin-coated at 1000 rpm for 60 seconds. Dried for 1 hour at room temperature. Furthermore, it was immersed in pure water for 24 hours and washed to prepare a base material coated with a block copolymer.
[Measurement of response temperature of block copolymer]
Measurement was performed in the same manner as in Example 1 [Wettability of block copolymer-coated substrate] except that the block copolymer-coated substrate was used.

Table 2 shows the water contact angles at 37°C, 20°C and 10°C. The contact angle of water at 20°C is lower than the contact angle of water at 37°C, and the base material coated with the block copolymer exhibits temperature responsiveness, and the response temperature is in the range of 20 to 37°C. I found out. Further, the water contact angle was similarly measured at intervals of 5°C in the range of 20°C to 45°C, and the response temperature was determined by the midpoint method.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Cultivation was performed according to Example 1 [Pluripotent stem cell culture evaluation and exfoliation evaluation] except that the base material coated with the block copolymer was used.

24 hours, 96 hours and 144 hours after seeding the cells, the state of the cells was observed under a phase-contrast microscope. After medium change 144 hours after cell seeding, 0.25 mM ethylenediaminetetraacetic acid solution was added, (1) when the substrate was cooled to 4° C., (2) when the substrate was cooled to 4° C. and repeated 20 times. Detachment of cells was confirmed in each of the case of tapping and (3) the case of cooling the substrate to 4° C. and pipetting 10 times. Under the condition (3), almost all cells were detached and recovered as single cells.

Example 8
[Synthesis of block copolymer]
In a test tube, 0.40 g (0.1 mmol) of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 7.11 g (50 mmol) of n-butyl methacrylate, azobis(isobutyronitrile) 33 mg (0.2 mmol) was added and dissolved in 50 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 70° C. for 24 hours. After completion of the reaction, the reaction solvent was distilled off under reduced pressure using a rotary evaporator to concentrate the reaction solution. The concentrate was poured into 250 mL of methanol, and the precipitated yellow oily substance was collected and dried under reduced pressure to obtain an n-butyl methacrylate polymer.

In a test tube, 0.9 g (0.3 mmol) of the n-butyl methacrylate polymer, 8.14 g (72 mmol) of N-isopropylacrylamide, and 5 mg (0.03 mmol) of azobisisobutyronitrile were added. Dissolved in 15 mL of dioxane. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 65° C. for 17 hours. After completion of the reaction, the reaction solvent was diluted with acetone and poured into 500 mL of hexane, and the precipitated pale yellow solid was collected and dried under reduced pressure to obtain a diblock copolymer of N-isopropylacrylamide and n-butyl methacrylate.
[Preparation of base material coated with block copolymer]
It was produced in the same manner as in Example 7 [Preparation of base material coated with block copolymer] except that the block copolymer was used.
[Measurement of response temperature of block copolymer]
Measurement was performed in the same manner as in Example 7 [Wettability of block copolymer-coated substrate] except that the block copolymer-coated substrate was used.

Table 2 shows the water contact angles at 37°C, 20°C and 10°C. The contact angle of water at 20°C is lower than the contact angle of water at 37°C, and the base material coated with the block copolymer exhibits temperature responsiveness, and the response temperature is in the range of 20 to 37°C. I found out. Further, the water contact angle was similarly measured at intervals of 5°C in the range of 20°C to 45°C, and the response temperature was determined by the midpoint method.
[Structure of substrate surface coated with block copolymer]
FIG. 4 shows an underwater AFM image at 37° C. of the substrate surface coated with the block copolymer. The block copolymer layer formed a phase-separated structure, and there were areas where the amount of the block copolymer was large and areas where the amount of the block copolymer was small.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Cultivation was performed according to Example 1 [Pluripotent stem cell culture evaluation and exfoliation evaluation] except that the base material coated with the block copolymer was used.

24 hours, 96 hours and 144 hours after seeding the cells, the state of the cells was observed under a phase-contrast microscope. After changing the medium 144 hours after seeding the cells, (1) when the substrate was cooled to 4 ° C., (2) when the substrate was cooled to 4 ° C. and tapped 20 times, (3) the substrate was cooled to 4° C. and pipetting was performed 10 times, and detachment of cells was confirmed. Under the conditions (2) and (3), almost all cells were detached and collected as aggregates.

Example 9
[Synthesis of block copolymer]
95 mg of propargyl ester of 4-cyanopentanoic acid dithiobenzoate, 6.8 g of N-isopropylacrylamide, and 8 mg of AIBN were added to a test tube equipped with a three-way cock and dissolved in 20 ml of 1,4-dioxane. After bubbling with nitrogen for 30 minutes, the mixture was stirred at 70° C. for 24 hours for polymerization. After the polymerization was completed, the reaction solution was poured into 500 mL of hexane, and the precipitated pink solid was collected by filtration and dried to obtain an N-isopropylacrylamide polymer having a terminal alkyne.

Separately, 0.18 g of 3-azidopropyl ester of 4-cyanopentanoic acid dithiobenzoate, 5.3 g of N-(4-hydroxyphenyl)methacrylamide, and 26 mg of AIBN were added to a test tube equipped with a three-way stopcock, and dissolved in 20 mL of DMF. bottom. After bubbling with nitrogen for 30 minutes, the mixture was stirred at 70° C. for 24 hours for polymerization. After the polymerization was completed, the reaction solution was poured into 500 mL of pure water, and the precipitated pink solid was collected by filtration and dried to obtain an N-(4-hydroxyphenyl)methacrylamide polymer having a terminal azide.

Subsequently, 0.65 g of the N-isopropylacrylamide polymer having a terminal alkyne and 0.30 g of the N-(4-hydroxyphenyl)methacrylamide polymer having a terminal azide were added to a test tube equipped with a three-way cock, Nitrogen substitution was performed. 9 mL of nitrogen-bubbled DMF was added and dissolved. A separately prepared solution of 14 mg of copper (I) bromide, 31 mg of 2,2′-bipyridyl, and 1 mL of DMF was added to the test tube under a nitrogen stream and allowed to react at room temperature for 48 hours. After completion of the reaction, the three-way cock was removed and air was exposed to deactivate the copper catalyst. The reaction solution was passed through a column packed with activated alumina to remove the copper catalyst, and the solution was concentrated using a rotary evaporator. The concentrate was dissolved in a methanol/acetone mixed solvent, hexane was added little by little, and the precipitated polymer was recovered. Reprecipitation purification was repeated three times to obtain a diblock copolymer of N-isopropylacrylamide and N-(4-hydroxyphenyl)methacrylamide.
[Preparation of base material coated with block copolymer]
It was produced in the same manner as in Example 7 [Preparation of base material coated with block copolymer] except that the block copolymer was used.
[Measurement of response temperature of block copolymer]
Measurement was performed in the same manner as in Example 1 [Wettability of block copolymer-coated substrate] except that the block copolymer-coated substrate was used.

Table 2 shows the water contact angles at 37°C, 20°C and 10°C. The contact angle of water at 20°C is lower than the contact angle of water at 37°C, and the base material coated with the block copolymer exhibits temperature responsiveness, and the response temperature is in the range of 20 to 37°C. I found out.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Cultivation was performed according to Example 1 [Pluripotent stem cell culture evaluation and exfoliation evaluation] except that the base material coated with the block copolymer was used.

24 hours, 96 hours and 144 hours after seeding the cells, the state of the cells was observed under a phase-contrast microscope. After medium change 144 hours after cell seeding, 0.25 mM ethylenediaminetetraacetic acid solution was added, (1) when the substrate was cooled to 4° C., (2) when the substrate was cooled to 4° C. and repeated 20 times. Detachment of cells was confirmed in each of the case of tapping and (3) the case of cooling the substrate to 4° C. and pipetting 10 times. Under the condition (3), almost all cells were detached and recovered as single cells.

Example 10
[Synthesis of block copolymer]
In a test tube, 0.40 g (0.1 mmol) of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 7.11 g (50 mmol) of n-butyl methacrylate, azobis(isobutyronitrile) 33 mg (0.2 mmol) was added and dissolved in 50 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 70° C. for 24 hours. After completion of the reaction, the reaction solvent was distilled off under reduced pressure using a rotary evaporator to concentrate the reaction solution. The concentrate was poured into 250 mL of methanol, and the precipitated yellow oily substance was collected and dried under reduced pressure to obtain an n-butyl methacrylate polymer.

0.9 g (0.3 mmol) of the n-butyl methacrylate polymer, 8.14 g (72 mmol) of Nn-propylacrylamide, and 5 mg (0.03 mmol) of azobisisobutyronitrile were added to a test tube. It was dissolved in 15 mL of 4-dioxane. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 65° C. for 17 hours. After completion of the reaction, the reaction solvent was diluted with acetone and poured into 500 mL of hexane, and the precipitated pale yellow solid was recovered and dried under reduced pressure to obtain a diblock copolymer of Nn-propylacrylamide and n-butyl methacrylate. .
[Preparation of base material coated with block copolymer]
It was produced in the same manner as in Example 7 [Preparation of base material coated with block copolymer] except that the block copolymer was used.
[LCST measurement of block segment (A)]
4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid 0.40 g (0.1 mmol), Nn-propylacrylamide 2.26 g (20 mmol) and azobis(isobutyronitrile) 33 mg ( 0.2 mmol) was dissolved in 20 mL of dioxane. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 70° C. for 24 hours. After completion of the reaction, the reaction solvent was distilled off under reduced pressure using a rotary evaporator to concentrate the reaction solution. The concentrate was poured into 250 mL of hexane, and the precipitated white solid was collected and dried under reduced pressure to obtain an Nn-propylacrylamide polymer. An Nn-propylacrylamide polymer was dissolved in pure water to obtain a 0.6 wt % aqueous solution. This solution was placed in a quartz cell with an optical path length of 1 cm, and the transmittance of light with a wavelength of 500 nm was measured with a spectrophotometer (Hitachi UH-5300) while raising the temperature at a rate of 1° C./min. When the LCST was obtained by the midpoint method, the LCST was 17°C.
[Measurement of response temperature of block copolymer]
Measurement was performed in the same manner as in Example 1 [Wettability of block copolymer-coated substrate] except that the block copolymer-coated substrate was used.

Table 2 shows the water contact angles at 37°C, 20°C and 10°C. The contact angle of water at 10°C is lower than the contact angle of water at 20°C, and the base material coated with the block copolymer exhibits temperature responsiveness, and the response temperature is in the range of 10 to 20°C. I found out. Further, the contact angle of water was similarly measured at intervals of 5°C in the range of 5°C to 30°C, and the response temperature was found to be 18°C by the midpoint method.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Cultivation was performed according to Example 1 [Pluripotent stem cell culture evaluation and exfoliation evaluation] except that the base material coated with the block copolymer was used.

24 hours, 96 hours and 144 hours after seeding the cells, the state of the cells was observed under a phase-contrast microscope. After changing the medium 144 hours after seeding the cells, (1) when the substrate was cooled to 4 ° C., (2) when the substrate was cooled to 4 ° C. and tapped 20 times, (3) the substrate was cooled to 4° C. and pipetting was performed 10 times, and detachment of cells was confirmed. Under the conditions (2) and (3), almost all cells were detached and collected as aggregates.

Example 11
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
0.05 mL/cm ² of Matrigel solution (manufactured by Corning Incorporated) was added to the base material coated with the block copolymer prepared in Example 8, and left to stand for 1 hour at 37° C. in an environment with _a CO concentration of 5%. bottom. After removing the Matrigel solution, human iPS cell strain 201B7 was seeded at a density of 1300 cells/cm ² and cultured at 37° C. in a CO ₂ concentration of 5%. StemFit AK02N (manufactured by Ajinomoto) was added to the medium at 0.2 mL/cm ² . In addition, the cells were cultured using the above medium supplemented with Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) until 24 hours after seeding the cells.

24 hours, 96 hours and 144 hours after seeding the cells, the state of the cells was observed under a phase-contrast microscope. After changing the medium 144 hours after seeding the cells, (1) when the substrate was cooled to 4 ° C., (2) when the substrate was cooled to 4 ° C. and tapped 20 times, (3) the substrate was cooled to 4° C. and pipetting was performed 10 times, and detachment of cells was confirmed. Under the conditions (2) and (3), almost all cells were detached and collected as aggregates.

Example 12
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
0.63 mL/well of Vitronectin (VTN-N) solution (manufactured by Thermo Fisher Scientific) (diluted to about 50 μg/mL with PBS(−)) was added to the substrate coated with the block copolymer prepared in Example 8. and allowed to stand for 1 hour in an environment of 37° C. and a CO ₂ concentration of 5%. After that, the iMatrix-511 solution was removed, and human iPS cell line 201B7 was seeded at a density of 1300 cells/cm ² and cultured at 37° C. under a CO ₂ concentration of 5%. 0.2 mL/cm ² of StemFit AK02N (manufactured by Ajinomoto Co., Inc.) was added to the medium. In addition, until 24 hours after seeding the cells, the cells were cultured using the above medium supplemented with Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration 10 μM).

24 hours, 96 hours and 144 hours after seeding the cells, the state of the cells was observed under a phase-contrast microscope. After changing the medium 144 hours after seeding the cells, (1) when the substrate was cooled to 4 ° C., (2) when the substrate was cooled to 4 ° C. and tapped 20 times, (3) the substrate was cooled to 4° C. and pipetting was performed 10 times, and detachment of cells was confirmed. Under the conditions (2) and (3), almost all cells were detached and collected as aggregates.

Example 13
[Preparation of base material coated with block copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to obtain a 0.5 wt % solution. 50 μL of this solution was dropped on a porous membrane (manufactured by Corning Incorporated, trade name Falcon (registered trademark) culture insert, material: polyethylene terephthalate) having a pore diameter of 0.4 μm and pores of 1.8×10 ⁶ /cm ² . , 1000 rpm for 60 seconds. Dried for 1 hour at room temperature. Furthermore, it was immersed in pure water for 24 hours and washed to prepare a base material coated with a block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The base material coated with the block copolymer was immersed in a medium and fixed, and the human iPS cell 201B7 strain was seeded at a density of 1300 cells/cm ² and cultured at 37°C in an environment with _a CO concentration of 5%. . 0.2 mL/cm ² of StemFit AK02N (manufactured by Ajinomoto Co., Inc.) was added to the medium. In addition, until 24 hours after cell seeding, Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) and iMatrix-511 solution (manufactured by Nippi Co., Ltd.) (2.5 μL / mL) were added to the medium. was cultured using a medium supplemented with

24 hours, 96 hours and 144 hours after seeding the cells, the state of the cells was observed under a phase-contrast microscope. After changing the medium 144 hours after seeding the cells, (1) when the substrate was cooled to 4 ° C., (2) when the substrate was cooled to 4 ° C. and tapped 20 times, (3) the substrate was cooled to 4° C. and pipetting was performed 10 times, and detachment of cells was confirmed. Under all conditions (1) to (3), almost all cells were detached and collected as aggregates.

Example 14
[Preparation of base material coated with block copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to obtain a 0.5 wt % solution. 50 μL of this solution was dropped onto a porous membrane (manufactured by Corning Incorporated, trade name Falcon (registered trademark) culture insert, material: polyethylene terephthalate) having a pore diameter of 0.4 μm and pores of 1.1×10 ⁸ /cm ² . , 1000 rpm for 60 seconds. Dried for 1 hour at room temperature. Furthermore, it was immersed in pure water for 24 hours and washed to prepare a base material coated with a block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Cultivation was performed according to Example 13 [Evaluation of pluripotent stem cell culture and evaluation of detachment], except that the base material coated with the block copolymer was used.

24 hours, 96 hours, and 144 hours after seeding the cells, the medium was replaced with a new medium. After changing the medium 144 hours after seeding the cells, (1) when the substrate was cooled to 4 ° C., (2) when the substrate was cooled to 4 ° C. and tapped 20 times, (3) the substrate was cooled to 4° C. and pipetting was performed 10 times, and detachment of cells was confirmed. Under all conditions (1) to (3), almost all cells were detached and collected as aggregates.

Example 15
[Preparation of base material coated with block copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to obtain a 0.5 wt % solution. 50 μL of this solution was added dropwise to a porous membrane (manufactured by Corning Incorporated, trade name Falcon (registered trademark) culture insert, material: polycarbonate) having pores with a pore size of 0.4 μm and 1.0×10 ⁸ /cm ² , Spin coated at 1000 rpm for 60 seconds. Dried for 1 hour at room temperature. Furthermore, it was immersed in pure water for 24 hours and washed to prepare a base material coated with a block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Cultivation was performed according to Example 13 [Evaluation of pluripotent stem cell culture and evaluation of detachment], except that the base material coated with the block copolymer was used.

24 hours, 96 hours, and 144 hours after seeding the cells, the medium was replaced with a new medium. After changing the medium 144 hours after seeding the cells, (1) when the substrate was cooled to 4 ° C., (2) when the substrate was cooled to 4 ° C. and tapped 20 times, (3) the substrate was cooled to 4° C. and pipetting was performed 10 times, and detachment of cells was confirmed. Under all conditions (1) to (3), almost all cells were detached and collected as aggregates.

Example 16
[Preparation of base material coated with block copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to obtain a 0.5 wt % solution. 50 μL of this solution was dropped onto a porous membrane (manufactured by Corning Incorporated, trade name Falcon (registered trademark) culture insert, material: polyethylene terephthalate) having a pore diameter of 1 μm and pores of 1.8×10 ⁶ /cm ² , and the mixture was rotated at 1000 rpm. for 60 seconds. Dried for 1 hour at room temperature. Furthermore, it was immersed in pure water for 24 hours and washed to prepare a base material coated with a block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Cultivation was performed according to Example 13 [Evaluation of pluripotent stem cell culture and evaluation of detachment], except that the base material coated with the block copolymer was used.

24 hours, 96 hours and 144 hours after seeding the cells, the state of the cells was observed under a phase-contrast microscope. After changing the medium 144 hours after seeding the cells, (1) when the substrate was cooled to 4 ° C., (2) when the substrate was cooled to 4 ° C. and tapped 20 times, (3) the substrate was cooled to 4° C. and pipetting was performed 10 times, and detachment of cells was confirmed. Under the conditions of (3), almost all the cells were detached and collected as aggregates.

Example 17
[Preparation of base material coated with block copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to obtain a 0.5 wt % solution. 50 μL of this solution was added dropwise to a porous membrane (manufactured by Corning Incorporated, trade name Falcon (registered trademark) culture insert, material: polyethylene terephthalate) having a pore diameter of 3 μm and pores of 5.8×10 ⁵ /cm ² , and the mixture was rotated at 1000 rpm. for 60 seconds. Dried for 1 hour at room temperature. Furthermore, it was immersed in pure water for 24 hours and washed to prepare a base material coated with a block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Cultivation was performed according to Example 13 [Evaluation of pluripotent stem cell culture and evaluation of detachment], except that the base material coated with the block copolymer was used.

24 hours, 96 hours and 144 hours after seeding the cells, the state of the cells was observed under a phase-contrast microscope. After changing the medium 144 hours after seeding the cells, (1) when the substrate was cooled to 4 ° C., (2) when the substrate was cooled to 4 ° C. and tapped 20 times, (3) the substrate was cooled to 4° C. and pipetting was performed 10 times, and detachment of cells was confirmed. Under the conditions of (3), almost all the cells were detached and collected as aggregates.

Example 18
[Preparation of base material coated with block copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to obtain a 0.5 wt % solution. 50 μL of this solution was added dropwise to a porous membrane (manufactured by Greiner, trade name ThinCert™ Cell Culture Inserts, material: polyethylene terephthalate) having a pore diameter of 3 μm and pores of 1.7×10 ⁶ /cm ² , and rotated at 1000 rpm. for 60 seconds. Dried for 1 hour at room temperature. Furthermore, it was immersed in pure water for 24 hours and washed to prepare a base material coated with a block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Cultivation was performed according to Example 13 [Evaluation of pluripotent stem cell culture and evaluation of detachment], except that the base material coated with the block copolymer was used.

24 hours, 96 hours and 144 hours after seeding the cells, the state of the cells was observed under a phase-contrast microscope. After changing the medium 144 hours after seeding the cells, (1) when the substrate was cooled to 4 ° C., (2) when the substrate was cooled to 4 ° C. and tapped 20 times, (3) the substrate was cooled to 4° C. and pipetting was performed 10 times, and detachment of cells was confirmed. Under the conditions (2) and (3), almost all cells were detached and collected as aggregates.

Example 19
[Preparation of base material coated with block copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to obtain a 0.5 wt % solution. 50 μL of this solution was added dropwise to a porous membrane (manufactured by Corning Incorporated, trade name Falcon (registered trademark) culture insert, material: polyethylene terephthalate) having a pore size of 8 μm and pores of 6.2× ^{10 4} /cm ² and rotated at 1000 rpm. for 60 seconds. Dried for 1 hour at room temperature. Furthermore, it was immersed in pure water for 24 hours and washed to prepare a base material coated with a block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Cultivation was performed according to Example 13 [Evaluation of pluripotent stem cell culture and evaluation of detachment], except that the base material coated with the block copolymer was used.

24 hours, 96 hours and 144 hours after seeding the cells, the state of the cells was observed under a phase-contrast microscope. After changing the medium 144 hours after seeding the cells, (1) when the substrate was cooled to 4 ° C., (2) when the substrate was cooled to 4 ° C. and tapped 20 times, (3) the substrate was cooled to 4° C. and pipetting was performed 10 times, and detachment of cells was confirmed. Under the conditions of (3), almost all the cells were detached and collected as aggregates.

Example 20
[Preparation of base material coated with block copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to obtain a 0.5 wt % solution. 50 μL of this solution was dropped onto a porous membrane having pores with a pore size of 0.2 μm (manufactured by Teijin Limited, trade name Mirame, material: polyethylene) and spin-coated at 1000 rpm for 60 seconds. Dried for 1 hour at room temperature. Furthermore, it was immersed in pure water for 24 hours and washed to prepare a base material coated with a block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Cultivation was performed according to Example 13 [Evaluation of pluripotent stem cell culture and evaluation of detachment], except that the base material coated with the block copolymer was used.

24 hours, 96 hours, and 144 hours after seeding the cells, the medium was replaced with a new medium. After changing the medium 144 hours after seeding the cells, (1) when the substrate was cooled to 4 ° C., (2) when the substrate was cooled to 4 ° C. and tapped 20 times, (3) the substrate was cooled to 4° C. and pipetting was performed 10 times, and detachment of cells was confirmed. Under the conditions (2) and (3), almost all cells were detached and collected as aggregates.

Comparative example 1
Pluripotent stem cells were cultured in the same manner as in Example 1 except that the iMatrix-511 solution was not added in [Pluripotent stem cell culture evaluation and detachment evaluation]. Cells died without adhesion, and pluripotent stem cells could not be cultured.

Comparative example 2
[Synthesis of copolymer]
4.78 g (27 mmol) of N-(4-hydroxyphenyl)methacrylamide, 0.83 g (3 mmol) of condensate of 4-azidobenzoic acid and hydroxyethyl methacrylate, and 49 mg (0.3 mmol) of azobisisobutyronitrile were placed in a test tube. ) was added and dissolved in 18 mL of N,N'-dimethylformamide. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 70° C. for 69 hours. After completion of the reaction, the reaction solvent was distilled off under reduced pressure using a rotary evaporator to concentrate the reaction solution. The concentrated liquid was poured into 250 mL of methanol, and the precipitated yellow oily substance was collected and dried under reduced pressure to obtain an N-(4-hydroxyphenyl)methacrylamide/azido random copolymer.
[Preparation of substrate coated with copolymer]
It was produced in the same manner as in Example 1 [Preparation of base material coated with block copolymer] except that the above copolymer was used.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Cultivation was performed in the same manner as in Example 1 [Evaluation of pluripotent stem cell culture and evaluation of exfoliation], except that the substrate coated with the copolymer was used.

24 hours, 96 hours, and 144 hours after seeding the cells, the state of the cells was observed with a phase-contrast microscope. In each case, cell adhesion and proliferation were confirmed, and the medium was replaced with a new medium. After changing the medium 144 hours after seeding the cells, (1) when the substrate was cooled to 4 ° C., (2) when the substrate was cooled to 4 ° C. and tapped 20 times, (3) the substrate was cooled to 4° C. and pipetting was performed 10 times, and detachment of cells was confirmed. Cells did not detach in any of (1) to (3), and pluripotent stem cells could not be recovered by lowering the temperature in the absence of a layer of temperature-responsive polymer.

Comparative example 3
Evaluation was performed in the same manner as in Example 1 except that the substrate was not coated with the block copolymer, and a Corning Incorporated 6-well plate for cell culture was used as it was.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Cultivation was performed in the same manner as in Example 1 [Evaluation of pluripotent stem cell culture and evaluation of detachment], except that Corning Incorporated 6-well plates for cell culture were used as they were.

24 hours, 96 hours, and 144 hours after seeding the cells, the state of the cells was observed with a phase-contrast microscope. In each case, cell adhesion and proliferation were confirmed, and the medium was replaced with a new medium. After changing the medium 144 hours after seeding the cells, (1) when the substrate was cooled to 4 ° C., (2) when the substrate was cooled to 4 ° C. and tapped 20 times, (3) the substrate was cooled to 4° C. and pipetting was performed 10 times, and detachment of cells was confirmed. Cells were not detached in any of (1) to (3), and pluripotent stem cells could not be recovered by lowering the temperature in the absence of the block copolymer.

Comparative example 4
[Synthesis of polymer]
5.65 g (50 mmol) of N-isopropylacrylamide and 33 mg (0.2 mmol) of azobis(isobutyronitrile) were added to a test tube and dissolved in 50 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, reaction was carried out at 70° C. for 24 hours. After completion of the reaction, the reaction solvent was distilled off under reduced pressure using a rotary evaporator to concentrate the reaction solution. The concentrate was poured into 500 mL of hexane, and the precipitated solid was collected and dried under reduced pressure to obtain an N-isopropylacrylamide polymer.
[Preparation of substrate coated with polymer]
It was produced in the same manner as in Example 1 [Preparation of base material coated with block copolymer] except that the above polymer was used. However, when it was immersed in pure water for 24 hours and washed, most of the polymer dissolved. When the polymer dissolves in water, the polymer is eluted into the culture medium, contaminating the cells.

REFERENCE SIGNS LIST 1 base material 2 block copolymer 3 biological material 10 culture base material

Claims (7)

A block copolymer containing at least the following block segments (A) and (B), having a response temperature to water in the range of 15° C. to 35° C., and having a polydispersity of 1 to 1. It has three layers of temperature-responsive polymers, and at least one biological substance selected from the group consisting of matrigel, laminin, fibronectin, vitronectin, and collagen is immobilized on the layer. Culture substrate for pluripotent stem cells.
(A) A temperature-responsive polymer block segment containing N-isopropylacrylamide or N-propylacrylamide.
(B) A block segment containing at least one type of repeating unit among repeating units represented by the following general formula (1).

[Wherein, R ¹ is a hydrogen atom or a methyl group, Q is a divalent bond selected from an ester bond, an amide bond, a urethane bond or an ether bond, and R ² is represented by the following general formula (2), ( 3), (4), (5), (6), (7) or (8), a hydrocarbon group having 1 to 30 carbon atoms, or a hydrogen atom.

(In the formula, R ³ is a divalent hydrocarbon group having 1 to 10 carbon atoms, and R ⁴ and R ⁵ are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms.)

(wherein R ⁶ is a divalent hydrocarbon group having 1 to 10 carbon atoms, R ⁷ is a divalent hydrocarbon group having 1 to 4 carbon atoms, and R ⁸ and R ⁹ are each independently , a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms, and X is a sulfonate anion group, a carboxylate anion group or a phosphate anion group.)

(Wherein, R ¹⁰ is a hydrogen atom or an alkyl group having 1 to 30 carbon atoms, i is an integer of 1 to 300, and j is an integer of 0 to 60.)

(In the formula, R ¹¹ is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms.)

(In the formula, R ¹² is a divalent hydrocarbon group having 1 to 12 carbon atoms or a (poly)oxyethylene group, R ¹³ is a divalent hydrocarbon group having 1 to 4 carbon atoms, ¹⁴ , R ¹⁵ and R ¹⁶ are each independently a hydrogen atom or a hydrocarbon group having 1 to 2 carbon atoms.)

(In the formula, A is an ether bond or an ester bond, R ¹⁷ represents a divalent hydrocarbon group having 1 to 5 carbon atoms, R ¹⁸ represents a fluorine atom, and n represents an integer of 0 to 4. )

(In the formula, R ¹⁹ represents a divalent hydrocarbon group having 1 to 5 carbon atoms, but may be absent. R ²⁰ , R ²¹ , R ²² , R ²³ and R ²⁴ are each independently hydrogen represents an atom, a hydroxyl group, a carboxyl group, an amino group, or a hydrocarbon group having 1 to 4 carbon atoms.)] Claim 1, wherein the block copolymer containing the block segments (A) and (B) is a block copolymer having a structural unit ratio of the block segment (A) of 80 to 99 wt%. A culture substrate for the described pluripotent stem cells. 3. The substrate for culturing pluripotent stem cells according to claim 1 or 2, wherein the temperature-responsive polymer is a block copolymer of one type or a mixture of two types of block copolymers. The pluripotent according to any one of claims 1 to 3, wherein the substrate is a porous substrate, and the pore size of the porous substrate is smaller than the pluripotent stem cells to be cultured. Culture substrate for sex stem cells. The substrate for culturing pluripotent stem cells according to any one of claims 1 to 4 , characterized by having a laminin adsorption rate of 10% or more according to the following test.
A solution obtained by adding 2 to 2.5 μL of a laminin 511-E8 fragment solution having a concentration of 0.5 mg/mL to 1 mL of phosphate buffered saline was added to the base material in an amount of 0.5 μL per unit area of the base material area. Laminin adsorption rate obtained from the following formula when dropped onto a 2 mL/cm2 substrate and allowed to stand at 37°C for 24 hours.

The substrate for culturing pluripotent stem cells according to any one of claims 1 to 5, wherein the pluripotent stem cells are human induced pluripotent stem cells. A method for producing pluripotent stem cells, comprising producing undifferentiated pluripotent stem cells through the following steps [1] to [3].
[1] A step of seeding pluripotent stem cells on the culture substrate according to any one of claims 1 to 6.
[2] A step of culturing the pluripotent stem cells seeded on the culture substrate in a liquid at a temperature equal to or higher than the response temperature of the temperature-responsive polymer.
[3] A step of cooling the culture substrate to a temperature lower than the response temperature of the temperature-responsive polymer and exfoliating the pluripotent stem cells cultured in the liquid from the substrate.

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