WO2019035436A1

WO2019035436A1 - Culture substrate for pluripotent stem cell and method for producing pluripotent stem cell

Info

Publication number: WO2019035436A1
Application number: PCT/JP2018/030175
Authority: WO
Inventors: 佐藤雪絵; 近藤聡; 久野豪士
Original assignee: 東ソー株式会社
Priority date: 2017-08-16
Filing date: 2018-08-13
Publication date: 2019-02-21

Abstract

Provided is a culture substrate capable of culturing pluripotent stem cells in a feeder-free environment and capable of detaching pluripotent stem cells from the substrate without using a protease by changing the temperature of the substrate. Provided is a culture substrate for pluripotent stem cells, the culture substrate being characterized by having a layer composed of a temperature-responsive polymer having a response temperature to water in the range of 0-50°C on the surface of the substrate, wherein at least one organism-derived material selected from the group consisting of Matrigel, laminin, fibronectin, vitronectin and collagen is immobilized on the layer.

Description

Culture base for pluripotent stem cells and method for producing pluripotent stem cells

The present invention is a culture substrate which can exfoliate pluripotent stem cells by temperature change by having a layer of a temperature responsive polymer on the substrate, and is excellent in mass productivity and without damaging the 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 having the ability (differentiation pluripotency) to differentiate into various tissues of the living body, and cells in the field of regenerative medicine Much attention has been drawn as a source. In order to apply pluripotent stem cells to regenerative medicine, it is essential to grow the necessary number of pluripotent stem cells in an undifferentiated state and collect them as single cells or cell aggregates (except for cell sheet). is there.

In order to maintain the undifferentiated state of pluripotent stem cells, there is a method of culturing mouse embryo-derived fibroblasts as a scaffold by feeder cells prepared by gamma irradiation or antibiotic administration. However, the culture method of pluripotent stem cells using feeder cells complicates the process of culture, and infection of endogenous virus between different animals can occur. In particular, in medical applications, a culture method which avoids the use of feeder cells between different species of animals as much as possible is required.

In order to solve the above problems, a culture substrate for iPS cells coated with an extracellular matrix such as laminin (Patent Document 1) is known. According to such a substrate, it is possible to culture pluripotent stem cells in an undifferentiated state without using feeder cells, that is, in a feeder-free environment. However, when the pluripotent stem cells grown on such a substrate are detached from the substrate, a proteolytic enzyme is used. Proteolytic enzymes degrade proteins on multicellular surfaces, and play a role in cleaving the bond between pluripotent stem cells and a substrate and the bond between pluripotent stem cells. On the other hand, proteolytic enzymes are known to adversely affect cell viability, and a method of separating cells from a substrate without using proteolytic enzymes is important as a method that does not damage cells.

As a method of detaching cells without using a proteolytic enzyme, a method using a substrate having temperature responsiveness is known. For example, Patent Document 2 uses a cell culture support coated with a thermoresponsive polymer having an upper limit or lower limit critical solution temperature of 0 to 80 ° C. in water, peels cells by temperature change, and recovers a cell sheet Methods are disclosed. However, the method described in Patent Document 2 has a problem that it can not be applied to pluripotent stem cells. Furthermore, Patent Document 2 mainly aims at recovery of a cell sheet, and does not obtain single cells or clumps of cells.

Japanese Patent Laid-Open Publication No. 2015-178526 Japan JP 2009-131275 gazette

An object of the present invention is to culture pluripotent stem cells in a feeder-free environment, and by changing the temperature of the culture substrate, it is possible to detach pluripotent stem cells from the culture substrate without using a proteolytic enzyme. An object of the present invention is to provide a culture substrate capable of recovering pluripotent stem cells as single cells or cell aggregates.

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

In view of the above points, the present inventors have conducted intensive studies, and as a result, it is possible that the culture substrate having a layer made of a thermoresponsive polymer and the method for producing a specific pluripotent stem cell can solve the above-mentioned problems. The present invention has been completed.

That is, the present invention resides in the following [1] to [11].
[1] The substrate 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 selected from the group consisting of matrigel, laminin, fibronectin, vitronectin and collagen A culture substrate for pluripotent stem cells, characterized in that at least one type of bio-derived substance is immobilized.
[2] The pluripotent stem cell according to the above [1], wherein the thermoresponsive polymer is a block copolymer containing at least the following block segments (A) and (B): Culture substrate.
(A) Temperature-responsive polymer block segment having a lower limit critical solution temperature (LCST) to water in the range of 0 ° C. to 80 ° C.
(B) Block segment having pluripotent stem cell adhesion.
[3] The polymer according to the above [1] or [2], wherein the thermoresponsive polymer is a block copolymer containing at least the following block segments (A) and (B): Culture substrate for functional stem cells.
(A) Temperature-responsive polymer block segment having an LCST in the range of 0 ° C. to 80 ° C.
(B) Water insoluble block segment.
[4] The block segment of the above (B) is a block polymer comprising at least one kind of repeating unit among repeating units represented by the following general formula (1): Or the culture substrate of the pluripotent stem cells as described in [3].

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 a group represented by the following general formula (2), ( And 3) a substituent represented by (4), (5), (6), (7) or (8), a hydrocarbon group having 1 to 30 carbon atoms or a hydrogen atom.

(Wherein, 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 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).

(Wherein, 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.

(Wherein, A represents 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). )

(Wherein, R ¹⁹ represents a divalent hydrocarbon group of 1 to 5 carbon atoms, which 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.
[5] The block copolymer containing the block segment of (A) and (B) is a block copolymer having a constituent unit ratio of 80 to 99 wt% of the block segment of (A) A culture substrate for pluripotent stem cells according to any one of [2] to [4].
[6] The polymer according to any one of the above [1] to [5], wherein the temperature responsive polymer is one block copolymer or a mixture of two block copolymers. Culture substrate for functional stem cells.
[7] The multifunctional according to any one of the above [1] to [6], wherein the substrate is a porous substrate, and the pore diameter of the porous substrate is 0.01 to 10 μm. Stem cell culture substrate.
[8] The substrate according to any one of the above [1] to [7], wherein the substrate is a porous substrate, and the porosity of the porous substrate is 0.01 to 30%. Culture substrate for pluripotent stem cells.
[9] The culture substrate for pluripotent stem cells according to any one of the above [1] to [8], which has a laminin adsorption rate of 10% or more according to the following test.

A solution prepared by adding 2-2.5 μL of a laminin 511-E8 fragment solution at a concentration of 0.5 mg / mL to 1 mL of phosphate buffered saline was added in an amount of 0. When dropped on a ² mL / cm ² base material and allowed to stand at 37 ° C. for 24 hours, the laminin adsorption rate determined from the following formula.

[10] The culture substrate for pluripotent stem cells according to any one of the above [1] to [9], wherein the pluripotent stem cells are human induced pluripotent stem cells.
[11] 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 the above [1] to [10].
(2) culturing the pluripotent stem cells seeded on the culture substrate in a liquid at a temperature 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 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 pluripotent stem cells can be detached from the substrate without using a proteolytic enzyme by changing the temperature of the culture substrate, Pluripotent stem cells can be provided as a culture substrate that can be recovered as single cells or cell aggregates. In addition, it is possible to provide a method for producing pluripotent stem cells which is excellent in mass productivity and can produce pluripotent stem cells as single cells or cell aggregates without damaging the cells.

Schematic drawing showing the components of the culture substrate of the present invention Example of measurement result of LCST by permeability measurement of aqueous solution Example of measurement result of response temperature by water contact angle measurement Underwater AFM image of the culture substrate surface of Example 8

Hereinafter, modes for carrying out the present invention (hereinafter, simply referred to as “the present embodiment”) will be described in detail. The following embodiment is an example for describing the present invention, and is not intended to limit the present invention to the following contents. The present invention can be appropriately modified and implemented within the scope of the spirit of the present invention.

As used herein, “temperature responsive polymer” refers to a polymer whose degree of hydrophilicity / hydrophobicity changes with temperature change. In addition, when raising the temperature of the polymer from low temperature to high temperature in the presence of water molecules, the degree of hydrophilicity / hydrophobicity of the polymer changes to be more hydrophobic at a certain temperature, and it becomes water soluble There exists a temperature responsive polymer which changes from water to water insoluble, and this boundary temperature is referred to as “lower critical solution temperature (LCST)”. Generally, when a temperature responsive polymer dissolves in water at a temperature lower than LCST, it becomes insoluble in water at a temperature higher than LCST. When the temperature responsive polymer is water insoluble, it does not have LCST, but has a response temperature in which the degree of hydrophilicity / hydrophobicity changes with temperature change.

Further, in the present specification, the “biologically-derived substance” is a substance present in the body of an organism, but may be a natural product, or may be artificially synthesized by genetic engineering or the like. In addition, it may be a substance chemically synthesized based on the aforementioned biological substance. There are no particular limitations on the substance derived from a living organism, but for example, nucleic acids, proteins, polysaccharides which are basic materials constituting the living body, nucleotides and nucleosides which are components thereof, amino acids, various sugars, lipids, vitamins and hormones is there.

Moreover, in the present specification, "pluripotent stem cell adhesion" refers to adhesion of pluripotent stem cells to a culture substrate at a temperature at which pluripotent stem cells are cultured.

Further, in the present specification, “water-insoluble” in the block segment indicates that at least a part of the homopolymer consisting only of monomer units constituting the block segment is insoluble in water.

Furthermore, in the present specification, “pluripotent stem cell proliferative” refers to the pliability of pluripotent stem cells at culture temperature, and having proliferative refers to culture of pluripotent stem cells at culture temperature. It adheres to the material and shows that it can grow. Furthermore, high proliferative means that the cells proliferate to more pluripotent stem cells when compared in the same culture period.

Moreover, in the present specification, “pluripotent stem cell releasability” indicates the removability of pluripotent stem cells grown on the culture substrate from the culture substrate, and “with releasability” Fig. 6 shows that pluripotent stem cells grown on a culture substrate can be detached from the culture substrate by an external stimulus. Furthermore, "high releasability" indicates that pluripotent stem cells are detached from the culture substrate by a weaker external stimulus such as short-time cooling or weak stress. Further, "having a cold peelability" indicates that it has peelability, and further cooling the culture substrate increases peelability as compared to the case without cooling. Here, in the present specification, the term "external stimulation" refers to mechanical stimulation such as ultrasound, vibration and convection, electromagnetic stimulation such as light, electricity and magnetism, and thermodynamic stimulation such as heating and cooling. , Excluding those due to biological reactions such as enzyme reactions.

Furthermore, in the present specification, the “undifferentiated maintenance rate” of pluripotent stem cells indicates the proportion of undifferentiated pluripotent stem cells contained in cultured cells, and it is possible to stain undifferentiated markers of pluripotent stem cells. , Can be measured by a flow cytometer. When the undifferentiated maintenance rate is high, it means that the purity of pluripotent stem cells is high, which is preferable. The culture substrate of the present invention has a layer of a temperature responsive polymer having a response temperature to water 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 the ability to cool off pluripotent stem cells. In addition, by having a layer with a temperature responsive polymer, it is possible to suppress damage to cells when detaching pluripotent stem cells from a substrate, and maintain undifferentiated maintenance in pluripotent stem cell passaging The rate is high. When it does not have a layer by a temperature responsive polymer, it does not have the cooling peelability of pluripotent stem cells. Here, in the present invention, the "culture substrate" refers to the entire article (for example, the portion shown by reference numeral 10 in FIG. 1) for culturing pluripotent stem cells, and is based on the substrate and the temperature responsive polymer Including layers. Further, in the present invention, “base material” indicates a base material (for example, a portion shown by reference numeral 1 in FIG. 1) coated with a layer of a temperature responsive polymer.

In the present invention, the temperature responsive polymer has a response temperature in the range of 0 ° C. to 50 ° C. When 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 proliferation near body temperature (37 ° C.) and a temperature range which does not damage cells. Have a cold peelability. If it does not have a response temperature, it does not have cold peelability. The response temperature is in the range of 10 ° C to 40 ° C because it is suitable for imparting pluripotent stem cell adhesiveness and proliferation near the body temperature and for imparting cooling peelability in a temperature range that does not damage cells. Is more preferably in the range of 15 ° C. to 35 ° C., particularly preferably 15 ° C. to 30 ° C., and most preferably 15 ° C. to 25 ° C. At a temperature higher than the response temperature, the temperature-responsive polymer has hydrophobicity, so that proteins are easily adsorbed, and the adsorbed proteins can be used as a scaffold to allow adhesion culture of cells. On the other hand, when the temperature is lowered, the change to hydrophilicity promotes cell detachment. If the response temperature is less than 0 ° C., it becomes difficult to impart cooling peelability in a temperature range that does not damage the cells, and if it exceeds 50 ° C., there is no pluripotent stem cell adhesiveness and proliferation near body temperature. , Cell culture becomes difficult. In the case of using a room temperature medium at the time of medium exchange in culture operation, the response temperature is in the range of 0 ° C. to 30 ° C. because it is suitable for suppressing cell detachment at the time of medium exchange. Is more preferably in the range of 5 ° C. to 25 ° C., particularly preferably 5 ° C. to 20 ° C., and most preferably 10 ° C. to 20 ° C.

In the present invention, the type of the thermoresponsive polymer is not particularly limited, but a block copolymer coated on a base material, a copolymer weight immobilized on a base material via a reactive group such as an azide group, etc. A polymer etc. which were fix | immobilized on the base material can be used suitably by apply | coating a monomer to a base material and performing electron beam polymerization or radical polymerization on a base material.

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

Since the block copolymer contains the block segment (A), it is easy to impart pluripotent stem cell adhesiveness and proliferation to the culture substrate of the present invention at around body temperature, and a temperature range which does not damage the cells. It is easy to give cold

Here, when it is described as "LCST of block segment (A)" in the present invention, LCST of a homopolymer consisting only of repeating units constituting the block segment (A) is shown. The LCST of the block segment (A) is a temperature at which the homopolymer is insolubilized in water, for example, while heating the aqueous solution at a rate of 1 ° C./min in an aqueous solution in which 0.6 wt% of the homopolymer is dissolved. It can obtain | require by measuring the transmittance | permeability of the light of wavelength 500nm. From the low temperature to reaching the LCST, the transmittance is approximately constant, but the transmittance is sharply reduced due to the white turbidity near the LCST, and thereafter, the transmittance becomes substantially constant again (for example, FIG. 2) ). In this curve, the LCST can be determined by determining the transmittance at a temperature lower than the LCST and the temperature at which the transmittance of the average value of the transmittances at a temperature higher than the LCST is obtained (midpoint method). The temperature range to be measured is a range including a temperature range of 5 ° C. or more in which the transmittance is substantially constant at a temperature less than LCST, and further includes a temperature range of 5 ° C. or more in which the transmittance is constant at a temperature of LCST or more.

In addition, when the block copolymer of the present invention has a water-insoluble block segment, the response temperature is measured by finding a temperature which is insoluble in water but the degree of hydrophilicity / hydrophobicity changes due to temperature change. be able to. For example, the degree of hydrophilicity / hydrophobicity of the block copolymer is measured by immersing the block copolymer-coated substrate 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 water, and after waiting for the temperature to stabilize, the contact angle at various temperatures is determined by measuring the contact angle of the bubble again. . Below the response temperature, the bubble contact angle is generally constant at a large value (small water contact angle), but the bubble contact angle becomes a small value (large water contact angle) at the response temperature. 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 contact angle at a temperature lower than the response temperature and the contact angle at an average value of the contact angles at a temperature higher than the response temperature (midpoint method). The temperature range to be measured includes a temperature range of 10 ° C. or more at which the contact angle becomes substantially constant at a temperature lower than the response temperature, and further includes a temperature range at 10 ° C. or more at which the contact angle becomes substantially constant at a temperature higher than the response temperature. It is.

As a typical control method of the response temperature of the temperature responsive polymer, there are those which adjust the property of the copolymerizable monomer and the copolymerization rate. The temperature responsive polymer copolymerized with a hydrophilic monomer shifts the response temperature to the high temperature side as the composition increases. On the other hand, in the case of a temperature responsive polymer copolymerized with a hydrophobic monomer, the response temperature shifts to the low temperature side as the composition increases. From such a point of view, although the suitable LCST of the block segment (A) changes depending on the nature of the monomer of the block segment (B) to be copolymerized, the response temperature of the temperature responsive polymer is 0 to 50 ° C. As being suitable, the LCST of the block segment (A) is preferably 10 to 75 ° C., more preferably 10 to 60 ° C., particularly preferably 20 to 50 ° C.

The monomer unit constituting the block segment (A) is not particularly limited. For example, (meth) acrylamide compounds such as acrylamide and methacrylamide; N, N-diethylacrylamide, N-ethylacrylamide, Nn -Propyl Acrylamide, Nn-Propyl Methacrylamide, N-Isopropyl Acrylamide, N-Isopropyl Methacrylamide, N-Cyclopropyl Acrylamide, N-Cyclopropyl Methacrylamide, N-Ethoxyethyl Acrylamide, N-Ethoxyethyl Methacrylamide, N N-alkyl substituted (meth) acrylamide derivatives such as -tetrahydrofurfuryl acrylamide, N-tetrahydrofurfuryl methacrylamide; N, N-dimethyl (meth) acrylamide, N, N-ethy N, N-dialkyl-substituted (meth) acrylamide derivatives such as methyl acrylamide, N, N-diethyl acrylamide; 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 (Meth) acrylamide derivatives having a cyclic group such as 4- (1-oxo-2-methyl-2-propenyl) -morpholine; vinyl ethers such as methyl vinyl ether; proline derivatives such as N-proline methyl ester acrylamide And is suitable for setting the response temperature of the block copolymer to 0 to 50 ° C. , N-diethyl acrylamide, N-n-propyl acrylamide, N-isopropyl acrylamide, N-n-propyl methacrylamide, N-ethoxyethyl acrylamide, N-tetrahydrofurfuryl acrylamide, N-tetrahydrofurfuryl methacrylamide, N More preferred are n-propyl acrylamide and N-isopropyl acrylamide, and particularly preferred is N-isopropyl acrylamide. In addition, when a medium at room temperature is used at the time of medium exchange in the culture operation, it is suitable for setting the response temperature of the block copolymer to a temperature lower than room temperature, so N-isopropylacrylamide, N-proline Methyl ester acrylamide is preferred.

In the present invention, when the block copolymer contains the water-insoluble block segment (B), mixing of the temperature-responsive polymer into the culture solution can be easily suppressed, which is preferable.

Since the block segment (B) in the present invention is also suitable for enhancing the pluripotent stem cell adhesion of the culture substrate, the homopolymer comprising the monomer unit of the block segment (B) is a pluripotent stem cell It is preferable to have adhesiveness. There are no particular limitations on those having pluripotent stem cell adhesiveness, but for example, those having an ionic group, a hydrophilic group, a hydrophobic group, etc., and after covering them on the substrate surface, What surface-treated by gamma irradiation, plasma treatment, corona treatment etc. is mentioned.

The block segment (B) in the present invention is also a block that contributes to controlling the response temperature of the block copolymer because it is suitable for setting the response temperature of the block copolymer to the range of 0 to 50 ° C. Is preferred. Since the block copolymer (B) 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 in the range of 0 to 50 ° C. be able to. When the block copolymer has the block segment (B), the response temperature of the block copolymer can be controlled, and various monomers can be used as monomer units constituting the block segment (B). is there. For controlling the response temperature of the block copolymer, for example, hydrophilic monomers, hydrophobic monomers, or both may be used for the block segment (B).

In addition, since the block segment (B) in the present invention is suitable for enhancing the releasability of pluripotent stem cells, it is a block that contributes to shortening the cooling time necessary for the exfoliation of pluripotent stem cells. preferable. Since the block copolymer has the block segment (B), when the culture substrate is at a temperature lower than the response temperature of the block copolymer, water easily enters the block copolymer, and the cell and the block copolymer The adhesion of the cells is apt to weaken, and the time required for detachment of pluripotent stem cells can be shortened. For this reason, when the block copolymer has the block segment (B), the time required for detachment of pluripotent stem cells can be shortened. It is preferable to use, for example, a hydrophilic monomer for the block segment (B) in order to shorten the cooling time required for the detachment of pluripotent stem cells.

Furthermore, the block segment (B) in the present invention is preferably a block that contributes to adhesion to a substrate because 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 culture substrate is at a temperature lower than the response temperature of the block copolymer You can continue. For this reason, since the block segment (B) is a block that contributes to adhesion to the substrate, the immobilization of the block copolymer on the substrate is not limited to chemical coating, but is physically covered. You can also In order to enhance the adhesion between the block copolymer and the substrate, it is preferable to use, for example, a hydrophobic monomer or a monomer having a reactive functional group as the block segment (B).

In the present invention, the block segment (B) is a block weight comprising at least one repeating unit of repeating units represented by the following general formula (1) since it is suitable for enhancing water insolubility. It is preferable to be combined.

(Wherein, A represents an ether bond or a methylene group, R ¹⁷ represents a C 3 -C 5 divalent hydrocarbon group, R ¹⁸ represents a fluorine atom, and n represents an integer of 0 to 4). )

(Wherein, R ¹⁹ represents a divalent hydrocarbon group of 1 to 5 carbon atoms, which 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 the general formula (1) in the present invention, 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, preferably an ester bond or an amide bond, particularly preferably an ester bond. In the general formula (1), R ² represents a substituent represented by the general formula (2), (3), (4), (5), (6), (7) or (8), having 1 to carbon atoms 30 indicates a hydrocarbon group or a hydrogen atom.

In the general formula (1), R ² enhances water insolubility, enhances pluripotent stem cell adhesion and proliferation, shifts the response temperature of the block copolymer to the high temperature side, or alternatively, pluripotent stem cells It is preferable to use a substituent represented by the general formula (2) because it is suitable for enhancing the removability of In the general formula (2), R ³ is a divalent hydrocarbon group having 1 to 10 carbon atoms, which is suitable for enhancing the releasability of pluripotent stem cells, and preferably has 1 to 6 carbon atoms. A divalent hydrocarbon group, in particular an alkylene group, is used. A methylene group, ethylene group, a propylene group, a butylene group, a pentamethylene group, a hexamethylene group etc. are illustrated as such an alkylene group, Preferably it is an ethylene group. R ⁴ and R ⁵ are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms, and since R ⁴ and R ⁵ are simultaneously suitable for enhancing the peelability of pluripotent stem cells, R ⁴ and R ⁵ are simultaneously It is preferably a methyl group or an ethyl group, and particularly preferably a methyl group.

As the substituent represented by the general formula (2) in the present invention, an aminomethyl group, an N, N-dimethylaminomethyl group, an N, N-diethylaminomethyl group, an aminoethyl group, an N, N-dimethylaminoethyl group And N, N-diethylaminoethyl group, 3-aminopropyl group, 3- (N, N-dimethylamino) -propyl group, 3- (N, N-diethylamino) -propyl group, etc. Among them, preferred are N, N-dimethylaminomethyl group, N, N-dimethylaminoethyl group and N, N-diethylaminoethyl group since they are suitable for enhancing the exfoliation of stem cells.

As the monomer units in the general formula (1) R ² is represented by the general formula (2) is not particularly limited, for example, 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 the general formula (1), R ² enhances water insolubility, enhances pluripotent stem cell adhesion and proliferation, shifts the response temperature of the block copolymer to the high temperature side, or alternatively, pluripotent stem cells The substituent represented by the general formula (3) can be used because it is suitable for enhancing the removability of In the general formula (3), since R ⁶ is a divalent hydrocarbon group having 1 to 10 carbon atoms and is suitable for enhancing the releasability of pluripotent stem cells, it is preferably a methylene group or an ethylene group. And a divalent alkylene group having 1 to 6 carbon atoms such as a propylene group, a butylene group, a pentamethylene group or a hexamethylene group, and more preferably an ethylene group. R ⁷ is a divalent hydrocarbon group having 1 to 4 carbon atoms, and is preferably an alkylene group such as a methylene group, an ethylene group, a propylene group or a butylene group in order to enhance the peelability of the pluripotent stem cells. More preferably, it is an ethylene group. R ⁸ and R ⁹ each independently represent a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms, and since R ⁸ and R ⁹ are each suitable for enhancing the peelability of pluripotent stem cells, R ⁸ and ⁹ are preferably At the same time, it is a hydrogen atom or a methyl group, more preferably a methyl group. X is a sulfonate anion group, a carboxylate anion group or a phosphate anion group.

As the substituent represented by the general formula (3) in the present invention, 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, etc. Preferably it is dimethyl because it is suitable for Ethyl) (carboxylatomethyl) aminium group, dimethyl (ethyl) (2-carboxylatoethyl) aminium group, dimethyl (propyl) (3-sulfonatopropyl) aminium group, dimethyl (propyl) (4-sulfonatobutyl) aminium group, It is a dimethyl (ethyl) (2-sulfonatoethyl) aminium group.

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

In the general formula (1), R ² enhances water insolubility, enhances pluripotent stem cell adhesion and proliferation, shifts the response temperature of the block copolymer to high temperature side or low temperature side, or exfoliates It is preferable to use the substituent represented by General formula (4) from being suitable in order to improve property. In the general formula (4), R ¹⁰ is a hydrogen atom or an alkyl group having 1 to 30 carbon atoms, and is methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, tert. -Butyl group, n-hexyl group, isohexyl group etc. can be exemplified, but methyl group, ethyl group, n-propyl group, isopropyl group is preferable because it is suitable for enhancing the releasability of pluripotent stem cells. is there. However, in the general formula (4), i is an integer of 1 to 300, and j is an integer of 0 to 60.

As a substituent represented by General formula (4) in this invention, a polyethylene glycol group, 2-hydroxyethyl group, 2-hydroxyethyl group, a hydroxymethyl group, 2-methoxyethyl group, a furfuryl group, tetrahydrofurfuryl group And the like, but is preferably a polyethylene glycol group, a 2-methoxyethyl group or a tetrahydrofurfuryl group because it is suitable for enhancing the releasability of pluripotent stem cells.

In the general formula (1), the monomer unit in which R ² is represented by the general formula (4) is not particularly limited. For example, hydroxyethyl acrylate, hydroxyethyl methacrylate, N- (2-hydroxyethyl) Acrylamide, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, polypropylene glycol monomethacrylate, methoxypolyethylene glycol monoacrylate, methoxypolyethylene glycol monomethacrylate, diethylene glycol monomethyl ether acrylate, diethylene glycol monomethyl ether methacrylate, diethylene glycol monoethyl ether acrylate Diethylene glycol monoethyl ether Tacrylate, 2-methoxyethyl acrylate, 2-methoxyethyl methacrylate, 2-ethoxyethyl acrylate, 2-ethoxyethyl methacrylate, 3-butoxyethyl acrylate, 3-butoxyethyl methacrylate, 3-butoxyethyl acrylamide, furfuryl acrylate, furfuryl Methacrylate, tetrahydrofurfuryl acrylate, tetrahydrofurfuryl methacrylate and the like can be mentioned.

In the general formula (1), in order to enhance water insolubility, to enhance pluripotent stem cell adhesiveness and proliferation, R ² shifts the response temperature of the block copolymer to the high temperature side or the low temperature side, or The substituent represented by the general formula (5) can be used because it is suitable for enhancing the releasability of functional stem cells. In the general formula (5), R ¹¹ is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms.

In the general formula (1), the monomer unit in which R ² is represented by the general formula (5) is not particularly limited. For example, 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 the general formula (1), R ² enhances water insolubility, enhances pluripotent stem cell adhesion and proliferation, shifts the response temperature of the block copolymer to the high temperature side, or alternatively, pluripotent stem cells The substituent represented by the general formula (6) can be used because it is suitable for enhancing the removability of In the 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 to detach pluripotent stem cells It is preferably a divalent hydrocarbon group having 1 to 6 carbon atoms, particularly an alkylene group. A methylene group, ethylene group, a propylene group, a butylene group, a pentamethylene group, a hexamethylene group etc. are illustrated as such an alkylene group, Preferably it is an 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 is preferably an alkylene having 1 to 4 carbon atoms Examples thereof include a methylene group, an ethylene group, a propylene group, a butylene group and the like, and an ethylene group is particularly preferable.

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

As a substituent represented by General formula (6) in this invention, 2-ethyl phosphoryl choline group, 3-propyl phosphoryl choline group, 4-butyl phosphoryl choline group, 6-hexyl phosphoryl choline group, 10-decyl phosphoryl choline group, omega- ( Although a poly) oxyethylene phosphoryl choline group etc. can be illustrated, since it is suitable in order to raise the exfoliation nature of pluripotent stem cells, preferably a 2-ethyl phosphoryl choline group is used.

In the general formula (1), the monomer unit in which R ² is represented by the general formula (6) is not particularly limited. For example, 2-methacryloyloxyethyl phosphorylcholine, 2-acryloyloxyethyl phosphorylcholine, 3- (Meth) acryloyloxypropyl phosphorylcholine, 4- (meth) acryloyloxybutyl phosphorylcholine, 6- (meth) acryloyloxyhexyl phosphorylcholine, 10- (meth) acryloyloxydecyl phosphorylcholine, ω- (meth) acryloyl (poly) oxyethylene phosphorylcholine , 2-acrylamidoethyl phosphoryl choline, 3-acrylamido propyl phosphoryl choline, 4-acrylamido butyl phosphoryl choline, 6-acrylamido hexyl phosphoryl choline, 10-acryl amide Decyl phosphorylcholine, .omega. (meth) can be mentioned acrylamide (poly) oxyethylene phosphoryl choline.

In the general formula (1), R ² enhances water insolubility, enhances pluripotent stem cell adhesion and proliferation, shifts the response temperature of the block copolymer to the low temperature side, or blocks Since it is suitable for covering a polymer by a chemical bond, the substituent which has a phenyl azide group represented by General formula (7) can be used. In the 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, and examples thereof include 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 to 4.

In the general formula (1), the monomer unit in which R ² is represented by the general formula (7) is not particularly limited. For example, 4-azidophenyl acrylate, 4-azidophenyl methacrylate, 2-(( 4-azidobenzoyl) oxy) ethyl acrylate, 2-((4-azidobenzoyl) oxy) ethyl methacrylate and the like can be mentioned.

In the general formula (1), R ² is used as a substrate to shift the response temperature of the block copolymer to the high temperature side or the low temperature side in order to enhance water insolubility and to enhance pluripotent stem cell adhesion and proliferation. It is represented by the general formula (8) because it is suitable for covering a block copolymer by physical interaction or for covering a block copolymer on a substrate by chemical bonding. It is preferable to use a substituent having an aromatic ring. In the general formula (8), R ¹⁹ represents a divalent hydrocarbon group having 1 to 5 carbon atoms, which may not be present. 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 the general formula (1), the monomer unit in which R ² is represented by the general formula (8) is not particularly limited. For example, 2-hydroxyphenyl acrylate, 2-hydroxyphenyl methacrylate, 3-hydroxyphenyl Acrylate, 3-hydroxyphenyl methacrylate, 4-hydroxyphenyl acrylate, 4-hydroxyphenyl methacrylate, N- (2-hydroxyphenyl) acrylamide, N- (2-hydroxyphenyl) methacrylamide, N- (3-hydroxyphenyl) acrylamide And N- (3-hydroxyphenyl) methacrylamide, N- (4-hydroxyphenyl) acrylamide, N- (4-hydroxyphenyl) methacrylamide and the like.

In the general formula (1), R ² enhances water insolubility, enhances pluripotent stem cell adhesion and proliferation, shifts the response temperature of the block copolymer to the low temperature side, or blocks It is preferable to use a hydrocarbon group having 1 to 30 carbon atoms because it is suitable for covering the polymer by physical interaction, and in order to stably coat the block copolymer on the substrate, it is preferable. It is a hydrocarbon group having 4 to 15 carbon atoms. The monomer unit represented by hydrocarbon group having 1 to 30 carbon atoms as R ² is not particularly limited, and examples thereof include n-butyl acrylate, n-butyl methacrylate, isobutyl acrylate, isobutyl methacrylate, and 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 Examples include decyl acrylate, n-tetradecyl methacrylate and the like.

The structure of the block copolymer containing the block segments of (A) and (B) in the present invention is not particularly limited, but a diblock copolymer having at least the block segments of (A) and (B) It is preferable to be combined. Also, the block segment (A) and the block segment (B) may be directly bonded or may be bonded via a spacer. In addition, when the repeating unit which comprises a block segment (B) is 2 or more types, those arrangement | sequences may be block arrangement, and random arrangement and alternate arrangement may be sufficient.

The constituent unit ratio of the block segment (A) in the block copolymer containing the block segments of (A) and (B) in the present invention is preferably from 40 to 40 from the viewpoint of enhancing the cold peelability of pluripotent stem cells. It is 99 wt%, more preferably 60 to 99 wt%, particularly preferably 80 to 99 wt%, and most preferably more than 90 wt%. In addition, when the constituent unit ratio of the block segment (A) is within the above range, undifferentiated pluripotent stem cells can be selectively exfoliated, and the cells exfoliated and recovered in the passage culture of pluripotent stem cells Undifferentiated maintenance rate can be increased. On the other hand, from the viewpoint of enhancing the water insolubility of the block copolymer, the constituent unit ratio of the block segment (B) is preferably 1 to 50 wt%, preferably 1 to 25 wt%, and more preferably 3 to 15 wt %, Most preferably 5 to 15 wt%.

The molecular weight of the thermoresponsive polymer in the present invention is not particularly limited, but is preferably 1000 to 1,000,000 in number average molecular weight, and preferably 2000 to 500,000 because it is suitable for increasing the strength of the block copolymer. Is more preferably, 5,000 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 and the like as necessary. The chain transfer agent is not particularly limited, and commonly used ones can be suitably used. For example, dithiobenzoate, trithiocarbonate, 4-cyano-4-[(dodecylsulfonylthiocarbonyl) sulfonyl] pentan Noic acid, 2-cyanopropan-2-yl N-methyl-N- (pyridin-4-yl) carbamodithioate, methyl methyl 2-propionate (4-pyridinyl) carbamodithioate can be mentioned . The polymerization initiator is not particularly limited, and commonly used ones can be suitably used. For example, azobisisobutyronitrile, 1,1'-azobis (cyclohexanecarbonitrile), di-tert And butyl peroxide, tert-butyl hydroperoxide, hydrogen peroxide, potassium peroxodisulfate, benzoyl peroxide, triethyl borane, diethyl zinc and the like. Further, the polymerization inhibitor is not particularly limited, and commonly used ones can be suitably used, but hydroquinone, p-methoxyphenol, triphenylferdadil, 2,2,6,6-tetramethyl piperidine Examples include nyl-1-oxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl and the like.

There is no particular limitation on the method of synthesizing the temperature responsive polymer in the present invention, but there may be provided a "radical polymerization handbook", p. The living radical polymerization techniques described in 161-225 (2010) can be used.

In the present invention, the layer of the thermoresponsive polymer preferably has a layer thickness of 5 to 1000 nm, preferably 5 to 200 nm, because it is suitable for enhancing pluripotent stem cell adhesion and proliferation and exfoliation. 20 to 100 nm is particularly preferable, and 35 to 80 nm is most preferable.

In the present invention, although the type of the thermoresponsive polymer is not particularly limited, it is suitable for uniforming the state of pluripotent stem cells to be cultured without causing any variation in the coating, and thus one type of block It is preferable that it is a copolymer or a mixture of 2 types of block copolymers, and it is more preferable that it is 1 type of block copolymer. Here, in the present invention, “type of temperature responsive polymer” is regarded as the same type of block copolymer when all the blocks constituting the block copolymer are the same. Here, that the blocks are the same means that, when the blocks are mainly composed of one kind of monomer unit, the case in which the monomer units contained most in wt% ratio are the same, and When the block is composed of two or more types of monomer units, the case is shown where the upper two monomer units having a large wt% ratio are the same. It is preferable that the polydispersity (weight-average molecular weight M _w / number-average molecular weight M _n ) of each block is 1 to 20, since it is more preferable to make the state of pluripotent stem cells to be cultured uniform. It is more preferably 1 to 10, particularly preferably 1 to 5, and most preferably 1 to 2.

In the present invention, the layer of the thermoresponsive 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. At least one type of

The culture substrate of the present invention can culture pluripotent stem cells by having a bioderived substance immobilized in a layer composed of a temperature responsive polymer. When the layer of the thermoresponsive polymer does not have a bio-derived substance, the adhesion of pluripotent stem cells to the culture substrate is weak, and pluripotent stem cells can not be cultured.

In addition, since it is possible to suppress degeneration of a biological substance and to enhance the proliferation of pluripotent stem cells, it is preferable that the biological substance be immobilized not by covalent bond but by noncovalent bond. Here, in the present invention, “non-covalent bond” means electrostatic interaction, hydrophobic interaction, hydrogen bond, π-π interaction, dipole-dipole interaction, London dispersion force, other van der Waals. It shows a bonding force other than covalent bond derived from intermolecular force such as interaction. Immobilization of the bioderived substance to the block copolymer may be by a single bond or by a combination of two or more.

In the present invention, the method for immobilizing the bio-derived substance is not particularly limited. For example, the solution of the bio-derived substance is applied for a predetermined time to the substrate having the layer of temperature responsive polymer. A method or a method of immobilizing by adding a biologically-derived substance to a culture solution for culturing pluripotent stem cells can be suitably used.

In the present invention, the biological material is at least one selected from the group consisting of matrigel, laminin, vitronectin, fibronectin and collagen. The culture substrate of the present invention has pluripotent stem cell adhesiveness and proliferation since the bio-derived substance is Matrigel, laminin, fibronectin, vitronectin, collagen or a combination thereof. When the biomaterial is not Matrigel, laminin, vitronectin, fibronectin, or collagen, it does not have pluripotent stem cell adhesion and proliferation. More preferably, it is a combination of at least four laminins, because it is suitable for imparting pluripotent stem cell adhesion and proliferation, either laminin and matrigel, laminin and fibronectin, or laminin and collagen. It is particularly preferred that it is a combination of and most preferably only laminin.

These biological substances may be natural products, or may be artificially synthesized by genetic recombination technology etc., fragments cleaved with restriction enzymes etc., and synthetic proteins based on these biological substances Alternatively, it may be a synthetic peptide.

In the present invention, Matrigel (manufactured by Corning Incorporated) or Geltrex (manufactured by Thermo Fisher Scientific) can be suitably used as a commercially available product, for example, as the Matrigel as the matrigel.

The type of laminin is not particularly limited. For example, laminin 511, laminin 521 or laminin 511 which has been reported to exhibit high activity against α6β1 integrin expressed on the surface of human iPS cells E8 fragments can be used. The laminin may be a natural product, or may be artificially synthesized by genetic engineering or the like, or may be a synthetic protein or a synthetic peptide based on the laminin. From the viewpoint of availability, for example, iMatrix-511 (manufactured by Nippi Co., Ltd.) can be suitably used as a commercial product.

The vitronectin may be a natural product, may be artificially synthesized by genetic recombination technology or the like, and may be a synthetic protein or a synthetic peptide based on the vitronectin. From the viewpoint of availability, commercially available products such as vitronectin, derived from human plasma (manufactured by Wako Pure Chemical Industries, Ltd.), synthemax (manufactured by Corning Incorporated), 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 a synthetic peptide based on the fibronectin. From the viewpoint of easy availability, for example, fibronectin solution, human plasma derived (manufactured by Wako Pure Chemical Industries, Ltd.) and Retronectin (manufactured by Takara Bio Inc.) can be suitably used as commercial products.

The type of collagen is not particularly limited, and, 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. From the viewpoint of availability, 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 enhancing the proliferation and exfoliation of pluripotent stem cells. It is particularly preferably 20% to 80%, most preferably 30% to 60%.
[Laminin adsorption rate test]
A solution containing 2 to 2.5 μL of a laminin 511-E8 fragment solution at a concentration of 0.5 mg / mL per 1 mL of phosphate buffered saline is 0.2 mL / cm in an amount per unit area of culture substrate ² Laminin adsorption rate determined from the following formula when dropped on a substrate and allowed to stand at 37 ° C. for 24 hours.

Here, 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 is not particularly limited. using a fluorescence-labeled laminin 511-E8 fragment with ^{HiLyte Fluor TM 647 labeling Kit-NH} 2 ( manufactured by Dojin chemical Laboratory), there may be employed a method for obtaining the laminin adsorption ratio by fluorescence measurement.

In the present invention, the adsorption amount of the laminin 511-E8 fragment in the culture substrate is preferably 5 to 5000 ng / cm ² because it is suitable for enhancing the proliferation and exfoliation of pluripotent stem cells. It is 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 a laminin 511-E8 fragment, for example, iMatrix-511 (manufactured by Nippi Co., Ltd.) can be used as a commercial product.

The material of the substrate on which the layer of the thermoresponsive polymer is coated in the present invention is not particularly limited. However, not only substances such as glass and polystyrene which are usually used for cell culture but also form can generally be applied. For example, high molecular compounds such as polycarbonate, polyethylene terephthalate, polyvinylidene fluoride, polytetrafluoroethylene, polyethylene, polypropylene, and polymethyl methacrylate, ceramics, metals, and the like can be used. From the viewpoint of ease of culture operation, 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, polycarbonate It is further preferable to include at least one of polyethylene terephthalate and polyethylene, and polystyrene, polycarbonate, polyethylene terephthalate, and polyethylene are particularly preferable because they are suitable for enhancing flexibility.

Moreover, it is preferable that a base material has pluripotent stem cell adhesiveness. Examples of the base material having pluripotent stem cell adhesiveness include those obtained by subjecting the base material to plasma treatment, corona treatment, UV treatment and the like.

There is no restriction | limiting in particular as a shape of a base material, A planar shape like a board | plate and a film may be sufficient, A fiber, porous particle, a porous membrane, and a hollow fiber may be sufficient. Moreover, it may be a container (cell culture dish such as petri dish, flask, plate etc.) generally used for cell culture etc. From the viewpoint of easiness of culture operation, it is preferable to use a plate, a flat shape such as a film, or a flat porous membrane.

In the present invention, the base material is a porous base material, which is suitable for enhancing the releasability of pluripotent stem cells, and the pore diameter of the porous base material is smaller than that of pluripotent stem cells to be cultured. Is preferred. When the substrate is a porous substrate, the releasability of pluripotent stem cells can be enhanced. In addition, when the pore diameter of the porous substrate is smaller than that of pluripotent stem cells, pluripotent stem cell adhesion and proliferation can be enhanced. Furthermore, when the base material is a porous base material, nutrients can easily spread to cells during culture, and the undifferentiated maintenance rate of pluripotent stem cells can be increased in subculture. In addition, the pore diameter is more preferably 8 μm or less, particularly preferably 3 μm or less, and most preferably 1 μm or less because it is suitable for enhancing pluripotent stem cell adhesion and proliferation. . Furthermore, the pore diameter is preferably 0.01 μm or more, more preferably 0.05 μm or more, and more preferably 0.1 μm or more, because it is suitable for enhancing the releasability of pluripotent stem cells. Are particularly preferred, and most preferably 0.2 μm or more. Here, in the present invention, the “pore diameter” of the porous substrate means an average value of the diameters of the pores of the porous substrate along the in-plane direction of the porous substrate, and It can be calculated by measuring the diameters of pores of 20 or more points in a laser microscope image, a scanning electron microscope image, and a transmission electron microscope image, and determining an average value.

In the present invention, the pore density of the pores of the porous substrate is 10 to 10 ¹⁰ cells / cm ² because it is suitable for enhancing pluripotent stem cell adhesion and proliferation and exfoliation. Preferably, 10 ³ to 10 ⁹ particles / cm ² are more preferable, 10 ⁵ to 10 ⁹ particles / cm ² are particularly preferable, and 10 ⁵ to 10 ⁷ particles / cm ² are most preferable. In addition, the pore density of the pores of the porous substrate is 10 ⁷ cells / cm ² or less because it is suitable for enhancing the transparency of the porous substrate and facilitating observation of cells with a microscope. it is preferable, more preferably 10 ⁶ / cm ² or less, particularly preferably 10 ⁵ / cm ² or less, and 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 a laser microscope image or scanning of the porous substrate In a type electron microscope image and a transmission electron microscope image, it can be calculated by finding the number of pores in a square region having a side 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 means the surface area of one principal surface of the porous substrate when it is assumed that no pores are present in the porous substrate.

In the present invention, the porosity of the porous substrate is preferably 0.01 to 30%, and more preferably 0.01 to 20%, because it is suitable for enhancing the releasability of pluripotent stem cells. 0.01 to 5% is particularly preferable, and 0.01 to 1.5% is most preferable. Here, in the present invention, the "porosity" of the porous substrate is a value obtained by dividing the total area of the pore portion by the substrate area for one main surface of the surface of the porous substrate, and the area ratio is It indicates how much void space exists in the surface of the material, and in a laser microscope image, a scanning electron microscope image, and a transmission electron microscope image of the porous substrate, at least 200 times the pore diameter of the pores of the porous substrate It can measure by observing the square area which makes length one side.

The porosity is also preferably 80% or less, more preferably 50% or less, and particularly preferably 20% or less, because it is suitable for enabling observation of pluripotent stem cells by phase contrast microscopy. % Or less is most preferable.

Furthermore, the porosity is preferably 0.1% or more because it is suitable for rapidly permeating the components contained in the culture medium and evenly distributing nutrition to the cells. 1% or more is more preferable, 5% or more is particularly preferable, and 10% or more is most preferable.

In the present invention, the shape of the pores possessed by the porous substrate is not particularly limited, but it has flat portions and pores because it is suitable for enhancing pluripotent stem cell adhesion and proliferation and exfoliation. It is preferable that it is a flat membrane. Moreover, since it is suitable to enable observation of pluripotent stem cells by phase contrast microscopy, it is preferable that the pore which a porous base material has is a cylindrical shape, and the independent cylindrical shape It is further preferred that It is suitable for observing the shape of the cells of this surface in which the shape of the pore is a cylindrical shape.

As a method of forming a layer of a temperature responsive polymer on the surface of a substrate according to the present invention, a method of forming a layer by coating a temperature responsive polymer on a substrate by (1) chemical bonding, (2) A method of forming a layer by physical interaction can be performed alone or in combination. That is, (1) UV irradiation, electron beam irradiation, gamma ray irradiation, plasma treatment, corona treatment etc. can be used as a method by chemical bonding. Furthermore, when the temperature responsive polymer and the base material have appropriate reactive functional groups, generally used organic reactions such as radical reaction, anion reaction and cation reaction can be utilized. (2) As a method by physical interaction, a matrix having a good compatibility with a temperature responsive polymer and a good coatability is used as a medium, and coating, brushing, dip coating, spin coating, bar coating, It is possible to use various methods commonly known such as flow coating, spray coating, roll coating, air knife coating, blade coating, gravure coating, microgravure coating, slot die coating and the like.

In the present invention, the type of pluripotent stem cells is not particularly limited. For example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), somatic cell-derived embryonic stem cells (nuclear transfer ES) Cells or nt ES cells) can be used, particularly preferably induced pluripotent stem cells. Also, the animal from which pluripotent stem cells are derived is not particularly limited, but may be, for example, a mammal. Examples of mammals include rodents (mouse, rat, etc.), primates (monkey, human, etc.), and may be laboratory animals or companion animals. In the present invention, it is preferably of primate origin, particularly preferably of human origin.

The present invention also relates to a method of producing undifferentiated pluripotent stem cells using the culture substrate. The method is a method for producing pluripotent stem cells, which comprises producing undifferentiated pluripotent stem cells through the following steps (1) to (3).
(1) A step of seeding pluripotent stem cells on the culture substrate.
(2) culturing the pluripotent stem cells seeded on the culture substrate in a liquid at a temperature 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 detaching the pluripotent stem cells cultured in the liquid from the substrate.

Hereinafter, the steps (1) to (3) in the production method of the present invention will be described in detail.

The step (1) in the method for producing pluripotent stem cells of the present invention is a step of seeding pluripotent stem cells on the culture substrate using the culture substrate. In the present invention, "seeding cells" means that a medium in which the cells are dispersed (hereinafter referred to as "cell suspension") is applied onto the culture substrate, or injected into the culture substrate, etc. 7 shows contacting a cell suspension with a culture substrate. By using a substrate having a layer of a temperature responsive polymer having a response temperature in the range of 0 ° C. to 50 ° C., exfoliating pluripotent stem cells from the culture substrate by temperature change in step (3) described later Can. When the culture substrate does not have a layer of a temperature responsive polymer, it is not possible to detach pluripotent stem cells from the substrate due to temperature change in step (3). In addition, when the culture substrate has a bio-based substance immobilized in a layer of a temperature responsive polymer, pluripotent stem cells can be cultured in the step (2) described later. When the culture substrate does not have the bio-derived substance immobilized in the layer of the thermoresponsive polymer, the pluripotent stem cells can not be cultured in the step (2).

In the steps (1) to (3), the culture is carried out under conditions effective to maintain the undifferentiated nature of the pluripotent stem cells. Conditions effective for maintaining the undifferentiated nature are not particularly limited, but for example, it is preferable to set the density of pluripotent stem cells at the start of culture to the preferable range described as the cell density at the time of seeding, And the like in the presence of a liquid medium. As an effective culture medium for maintaining the undifferentiated nature of pluripotent stem cells, for example, insulin, transferrin, selenium, ascorbic acid, which is known as a factor for maintaining the undifferentiated nature of pluripotent stem cells A medium to which one or more of sodium bicarbonate, basic fibroblast growth factor, transforming growth factor β (TGFβ), CCL2, activin, and 2-mercaptomethanol is added can be suitably used. Insulin, transferrin, selenium, ascorbic acid, sodium bicarbonate, basic fibroblast growth factor, transforming growth factor beta (TGF beta), as it is particularly suitable for maintaining the undifferentiated nature of pluripotent stem cells It is more preferable to use a medium containing it, and it is most preferable to use a medium to which a basic fibroblast growth factor has been added.

The type of medium to which the basic fibroblast growth factor has been added is not particularly limited. For example, as a commercial product, DMEM (manufactured by Sigma-Aldrich Co. LLC), Ham's F12 (Sigma-Aldrich Co. LLC) D-MEM / Ham's F12 (Sigma-Aldrich Co. LLC), Primate ES Cell Medium (Reprocell Inc.), StemFit AK02N (Ajinomoto Co.), StemFit AK03 (Ajinomoto Co.) Made), mTeSR1 (made by STEMCELL TECHNOLOGIES), TeSR-E8 (made by STEMCELL TECHNOLOGIES), ReproNaive (made by REPROCELL), ReproXF (made) Made by EPROCELL, ReproFF (made by REPROCELL Co., Ltd.), ReproFF2 (made by REPROCELL Co., Ltd.), NutriStem (made by Biological Instries, Inc.), iSTEM (made by Takara Bio Inc.), GS2-M (Takara Bio (Takara Bio) And hPSC Growth Medium DXF (PromoCell Co., Ltd.) and the like. Primate ES Cell Medium (manufactured by REPROCELL Co., Ltd.), StemFit AK02N (manufactured by Ajinomoto Co.) or StemFit AK03 (manufactured by Ajinomoto Co., Ltd.) because it is suitable for maintaining the undifferentiated state of pluripotent stem cells. StemFit AK02N (manufactured by Ajinomoto Co., Ltd.) or StemFit AK03 (manufactured by Ajinomoto Co., Ltd.) is more preferable, and StemFit AK02N (manufactured by Ajinomoto Co., Ltd.) is particularly preferable.

Although there is no restriction | limiting in particular in the said (1) process in the seeding method of a pluripotent stem cell, For example, it can carry out by inject | pouring cell suspension into the said base material. The cell density at the time of seeding is not particularly limited, but 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 preferable, 1.0 × 10 ³ to 2.0 × 10 ⁵ cells / cm ² are particularly preferable, and 1.2 × 10 ³ to 1 The most preferable is .0 × 10 ⁵ cells / cm ² .

The medium used in the above step (1) is also suitable for maintaining the survival of pluripotent stem cells, so that the medium supplemented with the basic fibroblast growth factor is further added with a Rho-linked kinase inhibitor. It is preferable to use a culture medium. In particular, when human pluripotent stem cells are used and the cell density of human pluripotent stem cells is low, when a Rho-linked kinase inhibitor is added, survival of human pluripotent stem cells can be maintained. It may be effective. As a Rho binding kinase inhibitor, for example, (R)-(+)-trans-N- (4-pyridine) -4- (1-aminoethyl) -cyclohexanecarboxamide · 2HCl · H2O (manufactured by Wako Pure Chemical Industries, Ltd.) Y-27632), 1- (5-Isoquinolinessulfonyl) homopiperazine Hydrochloride (HA1077 manufactured by Wako Pure Chemical Industries, Ltd.) can be used. The concentration of the Rho-binding kinase inhibitor added to the culture medium is a range effective for maintaining the survival of human pluripotent stem cells and not affecting the undifferentiated state of human pluripotent stem cells, Preferably, it is 1 μM to 50 μM, more preferably 3 μM to 20 μM, still more preferably 5 μM to 15 μM, and most preferably 8 μM to 12 μM.

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

In the step (2) of the method for producing pluripotent stem cells of the present invention, the seeded pluripotent stem cells are cultured at a temperature higher than the response temperature of the temperature responsive polymer. When the culture temperature is equal to or higher than the response temperature of the thermoresponsive polymer, pluripotent stem cells can be proliferated. When the culture temperature is lower than the response temperature of the thermoresponsive polymer, pluripotent stem cells can not be grown. The culture temperature is preferably 30 to 42 ° C., more preferably 32 to 40 ° C., particularly preferably 36 to 38 ° C., and most preferably 37 ° C. because the culture temperature is suitable for maintaining the cell proliferation ability, physiological activity and function. It is.

It is preferable to perform the first medium replacement 22 to 26 hours after the start of the step (2). It is preferable to carry out a second medium exchange 48 to 72 hours later, and then exchange the medium every 24 to 48 hours. During this time, pluripotent stem cells proliferate and form a cell mass called a colony. The culture is continued until the size of the colonies is about 1 mm, and then the process proceeds to step (3).

In the step (3) of the method for producing pluripotent stem cells of the present invention, the culture substrate on which the pluripotent stem cells are cultured is cooled to a temperature lower than the response temperature of the temperature responsive polymer, Stem cells are detached from the culture substrate. The temperature at the time of cooling is preferably 0 to 30 ° C., more preferably 3 to 25 ° C., and still more preferably 5 to 50 ° C. in order to detach pluripotent stem cells in a short time and reduce damage by cooling. It is 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 method for cooling 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, It is possible to use a method in which the medium is replaced with a cooled medium or buffer solution and left for a predetermined time.

In the step (3), a step of causing convection in the liquid containing the cultured pluripotent stem cells at the time of cooling in order to detach the pluripotent stem cells in a short time and further reduce the damage caused by the cooling. May be included. The method of generating convection is not particularly limited. For example, other than the method of mechanically generating forced convection inside the liquid by a method such as pipetting the culture solution, using a pump, or a stirring blade, the culture There can be mentioned a method of applying physical vibration to the substrate and a method of generating natural convection such as Marangoni convection by giving a temperature difference.

Since the culture substrate of the present invention is suitable for simplifying the process of exfoliating cells and enhancing the mass productivity of cells when culturing a large amount of cells, it is preferable to exfoliate the cells by a weak external stimulus. It is further preferable to peel by cooling and pipetting, cooling and tapping, and it is particularly preferable to peel by cooling and tapping. It is most preferable to peel off only by cooling. Here, in the present invention, “pipetting” indicates an operation of causing convection in the culture environment by repeating suction and discharge of the culture solution using an instrument such as a pipetman. Moreover, "tapping" shows the operation which gives vibration to culture environment by tapping a culture container.

Since the culture substrate of the present invention is suitable for use in cell culture prepared from cultured pluripotent stem cells for preparation of undifferentiated maintenance culture or differentiation induction spheroids, pluripotent pluripotent cells are passaged. The undifferentiated maintenance rate of adult stem cells is preferably 70% or more, more preferably 80% or more, particularly preferably 90% or more, and most preferably 95% or more. The undifferentiated maintenance rate can be measured by a flow cytometer using cells stained with undifferentiated markers.

Hereinafter, the present invention will be described in detail by citing modes for carrying out the present invention, but it is an example for explaining the present invention, and the present invention is not intended to be limited to the following contents. Moreover, it can change suitably and can be implemented within the range of the summary of this invention. In addition, unless otherwise indicated, the reagent used the commercial item.
<Composition of Polymer>
Proton nuclear magnetic resonance spectroscopy ( ¹ H-NMR) spectrum analysis using a nuclear magnetic resonance measurement apparatus (manufactured by JEOL Ltd., trade name: JNM-GSX400) or a nuclear magnetic resonance measurement apparatus (trade name: AVANCE III HD 500, manufactured by Bruker) It was determined from carbon nuclear magnetic resonance spectroscopy ( ¹³ C-NMR) spectral analysis using
<Molecular weight of polymer, molecular weight distribution>
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 Corp., the column uses two TSKgel Super AWM-H manufactured by Tosoh Corp., the column temperature is set to 40 ° C., and the eluent contains 10 mM sodium trifluoroacetate. It was measured using 1,1,1,3,3,3-hexafluoro-2-isopropanol or N, N-dimethylformamide containing 10 mM lithium bromide. The measurement sample was prepared and measured at 1.0 mg / mL. The calibration curve of molecular weight used polymethyl methacrylate (made by Polymer Laboratories Ltd.) of known molecular weight.
<Surface structure>
The surface structure of the temperature-responsive polymer-coated substrate at the culture temperature was observed with an AFM in water (SPM-9600 manufactured by Shimadzu Corporation). The cantilever was measured using BL-AC40TS-C2 (manufactured by Olympus Corporation).

Example 1
[Synthesis of block copolymer]
1.50 g (5.1 mmol) of 2-methacryloyloxyethyl phosphoryl choline in a test tube, 43 mg (106 μmol, 4-cyano-4-[(dodecylsulfonylthiocarbonyl) sulfonyl] pentanoic acid as an RAFT agent, azobisiso as an initiator 1.7 mg (10 μmol) of butyronitrile was added and dissolved in 10.2 mL of 1,4-dioxane / ethanol = 1: 1 mixed solution 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 1 day to obtain a polymer of 2-methacryloyloxyethyl phosphorylcholine.

Into a test tube, 1.0 g of the above-mentioned 2-methacryloyloxyethyl phosphorylcholine polymer, 2.40 g (16.9 mmol) of n-butyl methacrylate and 2.5 mg (15 μmol) of azobisisobutyronitrile are added, and 1,4-dioxane / It was dissolved in 17 mL of an 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 is poured into 300 mL of hexane, and the precipitated pale yellow solid is filtered and dried under reduced pressure for 1 day to obtain a polymer of 2-methacryloyloxyethyl phosphorylcholine and n-butyl methacrylate (2-methacryloyloxyethyl phosphorylcholine / n Butyl methacrylate diblock polymer) was obtained.

Add 0.5 g of the above-mentioned 2-methacryloyloxyethyl phosphorylcholine / n-butyl methacrylate diblock polymer, 0.62 g (5.5 mmol) of N-isopropylacrylamide, 0.2 mg (1 μmol) of azobisisobutyronitrile to a test tube The solution was dissolved in 5.5 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 1 day to obtain a 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 a 0.2 wt% ethanol solution and 3 mL of ethanol were mixed to prepare a 0.05 wt% ethanol solution. This solution was added to each well of a cell culture 6-well plate (manufactured by Corning Incorporated, material: polystyrene) 0.42 mL each, and dried under reduced pressure for 2 hours. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[LCST measurement of block segment (A)]
0.40 g (0.1 mmol) of 4-cyano-4-[(dodecylsulfanylthiocarbonyl) sulfanyl] pentanoic acid, 2.26 g (20 mmol) of N-isopropylacrylamide and 33 mg of azobis (isobutyronitrile). 2 mmol) were dissolved in 20 mL dioxane. After degassing by nitrogen bubbling for 30 minutes, reaction was performed at 70 ° C. for 24 hours. After completion of the reaction, the reaction solvent was evaporated under reduced pressure with a rotary evaporator, and the reaction solution was concentrated. The concentrate was poured into 250 mL of hexane, and the precipitated white solid was recovered and dried under reduced pressure to obtain an N-isopropylacrylamide polymer. The N-isopropylacrylamide polymer was dissolved in pure water to form a 0.6 wt% aqueous solution. This solution is placed in a quartz cell of 1 cm in optical path length, and the temperature is raised at a rate of 1 ° C./min while the transmittance of light with a wavelength of 500 nm is measured with a spectrophotometer (UH-5300, manufactured by Hitachi High-Technologies Corp.) did. When LCST was calculated | required by the middle point method, LCST was 32 degreeC.
[Response temperature measurement of block copolymer]
The cell contact angles (θ) (°) at 37 ° C., 20 ° C. and 10 ° C. in water of the block copolymer-coated substrate are measured, and the water contact angles to 37 ° C., 20 ° C. and 10 ° C. (180-θ) (°) was calculated. The contact angle of 3 μl of bubbles in water was measured using a contact angle meter DM300 manufactured by Kyowa Interface Science Co., Ltd., for θ.

The water contact angles at 37 ° C., 20 ° C. and 10 ° C. are shown in Table 3. The contact angle to water at 20 ° C is lower than the contact angle to water at 37 ° C, the block copolymer-coated substrate exhibits temperature responsiveness, and the response temperature is in the range of 20 to 37 ° C. I found that. Further, when the contact angle to water was similarly measured at 5 ° C. intervals in the range of 20 to 45 ° C., and the response temperature was determined by the midpoint method, the response temperature was 34 ° C.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Human iPS cells 201B7 were seeded at a density of 1300 cells / cm ^{2 on} the block copolymer-coated substrate and cultured at 37 ° C. in an environment of 5% CO ₂ concentration. As the medium, 0.2 mL / cm ² of StemFitAK02N (manufactured by Ajinomoto Co., Ltd.) was added. In addition, until 24 hours after cell seeding, Y-27632 (Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) and iMatrix-511 solution (Nippi Co., Ltd.) (2.5 μL / mL) were added to the above medium. Was cultured using a medium supplemented with

After 24 hours, 96 hours and 144 hours after cell seeding, the appearance of the cells was observed with a phase-contrast microscope to confirm cell adhesion and proliferation in each case, and the fresh medium was replaced. After culture medium exchange 144 hours after cell seeding, (1) when the substrate is cooled to 4 ° C, (2) when the substrate is cooled to 4 ° C and tapping is performed 20 times, (3) substrate Were cooled to 4 ° C. and pipetting was performed 10 times, and cell detachment was confirmed in each case. Almost all the cells were detached under the condition (3) and recovered as aggregates. The detachment of the cells was confirmed by phase contrast microscopy.

Example 2
[Synthesis of block copolymer]
1.50 g (5.1 mmol) of 2-methacryloyloxyethyl phosphorylcholine in a test tube, 25.3 mg (63 μmol) of 4-cyano-4-[(dodecylsulfonylthiocarbonyl) sulfonyl] pentanoic acid as an RAFT agent, as an initiator 1 mg (3 μmol) of azobisisobutyronitrile was added and dissolved in 10.2 mL of a 1,4-dioxane / ethanol = 1: 1 mixed solution. 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 an acetone: methanol = 20: 1 mixed solution, and the precipitated yellow solid was filtered and dried under reduced pressure for 1 day to obtain a polymer of 2-methacryloyloxyethyl phosphorylcholine.

In a test tube, 1.50 g of the above-mentioned 2-methacryloyloxyethyl phosphorylcholine polymer, 1.71 g (12.0 mmol) of n-butyl methacrylate and 2 mg (13 μmol) of azobisisobutyronitrile are added, 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 1 day to obtain 2-methacryloyloxyethyl phosphorylcholine / n-butyl methacrylate diblock polymer.

In a test tube, 0.75 g of the above-mentioned 2-methacryloyloxyethyl phosphorylcholine / n-butyl methacrylate diblock copolymer, 0.93 g (8.2 mmol) of N-isopropylacrylamide, 0.3 mg (2 μmol) of azobisisobutyronitrile In addition, it was dissolved in 8.2 mL of a 1,4-dioxane / 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 200 mL of hexane, and the precipitated white solid was filtered and dried under reduced pressure for 1 day to obtain a triblock copolymer.
[Preparation of Base Material Coated with Block Copolymer]
It produced by the same method as Example 1 [preparation of the base material with which the block copolymer was covered] except having used the said block copolymer.
[Response temperature measurement of block copolymer]
It measured by the same method as Example 1 [Wettability of the base material with which the block copolymer was covered] except having used the base material with which the said block copolymer was covered.

The water contact angles at 37 ° C., 20 ° C. and 10 ° C. are shown in Table 3. The contact angle to water at 20 ° C is lower than the contact angle to water at 37 ° C, the block copolymer-coated substrate exhibits temperature responsiveness, and the response temperature is in the range of 20 to 37 ° C. I found that.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The culture was performed in Example 1 [Pluripotent Stem Cell Culture Evaluation and Detachment Evaluation] except that the base material coated with the block copolymer was used.

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

Add 1.50 g of the above 2-methacryloyloxyethyl phosphorylcholine / n-butyl methacrylate random copolymer, 1.85 g (16.4 mmol) of N-isopropylacrylamide, and 0.5 mg (3 μmol) of azobisisobutyronitrile to a test tube The mixture was dissolved in 12.6 mL of a 1,4-dioxane / 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 500 mL of hexane, and the precipitated white solid was filtered and dried under reduced pressure for 1 day to obtain a diblock copolymer.
[Preparation of Base Material Coated with Block Copolymer]
It produced by the same method as Example 1 [preparation of the base material with which the block copolymer was covered] except having used the said block copolymer.
[Response temperature measurement of block copolymer]
It measured by the same method as Example 1 [Wettability of the base material with which the block copolymer was covered] except having used the base material with which the said block copolymer was covered.

After 24 hours, 96 hours and 144 hours after cell seeding, the appearance of the cells was observed with a phase-contrast microscope to confirm cell adhesion and proliferation in each case, and the fresh medium was replaced. After culture medium exchange 144 hours after cell seeding, (1) when the substrate is cooled to 4 ° C, (2) when the substrate is cooled to 4 ° C and tapping is performed 20 times, (3) substrate Were cooled to 4 ° C. and pipetting was performed 10 times, and cell detachment was confirmed in each case. Almost all the cells were detached under the condition (3) and recovered as aggregates. The detachment of the cells was confirmed by phase contrast microscopy. Example 4
[Synthesis of block copolymer]
In a test tube equipped with a three-way cock, 0.57 g (1.8 mmol) of propargyl ester of 4-cyanopentanoic acid dithiobenzoate, 12.80 g (90 mmol) of n-butyl methacrylate, 60 mg of azobis (isobutyronitrile) 0.36 mmol) was added and dissolved in 90 mL of a 1,4-dioxane / ethanol = 1: 1 mixed solution. The test tubes were frozen by immersion in liquid nitrogen and vacuum degassed with a vacuum pump and allowed to return to room temperature. This operation was repeated three times to remove dissolved oxygen in the test tube, and then reacted at 65 ° C. for 24 hours. After completion of the reaction, the reaction solvent was evaporated under reduced pressure with a rotary evaporator, and the reaction solution was concentrated. The concentrate was poured into 300 mL of methanol, and the precipitated red oily substance was recovered 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 the above-mentioned 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 tubes were frozen by immersion in liquid nitrogen and vacuum degassed with a vacuum pump and allowed to return to room temperature. This operation was repeated three times to remove dissolved oxygen in the test tube, and then reacted at 65 ° C. for 43 hours. After completion of the reaction, the reaction solvent was evaporated under reduced pressure using a rotary evaporator, and the reaction solution was concentrated. The concentrate was poured into 1000 mL of hexane, and the precipitated pale red solid was recovered and dried under reduced pressure to obtain an N-isopropylacrylamide / n-butyl methacrylate diblock copolymer having a terminal alkyne.

On the other hand, 0.20 g (0.57 mmol) of 3-azidopropyl ester of 4-cyanopentanoic acid dithiobenzoate, 12.01 g (40 mmol) of polyethylene glycol methacrylate (manufactured by Aldrich, Mn = 300) in a test tube equipped with a three-way cock Then, 18.8 mg (0.11 mmol) of AIBN was added, and 28 mL of 1,4-dioxane was added and dissolved. The test tubes were frozen by immersion in liquid nitrogen and vacuum degassed with a vacuum pump and allowed to return to room temperature. This operation was repeated three times to remove the 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 evaporated under reduced pressure using a rotary evaporator, and the reaction solution was concentrated. The concentrate was poured into 500 ml of hexane and the red oil adhering to the bottom was recovered. The red oil obtained was washed twice with 300 mL of hexane and dried under vacuum to obtain a polyethylene glycol methacrylate polymer block having a terminal azide.

Subsequently, 0.50 g of the N-isopropyl acrylamide / n-butyl methacrylate diblock copolymer having the terminal alkyne and 0.94 g of a polyethylene glycol methacrylate polymer having the terminal azide are added to a test tube equipped with a three-way cock, Nitrogen substitution was performed. 9 mL of DMF subjected to nitrogen bubbling 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 a test tube under nitrogen flow and reacted 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 by 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). The obtained solid content was dissolved in 2 ml of methanol and poured again slowly into 50 ml of pure water, and the precipitated solid content was recovered by centrifugation (3000 rpm × 3 minutes). Vacuum drying was carried out to obtain a triblock copolymer consisting of a polyethylene glycol methacrylate polymer, an n-butyl methacrylate polymer and an N-isopropylacrylamide polymer block.
[Preparation of Base Material Coated with Block Copolymer]
It produced by the same method as Example 1 [preparation of the base material with which the block copolymer was covered] except having used the said block copolymer.
[Response temperature measurement of block copolymer]
It measured by the same method as Example 1 [Wettability of the base material with which the block copolymer was covered] except having used the base material with which the said block copolymer was covered.

The water contact angles at 37 ° C., 20 ° C. and 10 ° C. are shown in Table 3. The contact angle to water at 20 ° C is lower than the contact angle to water at 37 ° C, the block copolymer-coated substrate exhibits temperature responsiveness, and the response temperature is in the range of 20 to 37 ° C. I found that. Further, when the contact angle to water was similarly measured at 5 ° C. intervals in the range of 20 to 45 ° C., and the response temperature was determined by the midpoint method, the response temperature was 34 ° C.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The culture was performed in Example 1 [Pluripotent Stem Cell Culture Evaluation and Detachment Evaluation] except that the base material coated with the block copolymer was used.

After 24 hours, 96 hours and 144 hours after cell seeding, the appearance of the cells was observed with a phase-contrast microscope to confirm cell adhesion and proliferation in each case, and the fresh medium was replaced. After medium change 144 hours after cell seeding, 0.25 mM ethylenediaminetetraacetic acid solution is added and (1) substrate is cooled to 4 ° C, (2) substrate is cooled to 4 ° C and 20 times When tapping was performed, (3) the substrate was cooled to 4 ° C., and peeling of cells was confirmed in each case of pipetting 10 times. Almost all cells were detached under the condition of (3) and recovered as single cells. The detachment of the cells was confirmed by phase contrast microscopy.

Example 5
[Synthesis of block copolymer]
Synthesis was carried out in the same manner as Example 4 [Synthesis of block copolymer] except that polymerization was carried out at 65 ° C. for 9 hours using 5.20 g (40 mmol) of 2-methoxyethyl acrylate instead of 12.01 g of polyethylene glycol methacrylate. To synthesize a diblock copolymer.
[Preparation of Base Material Coated with Block Copolymer]
It produced by the method similar to Example 1 [preparation of the base material with which the block copolymer was coated] except having used the said block copolymer.
[Response temperature measurement of block copolymer]
It measured by the same method as Example 1 [Wettability of the base material with which the block copolymer was covered] except having used the base material with which the said block copolymer was covered.

The water contact angles at 37 ° C., 20 ° C. and 10 ° C. are shown in Table 3. The contact angle to water at 20 ° C is lower than the contact angle to water at 37 ° C, the block copolymer-coated substrate exhibits temperature responsiveness, and the response temperature is in the range of 20 to 37 ° C. I found that. Further, when the contact angle to water was similarly measured at 5 ° C. intervals in the range of 20 to 45 ° C., and the response temperature was determined by the midpoint method, the response temperature was 33 ° C.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The culture was performed in Example 1 [Pluripotent Stem Cell Culture Evaluation and Detachment Evaluation] except that the base material coated with the block copolymer was used.

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) Were dissolved in 9 mL of a 1,4-dioxane / ethanol = 1: 1 mixed solution. 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 evaporated under reduced pressure with a rotary evaporator, and the reaction solution was concentrated. The concentrate was poured into 250 mL of methanol, and the precipitated yellow oily substance was recovered and dried under reduced pressure to obtain an n-butyl methacrylate polymer.

In a test tube, 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 are added. 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 evaporated 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 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 produced by the method similar to Example 1 [preparation of the base material with which the block copolymer was coated] except having used the said block copolymer.
[Response temperature measurement of block copolymer]
It measured by the same method as Example 1 [Wettability of the base material with which the block copolymer was covered] except having used the base material with which the said block copolymer was covered.

The water contact angles at 37 ° C., 20 ° C. and 10 ° C. are shown in Table 3. The contact angle to water at 20 ° C is lower than the contact angle to water at 37 ° C, the block copolymer-coated substrate exhibits temperature responsiveness, and the response temperature is in the range of 20 to 37 ° C. I found that. Further, when the contact angle to water was similarly measured at 5 ° C. intervals in the range of 20 to 45 ° C., and the response temperature was determined by the midpoint method, the response temperature 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 block copolymer-coated substrate at 37 ° C. It was allowed to stand for 1 hour in an environment of 5% CO ₂ concentration. Thereafter, the iMatrix-511 solution was removed, and the human iPS cell 201B7 strain was seeded at a density of 1300 cells / cm ² and cultured at 37 ° C. in a 5% CO ₂ environment. As the medium, 0.2 mL / cm ² of StemFitAK02N (manufactured by Ajinomoto Co., Ltd.) was added. In addition, until 24 hours after cell seeding, Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) was added to the above-mentioned medium and cultured.

The culture was performed in Example 1 [Pluripotent Stem Cell Culture Evaluation and Detachment Evaluation] except that the base material coated with the block copolymer was used.

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 performed at 70 ° C. for 24 hours. After completion of the reaction, the reaction solvent was evaporated under reduced pressure with a rotary evaporator, and the reaction solution was concentrated. The concentrate was poured into 250 mL of methanol, and the precipitated yellow oily substance was recovered 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, 2.65 g (23.5 mmol) of N-isopropylacrylamide, and 5 mg (0.03 mmol) of azobisisobutyronitrile are added. It was dissolved in 15 mL of 4-dioxane. After degassing by nitrogen bubbling for 30 minutes, reaction was performed at 65 ° C. for 17 hours. After completion of the reaction, the reaction solvent was diluted with acetone, 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 N-isopropylacrylamide and n-butyl methacrylate.
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer was dissolved in ethanol to make a 0.5 wt% solution. 50 μL of this solution was dropped to a 3.5 cm diameter dish (manufactured by Corning Incorporated, material: polystyrene), and spin coated at 1000 rpm for 60 seconds. Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Response temperature measurement of block copolymer]
It measured by the same method as Example 1 [Wettability of the base material with which the block copolymer was covered] except having used the base material with which the said block copolymer was covered.

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 performed at 70 ° C. for 24 hours. After completion of the reaction, the reaction solvent was evaporated under reduced pressure with a rotary evaporator, and the reaction solution was concentrated. The concentrate was poured into 250 mL of methanol, and the precipitated yellow oily substance was recovered 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-isopropyl acrylamide, 5 mg (0.03 mmol) of azobisisobutyronitrile are added, and 1,4- It was dissolved in 15 mL of dioxane. After degassing by nitrogen bubbling for 30 minutes, reaction was performed at 65 ° C. for 17 hours. After completion of the reaction, the reaction solvent was diluted with acetone, 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 N-isopropylacrylamide and n-butyl methacrylate.
[Preparation of Base Material Coated with Block Copolymer]
It produced by the method similar to Example 7 [preparation of the base material with which the block copolymer was coated] except having used the said block copolymer.
[Response temperature measurement of block copolymer]
It measured by the same method as Example 7 [Wettability of the base material with which the block copolymer was covered] except having used the base material with which the said block copolymer was covered.

The water contact angles at 37 ° C., 20 ° C. and 10 ° C. are shown in Table 3. The contact angle to water at 20 ° C is lower than the contact angle to water at 37 ° C, the block copolymer-coated substrate exhibits temperature responsiveness, and the response temperature is in the range of 20 to 37 ° C. I found that. Further, when the contact angle to water was similarly measured at 5 ° C. intervals in the range of 20 to 45 ° C., and the response temperature was determined by the midpoint method, the response temperature was 33 ° C.
[Structure of substrate surface coated with block copolymer]
An AFM image in water at 37 ° C. of the substrate surface coated with the block copolymer is shown in FIG. The layer of the block copolymer forms a phase separation structure, and a region with a large amount of block copolymer coverage and a small region are present.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The culture was performed in Example 1 [Pluripotent Stem Cell Culture Evaluation and Detachment Evaluation] except that the base material coated with the block copolymer was used.

After 24 hours, 96 hours and 144 hours after cell seeding, the appearance of the cells was observed with a phase-contrast microscope to confirm cell adhesion and proliferation in each case, and the fresh medium was replaced. After culture medium exchange 144 hours after cell seeding, (1) when the substrate is cooled to 4 ° C, (2) when the substrate is cooled to 4 ° C and tapping is performed 20 times, (3) substrate Were cooled to 4 ° C. and pipetting was performed 10 times, and cell detachment was confirmed in each case. Under the conditions of (2) and (3), almost all the cells were exfoliated and recovered as aggregates. The detachment of the cells was confirmed by phase contrast microscopy.

Example 9
[Synthesis of block copolymer]
In a test tube equipped with a three-way cock, 95 mg of propargyl ester of 4-cyanopentanoic acid dithiobenzoate, 6.8 g of N-isopropylacrylamide and 8 mg of AIBN were added and dissolved in 20 ml of 1,4-dioxane. After bubbling nitrogen for 30 minutes, the mixture was stirred at 70 ° C. for 24 hours for polymerization. After completion of the polymerization, 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.

On the other hand, 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 cock and dissolved in 20 mL of DMF did. After bubbling nitrogen for 30 minutes, the mixture was stirred at 70 ° C. for 24 hours for polymerization. After completion of the polymerization, 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 the terminal alkyne and 0.30 g of the N- (4-hydroxyphenyl) methacrylamide polymer having the terminal azide are added to a test tube equipped with a three-way cock, Nitrogen substitution was performed. 9 mL of DMF subjected to nitrogen bubbling was added and dissolved. A solution separately prepared of 14 mg of copper (I) bromide, 31 mg of 2,2'-bipyridyl and 1 mL of DMF was added to a test tube under nitrogen flow and reacted at room temperature for 48 hours. After completion of the reaction, the three-way cock was removed, and the air was made to touch to inactivate 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 by a rotary evaporator. The concentrate was dissolved in a mixed solvent of methanol and acetone, hexane was added little by little, and the precipitated polymer was recovered. The 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 produced by the method similar to Example 7 [preparation of the base material with which the block copolymer was coated] except having used the said block copolymer.
[Response temperature measurement of block copolymer]
It measured by the same method as Example 1 [Wettability of the base material with which the block copolymer was covered] except having used the base material with which the said block copolymer was covered.

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 performed at 70 ° C. for 24 hours. After completion of the reaction, the reaction solvent was evaporated under reduced pressure with a rotary evaporator, and the reaction solution was concentrated. The concentrate was poured into 250 mL of methanol, and the precipitated yellow oily substance was recovered 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 Nn-propyl acrylamide, 5 mg (0.03 mmol) of azobisisobutyronitrile are added, It was dissolved in 15 mL of 4-dioxane. After degassing by nitrogen bubbling for 30 minutes, reaction was performed at 65 ° C. for 17 hours. After completion of the reaction, the reaction solvent was diluted with acetone, 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-propyl acrylamide and n-butyl methacrylate. .
[Preparation of Base Material Coated with Block Copolymer]
It produced by the method similar to Example 7 [preparation of the base material with which the block copolymer was coated] except having used the said block copolymer.
[LCST measurement of block segment (A)]
0.40 g (0.1 mmol) of 4-cyano-4-[(dodecylsulfanylthiocarbonyl) sulfanyl] pentanoic acid, 2.26 g (20 mmol) of N-n-propylacrylamide and 33 mg of azobis (isobutyronitrile) 0.2 mmol) was dissolved in 20 mL dioxane. After degassing by nitrogen bubbling for 30 minutes, reaction was performed at 70 ° C. for 24 hours. After completion of the reaction, the reaction solvent was evaporated under reduced pressure with a rotary evaporator, and the reaction solution was concentrated. 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-n-propyl acrylamide polymer. The N-n-propyl acrylamide polymer was dissolved in pure water to form a 0.6 wt% aqueous solution. This solution was placed in a quartz cell with a light path length of 1 cm, and while raising the temperature at a rate of 1 ° C./min, the transmittance of light with a wavelength of 500 nm was measured with a spectrophotometer (UH-5300 manufactured by Hitachi High-Technologies Corporation). When LCST was calculated | required by the middle point method, LCST was 17 degreeC.
[Response temperature measurement of block copolymer]
It measured by the same method as Example 1 [Wettability of the base material with which the block copolymer was covered] except having used the base material with which the said block copolymer was covered.

The water contact angles at 37 ° C., 20 ° C. and 10 ° C. are shown in Table 3. The contact angle to water at 10 ° C is lower than the contact angle to water at 20 ° C, the block copolymer-coated substrate exhibits temperature responsiveness, and the response temperature is in the range of 10 to 20 ° C. I found that. Further, when the contact angle to water was similarly measured at 5 ° C. intervals in the range of 5 to 30 ° C. and the response temperature was determined by the midpoint method, the response temperature was 18 ° C.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The culture was performed in Example 1 [Pluripotent Stem Cell Culture Evaluation and Detachment Evaluation] except that the base material coated with the block copolymer was used.

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 block copolymer-coated substrate prepared in Example 8 and allowed to stand at 37 ° C., 5% CO ₂ environment for 1 hour did. Thereafter, the Matrigel solution was removed, and human iPS cell 201B7 strain was seeded at a density of 1300 cells / cm ² and cultured at 37 ° C. in an environment of 5% CO ₂ concentration. As the medium, 0.2 mL / cm ² of StemFitAK02N (manufactured by Ajinomoto Co., Ltd.) was added. In addition, until 24 hours after cell seeding, culture was performed using a medium to which Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) was added to the above-mentioned medium.

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 block copolymer-coated substrate prepared in Example 8 It was allowed to stand at 37 ° C. in a 5% CO ₂ environment for 1 hour. Thereafter, the iMatrix-511 solution was removed, and the human iPS cell 201B7 strain was seeded at a density of 1300 cells / cm ² and cultured at 37 ° C. in a 5% CO ₂ environment. As the medium, 0.2 mL / cm ² of StemFitAK02N (manufactured by Ajinomoto Co., Ltd.) was added. In addition, until 24 hours after cell seeding, culture was performed using a medium to which Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) was added to the above-mentioned medium.

Example 13
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to form a 0.5 wt% solution. This solution was a porous membrane with a pore size of 0.4 μm and a porosity of 0.2% and a pore density of 1.8 × 10 ⁶ / cm ² (manufactured by Corning Incorporated, trade name Falcon® culture insert, Material: 50 μL was dropped to polyethylene terephthalate) and spin coated at 1000 rpm for 60 seconds. Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The substrate coated with the block copolymer was immersed in a medium and fixed, and human iPS cell 201B7 strain was seeded at a density of 1300 cells / cm ² and cultured at 37 ° C. under an environment of 5% CO ₂ concentration. . As the medium, 0.2 mL / cm ² of StemFitAK02N (manufactured by Ajinomoto Co., Ltd.) was added. In addition, until 24 hours after cell seeding, Y-27632 (Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) and iMatrix-511 solution (Nippi Co., Ltd.) (2.5 μL / mL) were added to the above medium. Was cultured using a medium supplemented with

After 24 hours, 96 hours and 144 hours after cell seeding, the appearance of the cells was observed with a phase-contrast microscope to confirm cell adhesion and proliferation in each case, and the fresh medium was replaced. After culture medium exchange 144 hours after cell seeding, (1) when the substrate is cooled to 4 ° C, (2) when the substrate is cooled to 4 ° C and tapping is performed 20 times, (3) substrate Were cooled to 4 ° C. and pipetting was performed 10 times, and cell detachment was confirmed in each case. Almost all cells were exfoliated under all the conditions (1) to (3) and recovered as aggregates. The detachment of the cells was confirmed by phase contrast microscopy.

Example 14
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to form a 0.5 wt% solution. This solution is a 13.8% porous film having a pore diameter of 0.4 μm and a pore density of 1.1 × 10 ⁸ / cm ² (manufactured by Corning Incorporated, trade name Falcon® culture insert, Material: 50 μL was dropped to polyethylene terephthalate) and spin coated at 1000 rpm for 60 seconds. Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The culture was performed in Example 13 [multipotent stem cell culture evaluation and exfoliation evaluation] except that the base material coated with the block copolymer was used.

24 hours after cell seeding, 96 hours and 144 hours after, fresh medium was replaced. After culture medium exchange 144 hours after cell seeding, (1) when the substrate is cooled to 4 ° C, (2) when the substrate is cooled to 4 ° C and tapping is performed 20 times, (3) substrate Were cooled to 4 ° C. and pipetting was performed 10 times, and cell detachment was confirmed in each case. Almost all the cells were detached under the condition (3) and recovered as aggregates. The cell detachment was confirmed by measuring the number of cells in the culture medium before and after the detachment operation, in addition to visual observation by applying light to the culture substrate.

Example 15
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to form a 0.5 wt% solution. This solution is a porous film having a pore diameter of 0.4 μm and a porosity of 12.6% and a porosity of 1 × 10 ⁸ pores / cm ² (Corning Incorporated, trade name Falcon® culture insert, Material: 50 μL was dropped to polycarbonate) and spin coated at 1000 rpm for 60 seconds. Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The culture was performed in Example 13 [multipotent stem cell culture evaluation and exfoliation evaluation] except that the base material coated with the block copolymer was used.

Example 16
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to form a 0.5 wt% solution. This solution is a porous membrane with a pore diameter of 1 μm and a porosity of 1.4% having pores with a pore density of 1.8 × 10 ⁶ / cm ² (manufactured by Corning Incorporated, trade name Falcon® culture insert, material: 50 μL was dropped to polyethylene terephthalate) and spin coated at 1000 rpm for 60 seconds. Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The culture was performed in Example 13 [multipotent stem cell culture evaluation and exfoliation evaluation] except that the base material coated with the block copolymer was used.

Example 17
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to form a 0.5 wt% solution. This solution is a porous membrane having a pore size of 3 μm and a porosity of 4.1 × 10 ⁵ / cm ² and a porosity of 4.1% (manufactured by Corning Incorporated, trade name Falcon® culture insert, material: 50 μL was dropped to polyethylene terephthalate) and spin coated at 1000 rpm for 60 seconds. Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The culture was performed in Example 13 [multipotent stem cell culture evaluation and exfoliation evaluation] except that the base material coated with the block copolymer was used.

Example 18
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to form a 0.5 wt% solution. This solution is a porous membrane having a pore diameter of 3 μm and a porosity of 12.0% having pores with a pore density of 1.7 × 10 ⁶ / cm ² (manufactured by Greiner, trade name: ThinCert (trade name) Cell Culture Inserts, material: 50 μL was dropped to polyethylene terephthalate) and spin coated at 1000 rpm for 60 seconds. Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The culture was performed in Example 13 [multipotent stem cell culture evaluation and exfoliation evaluation] except that the base material coated with the block copolymer was used.

Example 19
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to form a 0.5 wt% solution. This solution is a porous membrane with a pore size of 8 μm and a pore density of 6.2 × 10 ⁴ / cm ² and a porosity of 3.1% (manufactured by Corning Incorporated, trade name Falcon® culture insert, material: 50 μL was dropped to polyethylene terephthalate) and spin coated at 1000 rpm for 60 seconds. Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The culture was performed in Example 13 [multipotent stem cell culture evaluation and exfoliation evaluation] except that the base material coated with the block copolymer was used.

Example 20
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to form a 0.5 wt% solution. 50 μL of this solution was added dropwise to a porous membrane (made by Teijin Ltd., trade name Milim, material: polyethylene) having a pore size of 0.2 μm, and spin-coated at 1000 rpm for 60 seconds. Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The culture was performed in Example 13 [multipotent stem cell culture evaluation and exfoliation evaluation] except that the base material coated with the block copolymer was used.

24 hours after cell seeding, 96 hours and 144 hours after, fresh medium was replaced. After culture medium exchange 144 hours after cell seeding, (1) when the substrate is cooled to 4 ° C, (2) when the substrate is cooled to 4 ° C and tapping is performed 20 times, (3) substrate Were cooled to 4 ° C. and pipetting was performed 10 times, and cell detachment was confirmed in each case. Under the conditions of (2) and (3), almost all the cells were exfoliated and recovered as aggregates. The cell detachment was confirmed by measuring the number of cells in the culture medium before and after the detachment operation, in addition to visual observation by applying light to the culture substrate.

Example 21
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to form a 0.6 wt% solution. A porous membrane having a pore size of 0.05 μm and a porosity of 1.2% having pores with a pore density of 6.0 × 10 ⁸ / cm ² (it 4 ip, product name: ipPORE, material: polycarbonate) having a diameter of 3.2 cm, It fixed by pinching with a ring of internal diameter 1.6 cm. Plasma irradiation (flowing gas: air, 20 Pa gas pressure, conductive current 20 mA, irradiation for 1 minute) was applied to the porous film, and 30 μL of an ethanol solution in which the block copolymer was dissolved was dropped and spin coated at 2000 rpm for 60 seconds . Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The substrate coated with the block copolymer was immersed in a medium and fixed, and human iPS cell 201B7 strain was seeded at a density of 1300 cells / cm ² and cultured at 37 ° C. under an environment of 5% CO ₂ concentration. . As the medium, StemFitAK02N (manufactured by Ajinomoto Co., Ltd.) was added in such an amount that the liquid level on the porous membrane was 2 mm (= 0.2 mL / cm ^{2 with} respect to the area of the porous membrane). In addition, until 24 hours after cell seeding, Y-27632 (Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) and iMatrix-511 solution (Nippi Co., Ltd.) (2.5 μL / mL) were added to the above medium. Was cultured using a medium supplemented with

Example 22
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to form a 0.6 wt% solution. Porous membrane (made by it4ip, trade name: ipPORE, material: polyethylene terephthalate) having a pore diameter of 0.1 μm and a porosity of 4.7% having pores with a pore density of 6.0 × 10 ⁸ / cm ² and a diameter of 3.2 cm It was fixed by being pinched by a ring with an inner diameter of 1.6 cm. Plasma irradiation (flowing gas: air, 20 Pa gas pressure, conductive current 20 mA, irradiation for 1 minute) was applied to the porous film, and 30 μL of an ethanol solution in which the block copolymer was dissolved was dropped and spin coated at 2000 rpm for 60 seconds . Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The substrate coated with the block copolymer was immersed in a medium and fixed, and human iPS cell 201B7 strain was seeded at a density of 1300 cells / cm ² and cultured at 37 ° C. under an environment of 5% CO ₂ concentration. . As the medium, StemFitAK02N (manufactured by Ajinomoto Co., Ltd.) was added in such an amount that the liquid level on the porous membrane was 2 mm (= 0.2 mL / cm ^{2 with} respect to the area of the porous membrane). In addition, until 24 hours after cell seeding, Y-27632 (Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) and iMatrix-511 solution (Nippi Co., Ltd.) (2.5 μL / mL) were added to the above medium. Was cultured using a medium supplemented with

Example 23
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to form a 0.6 wt% solution. A porous film (product made by it4ip, trade name: ipPORE, material: polyethylene terephthalate) having a pore diameter of 0.2 μm and a pore density of 5.0 × 10 ⁸ pores / cm ² and having a diameter of 3.2 cm It was fixed by being pinched by a ring with an inner diameter of 1.6 cm. Plasma irradiation (flowing gas: air, 20 Pa gas pressure, conductive current 20 mA, irradiation for 1 minute) was applied to the porous film, and 30 μL of an ethanol solution in which the block copolymer was dissolved was dropped and spin coated at 2000 rpm for 60 seconds . Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The substrate coated with the block copolymer was immersed in a medium and fixed, and human iPS cell 201B7 strain was seeded at a density of 1300 cells / cm ² and cultured at 37 ° C. under an environment of 5% CO ₂ concentration. . As the medium, StemFitAK02N (manufactured by Ajinomoto Co., Ltd.) was added in such an amount that the liquid level on the porous membrane was 2 mm (= 0.2 mL / cm ^{2 with} respect to the area of the porous membrane). In addition, until 24 hours after cell seeding, Y-27632 (Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) and iMatrix-511 solution (Nippi Co., Ltd.) (2.5 μL / mL) were added to the above medium. Was cultured using a medium supplemented with

Example 24
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to form a 0.6 wt% solution. Porous membrane (product made by it4ip, trade name: ipPORE, material: polyethylene terephthalate) having a pore size of 0.4 μm and a pore density of 1.5 × 10 ⁸ pores / cm ² and having a diameter of 3.2 cm It was fixed by being pinched by a ring with an inner diameter of 1.6 cm. Plasma irradiation (flowing gas: air, 20 Pa gas pressure, conductive current 20 mA, irradiation for 1 minute) was applied to the porous film, and 30 μL of an ethanol solution in which the block copolymer was dissolved was dropped and spin coated at 2000 rpm for 60 seconds . Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The substrate coated with the block copolymer was immersed in a medium and fixed, and human iPS cell 201B7 strain was seeded at a density of 1300 cells / cm ² and cultured at 37 ° C. under an environment of 5% CO ₂ concentration. . As the medium, StemFitAK02N (manufactured by Ajinomoto Co., Ltd.) was added in such an amount that the liquid level on the porous membrane was 2 mm (= 0.2 mL / cm ^{2 with} respect to the area of the porous membrane). In addition, until 24 hours after cell seeding, Y-27632 (Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) and iMatrix-511 solution (Nippi Co., Ltd.) (2.5 μL / mL) were added to the above medium. Was cultured using a medium supplemented with

Example 25
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to form a 0.6 wt% solution. Porous membrane (product made by it4ip, trade name: ipPORE, material: polyethylene terephthalate) having a pore diameter of 0.45 μm and a pore density of 4.0 × 10 ⁶ / cm ^{2 and} a pore diameter of 3.2 cm It was fixed by being pinched by a ring with an inner diameter of 1.6 cm. Plasma irradiation (flowing gas: air, 20 Pa gas pressure, conductive current 20 mA, irradiation for 1 minute) was applied to the porous film, and 30 μL of an ethanol solution in which the block copolymer was dissolved was dropped and spin coated at 2000 rpm for 60 seconds . Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The substrate coated with the block copolymer was immersed in a medium and fixed, and human iPS cell 201B7 strain was seeded at a density of 1300 cells / cm ² and cultured at 37 ° C. under an environment of 5% CO ₂ concentration. . As the medium, StemFitAK02N (manufactured by Ajinomoto Co., Ltd.) was added in such an amount that the liquid level on the porous membrane was 2 mm (= 0.2 mL / cm ^{2 with} respect to the area of the porous membrane). In addition, until 24 hours after cell seeding, Y-27632 (Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) and iMatrix-511 solution (Nippi Co., Ltd.) (2.5 μL / mL) were added to the above medium. Was cultured using a medium supplemented with

Example 26
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to form a 0.6 wt% solution. A porous membrane (made by it4ip, trade name: ipPORE, material: polycarbonate) having a pore diameter of 0.6 μm and a porosity of 11.3% having pores with a pore density of 4.0 × 10 ⁷ / cm ² and a diameter of 3.2 cm, It fixed by pinching with a ring of internal diameter 1.6 cm. Plasma irradiation (flowing gas: air, 20 Pa gas pressure, conductive current 20 mA, irradiation for 1 minute) was applied to the porous film, and 30 μL of an ethanol solution in which the block copolymer was dissolved was dropped and spin coated at 2000 rpm for 60 seconds . Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The substrate coated with the block copolymer was immersed in a medium and fixed, and human iPS cell 201B7 strain was seeded at a density of 1300 cells / cm ² and cultured at 37 ° C. under an environment of 5% CO ₂ concentration. . As the medium, StemFitAK02N (manufactured by Ajinomoto Co., Ltd.) was added in such an amount that the liquid level on the porous membrane was 2 mm (= 0.2 mL / cm ^{2 with} respect to the area of the porous membrane). In addition, until 24 hours after cell seeding, Y-27632 (Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) and iMatrix-511 solution (Nippi Co., Ltd.) (2.5 μL / mL) were added to the above medium. Was cultured using a medium supplemented with

Example 27
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to form a 0.6 wt% solution. Porous membrane (product made by it4ip, trade name: ipPORE, material: polyethylene terephthalate) having a pore size of 0.8 μm and a pore density of 4.0 × 10 ⁷ / cm ^{2 and} a pore diameter of 3.2 cm It was fixed by being pinched by a ring with an inner diameter of 1.6 cm. Plasma irradiation (flowing gas: air, 20 Pa gas pressure, conductive current 20 mA, irradiation for 1 minute) was applied to the porous film, and 30 μL of an ethanol solution in which the block copolymer was dissolved was dropped and spin coated at 2000 rpm for 60 seconds . Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The substrate coated with the block copolymer was immersed in a medium and fixed, and human iPS cell 201B7 strain was seeded at a density of 1300 cells / cm ² and cultured at 37 ° C. under an environment of 5% CO ₂ concentration. . As the medium, StemFitAK02N (manufactured by Ajinomoto Co., Ltd.) was added in such an amount that the liquid level on the porous membrane was 2 mm (= 0.2 mL / cm ^{2 with} respect to the area of the porous membrane). In addition, until 24 hours after cell seeding, Y-27632 (Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) and iMatrix-511 solution (Nippi Co., Ltd.) (2.5 μL / mL) were added to the above medium. Was cultured using a medium supplemented with

Example 28
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to form a 0.6 wt% solution. Porous membrane with a pore size of 2 μm and a porosity of 9.4% having pores with a pore density of 3.0 × 10 ⁶ / cm ² (it 4 ip product, trade name ipPORE, material: polyethylene terephthalate), diameter 3.2 cm, inner diameter It fixed by pinching with a 1.6 cm ring. Plasma irradiation (flowing gas: air, 20 Pa gas pressure, conductive current 20 mA, irradiation for 1 minute) was applied to the porous film, and 30 μL of an ethanol solution in which the block copolymer was dissolved was dropped and spin coated at 2000 rpm for 60 seconds . Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The substrate coated with the block copolymer was immersed in a medium and fixed, and human iPS cell 201B7 strain was seeded at a density of 1300 cells / cm ² and cultured at 37 ° C. under an environment of 5% CO ₂ concentration. . As the medium, StemFitAK02N (manufactured by Ajinomoto Co., Ltd.) was added in such an amount that the liquid level on the porous membrane was 2 mm (= 0.2 mL / cm ^{2 with} respect to the area of the porous membrane). In addition, until 24 hours after cell seeding, Y-27632 (Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) and iMatrix-511 solution (Nippi Co., Ltd.) (2.5 μL / mL) were added to the above medium. Was cultured using a medium supplemented with

Example 29
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to form a 0.6 wt% solution. Porous membrane with a pore size of 5 μm and a porosity of 7.9% having pores with a pore density of 4.0 × 10 ⁵ / cm ² (it4ip product, trade name ipPORE, material: polycarbonate), diameter 3.2 cm, inner diameter 1 It was immobilized by pinching with a .6 cm ring. Plasma irradiation (flowing gas: air, 20 Pa gas pressure, conductive current 20 mA, irradiation for 1 minute) was applied to the porous film, and 30 μL of an ethanol solution in which the block copolymer was dissolved was dropped and spin coated at 2000 rpm for 60 seconds . Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The substrate coated with the block copolymer was immersed in a medium and fixed, and human iPS cell 201B7 strain was seeded at a density of 1300 cells / cm ² and cultured at 37 ° C. under an environment of 5% CO ₂ concentration. . As the medium, StemFitAK02N (manufactured by Ajinomoto Co., Ltd.) was added in such an amount that the liquid level on the porous membrane was 2 mm (= 0.2 mL / cm ^{2 with} respect to the area of the porous membrane). In addition, until 24 hours after cell seeding, Y-27632 (Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) and iMatrix-511 solution (Nippi Co., Ltd.) (2.5 μL / mL) were added to the above medium. Was cultured using a medium supplemented with

Example 30
[Preparation of Base Material Coated with Block Copolymer]
The block copolymer synthesized in Example 8 was dissolved in ethanol to form a 0.6 wt% solution. Porous membrane with a pore size of 10 μm and a porosity of 7.9% having pores with a pore density of 1.0 × 10 ⁵ / cm ² (it4ip product, trade name ipPORE, material: polyethylene terephthalate), diameter 3.2 cm, inner diameter It fixed by pinching with a 1.6 cm ring. Plasma irradiation (flowing gas: air, 20 Pa gas pressure, conductive current 20 mA, irradiation for 1 minute) was applied to the porous film, and 30 μL of an ethanol solution in which the block copolymer was dissolved was dropped and spin coated at 2000 rpm for 60 seconds . Dried at room temperature for 1 hour. Furthermore, the substrate was immersed in pure water for 24 hours and washed to prepare a substrate coated with the block copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The substrate coated with the block copolymer was immersed in a medium and fixed, and human iPS cell 201B7 strain was seeded at a density of 1300 cells / cm ² and cultured at 37 ° C. under an environment of 5% CO ₂ concentration. . As the medium, StemFitAK02N (manufactured by Ajinomoto Co., Ltd.) was added in such an amount that the liquid level on the porous membrane was 2 mm (= 0.2 mL / cm ^{2 with} respect to the area of the porous membrane). In addition, until 24 hours after cell seeding, Y-27632 (Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) and iMatrix-511 solution (Nippi Co., Ltd.) (2.5 μL / mL) were added to the above medium. Was cultured using a medium supplemented with

Example 31
[Passage culture and undifferentiated maintenance rate evaluation]
Medium StemFitAK02N the substrate block copolymer prepared was coated in Example 8 (manufactured by Ajinomoto (Ltd.)) 0.2mL / cm ² was added, further 260 human iPS cells 201B7 strain ^/ cm 2, ^iMatrix- A 511 solution (manufactured by Nippi Co., Ltd.) was added at a concentration of 2.5 μL / mL. The cells were cultured at 37 ° C. in a 5% CO ₂ environment. In addition, Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) was added to the medium until 24 hours after cell seeding.

After 24 hours, 96 hours and 144 hours after cell seeding, the appearance of the cells was observed with a phase-contrast microscope to confirm cell adhesion and proliferation in each case, and the fresh medium was replaced. After 168 hours of cell seeding, the cells were washed by removing all the media and then adding PBS buffer and aspirating off the PBS buffer. PBS buffer containing 0.25 mM ethylenediaminetetraacetic acid was added and incubated for 5 minutes at 37 ° C. After adding a culture medium containing Y-27632 (Wako Pure Chemical Industries, Ltd.) (concentration 10 μM), cool the substrate to 4 ° C, strike the side of the substrate to give vibration, and perform pipetting The cells were detached from the substrate according to to prepare a cell suspension. The cell suspension was used to perform passage by seeding cells on the block copolymer-coated substrate. Five passages were performed by repeating the same operation.

Five passaged cells are detached and collected, treated with PBS buffer containing 4% paraformaldehyde, and then stained with Alexa FluorTM 647 anti-human SSEA-4 Antibody. The undifferentiated maintenance rate of cells was measured by a flow cytometer. The undifferentiated maintenance rate was 96%.

Example 32
[Passage culture and undifferentiated maintenance rate evaluation]
Medium StemFitAK02N the substrate block copolymer prepared was coated with Example 13 (manufactured by Ajinomoto (Ltd.)) 0.2mL / cm ² was added, further 260 human iPS cells 201B7 strain ^/ cm 2, ^iMatrix- A 511 solution (manufactured by Nippi Co., Ltd.) was added at a concentration of 2.5 μL / mL. The cells were cultured at 37 ° C. in a 5% CO ₂ environment. In addition, Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) was added to the medium until 24 hours after cell seeding.

Five passaged cells are detached and collected, treated with PBS buffer containing 4% paraformaldehyde, and then stained with Alexa FluorTM 647 anti-human SSEA-4 Antibody. The undifferentiated maintenance rate of cells was measured by a flow cytometer. The undifferentiated maintenance rate was 98%. Comparative Example 1
In [Pluripotent stem cell culture evaluation and exfoliation evaluation], pluripotent stem cells were cultured in the same manner as in Example 1 except that the iMatrix-511 solution was not added. The cells did not adhere and died and pluripotent stem cells could not be cultured.

Comparative example 2
[Synthesis of copolymer]
In a test tube, 4.78 g (27 mmol) of N- (4-hydroxyphenyl) methacrylamide, 0.83 g (3 mmol) of a condensation product of 4-azidobenzoic acid and hydroxyethyl methacrylate, 49 mg (0.3 mmol) of azobisisobutyronitrile Was added and dissolved in 18 mL of N, N'-dimethylformamide. After degassing for 30 minutes by nitrogen bubbling, the reaction was carried out at 70 ° C. for 69 hours. After completion of the reaction, the reaction solvent was evaporated under reduced pressure with a rotary evaporator, and the reaction solution was concentrated. The concentrate was poured into 250 mL of methanol, and the precipitated yellow oily substance was recovered and dried under reduced pressure to obtain N- (4-hydroxyphenyl) methacrylamide / azido random copolymer.
[Preparation of base material coated with copolymer]
It produced by the method similar to Example 1 [preparation of the base material with which the block copolymer was coated] except having used the said copolymer.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
Culture was performed in the same manner as in Example 1 [Pluripotent Stem Cell Culture Evaluation and Detachment Evaluation] except that the above-described base material coated with the copolymer was used.

After 24 hours, 96 hours and 144 hours after cell seeding, the appearance of the cells was observed with a phase contrast microscope. In each case, cell adhesion and proliferation were confirmed, and the fresh medium was replaced. After culture medium exchange 144 hours after cell seeding, (1) when the substrate is cooled to 4 ° C, (2) when the substrate is cooled to 4 ° C and tapping is performed 20 times, (3) substrate Were cooled to 4 ° C. and pipetting was performed 10 times, and cell detachment was confirmed in each case. The cells were not detached in any of (1) to (3), and the pluripotent stem cells could not be recovered due to the temperature decrease in the absence of the layer of the thermoresponsive polymer. The detachment of the cells was confirmed by phase contrast microscopy.

Comparative example 3
The substrate was not coated with the block copolymer, and a 6-well plate for Corning Incorporated cell culture was used as it was, and the others were evaluated in the same manner as in Example 1.
[Pluripotent stem cell culture evaluation and exfoliation evaluation]
The culture was performed in the same manner as in Example 1 [Pluripotent Stem Cell Culture Evaluation and Detachment Evaluation] except that the 6-well plate for cell culture manufactured by Corning Incorporated was used as it was.

After 24 hours, 96 hours and 144 hours after cell seeding, the appearance of the cells was observed with a phase contrast microscope. In each case, cell adhesion and proliferation were confirmed, and the fresh medium was replaced. After culture medium exchange 144 hours after cell seeding, (1) when the substrate is cooled to 4 ° C, (2) when the substrate is cooled to 4 ° C and tapping is performed 20 times, (3) substrate Were cooled to 4 ° C. and pipetting was performed 10 times, and cell detachment was confirmed in each case. In any of (1) to (3), the cells were not detached, and in the absence of the block copolymer, it was not possible to recover pluripotent stem cells due to the temperature decrease. The detachment of the cells was confirmed by phase contrast microscopy.

Comparative example 4
[Synthesis of polymer]
In a test tube, 5.65 g (50 mmol) of N-isopropylacrylamide and 33 mg (0.2 mmol) of azobis (isobutyronitrile) were added and dissolved in 50 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, reaction was performed at 70 ° C. for 24 hours. After completion of the reaction, the reaction solvent was evaporated under reduced pressure with a rotary evaporator, and the reaction solution was concentrated. 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 polymer-coated substrate]
It produced by the method similar to Example 1 [preparation of the base material with which the block copolymer was coated] except having used the said polymer. However, when immersed and washed in pure water for 24 hours, most of the polymer was dissolved. When the polymer was dissolved in water, it was found that the N-isopropylacrylamide polymer can not be used for culture because the polymer is eluted into the culture solution and contaminates the cells.

Comparative example 5
The base material was not coated with the block copolymer, and a dish with a diameter of 3.5 cm (manufactured by Corning Incorporated, material: polystyrene) was used as it was.
[Passage culture and undifferentiated maintenance rate evaluation]
0.2 mL / cm ² of medium StemFitAK02N (manufactured by Ajinomoto Co., Ltd.) was added to the block copolymer-coated substrate, and further, human iPS cell 201 B7 strain was 1300 cells / cm ² , iMatrix-511 solution Nippi) was added at a concentration of 2.5 μL / mL. The cells were cultured at 37 ° C. in a 5% CO ₂ environment. In addition, Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) was added to the medium until 24 hours after cell seeding.

After 24 hours, 96 hours and 144 hours after cell seeding, the appearance of the cells was observed with a phase-contrast microscope to confirm cell adhesion and proliferation in each case, and the fresh medium was replaced. After 168 hours of cell seeding, the cells were washed by removing all the media and then adding PBS buffer and aspirating off the PBS buffer. PBS buffer containing 0.25 mM ethylenediaminetetraacetic acid was added and incubated for 5 minutes at 37 ° C. After adding a medium containing Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration 10 μM), all cells are detached from the substrate by rubbing the substrate surface with a cell scraper to obtain a cell suspension. The solution was made. The cell suspension was used to perform passage by seeding cells on the block copolymer-coated substrate. Twenty passages were performed by repeating the same operation.

20 passaged cells are detached and collected, treated with 4% paraformaldehyde in PBS buffer, and then stained with Alexa FluorTM 647 anti-human SSEA-4 Antibody; The undifferentiated maintenance rate of cells was measured by a flow cytometer. The undifferentiated maintenance rate was 36%.

The constitution of the culture substrate used in Examples 1 to 12 is shown in Table 1.

The constitutions of culture substrates used in Examples 13 to 30 and Comparative Examples 1 to 4 are shown in Table 2.

The evaluation results of the culture substrates of Examples 1 to 12 are shown in Table 3.

The evaluation results of the culture substrates of Examples 13 to 30 and Comparative Examples 1 to 4 are shown in Table 3.

While the invention has been described in detail and with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.

In addition, the specification, claims, drawings and abstracts of Japanese Patent Application Nos. 2017-157193 filed on August 16, 2017 and Japanese Patent Application No. 2018-142306 filed on July 30, 2018. The entire contents of which are incorporated herein by reference and incorporated as disclosure of the specification of the present invention.

1 substrate 2 block copolymer 3 biological substance 10 culture substrate

Claims

The substrate 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 includes at least one selected from the group consisting of matrigel, laminin, fibronectin, vitronectin and collagen. A culture substrate for pluripotent stem cells, characterized in that one type of biologically derived substance is immobilized.
The culture substrate for pluripotent stem cells according to claim 1, wherein the thermoresponsive polymer is a block copolymer containing at least the following block segments (A) and (B):
(A) Temperature-responsive polymer block segment having a lower limit critical solution temperature (LCST) to water in the range of 0 ° C. to 80 ° C.
(B) Block segment having pluripotent stem cell adhesion.
The culture medium for pluripotent stem cells according to claim 1 or 2, characterized in that the thermoresponsive polymer is a block copolymer containing at least the following block segments (A) and (B): Material.
(A) Temperature-responsive polymer block segment having an LCST in the range of 0 ° C. to 80 ° C.
(B) Water insoluble block segment.
4. The block polymer according to claim 2, wherein the block segment (B) is a block polymer comprising at least one kind of repeating unit among repeating units represented by the following general formula (1). A culture substrate for pluripotent stem cells as described above.

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 a group represented by the following general formula (2), ( And 3) a substituent represented by (4), (5), (6), (7) or (8), a hydrocarbon group having 1 to 30 carbon atoms or a hydrogen atom.

(Wherein, 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 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).

(Wherein, 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.

(Wherein, A represents 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). )

(Wherein, R 19 represents a divalent hydrocarbon group of 1 to 5 carbon atoms, which 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.
The block copolymer containing the block segments of (A) and (B) is a block copolymer having a constituent unit ratio of 80 to 99 wt% of the block segments of (A). A culture substrate for pluripotent stem cells according to any one of 4.
The culture medium for pluripotent stem cells according to any one of claims 1 to 5, wherein the temperature responsive polymer is one type of block copolymer or a mixture of two type of block copolymers. Material.
The culture substrate for pluripotent stem cells according to any one of claims 1 to 6, wherein the substrate is a porous substrate, and the pore diameter of the porous substrate is 0.01 to 10 μm. .
The culture of pluripotent stem cells according to any one of claims 1 to 7, wherein the base material is a porous base material, and the porosity of the porous base material is 0.01 to 30%. Base material.
The culture substrate for pluripotent stem cells according to any one of claims 1 to 8, wherein the laminin adsorption rate by the following test is 10% or more.
A solution prepared by adding 2-2.5 μL of a laminin 511-E8 fragment solution at a concentration of 0.5 mg / mL to 1 mL of phosphate buffered saline was added in an amount of 0. When dropped on a 2 mL / cm 2 base material and allowed to stand at 37 ° C. for 24 hours, the laminin adsorption rate determined from the following formula.
The culture substrate of pluripotent stem cells according to any one of claims 1 to 9, wherein the pluripotent stem cells are human induced pluripotent stem cells.
A method for producing pluripotent stem cells, which comprises 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 10.
(2) culturing the pluripotent stem cells seeded on the culture substrate in a liquid at a temperature 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 pluripotent stem cells cultured in the liquid from the substrate.