Cell separation substrate and cell separation method

Abstract

To provide a cell separation substrate that can adsorb a specific cell selectively, allows the cell to be easily desorbed from the cell separation substrate, and further can give a high-purity cell, and a cell separation method using the substrate.SOLUTION: A cell separation substrate has a substrate whose surface is fixed with a copolymer having at least an ureido group-containing repetitive sequence, which is a repeat of a specific ureido group-containing structure, and a charge-containing repetitive sequence, with its upper limit critical solution temperature (UCST) ranging from 4°C to 50°C.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a cell separation substrate for separating cells to be separated, a cell separation method, and the like. [Background technology]

[0002] In the field of regenerative medicine, a common technique is to extract healthy cells from a patient with a disease, cultivate them outside the body, and then transplant them back into the patient. However, it is difficult to extract specific cells from a patient's body, blood, or bodily fluids. Therefore, a technology to isolate specific cells is required. This cell separation method is also widely used in the fields of pathological diagnosis and clinical testing. Known cell separation methods include density gradient centrifugation, separation methods using magnetic particles, separation methods using substrates, and methods that combine flow cytometry with a cell sorter. Among these, methods that use substrates to separate specific cells have the advantage of being able to process a large number of cells and being easy to operate.

[0003] Furthermore, a recent method for detaching cells adsorbed to a substrate has been developed that uses temperature-responsive polymers, a method that does not require enzymes or other substances and does not damage the cells. For example, Patent Document 1 discloses a cell culture support material in which the temperature-responsive polymer poly[N-isopropylacrylamide (NIPAM)] is introduced onto the surface by electron beam irradiation, as a technique for recovering cultured cell structures from a cell culture vessel. However, the NIPAM polymer immobilized on this cell culture vessel does not contain an electric charge, which poses the problem of being unable to selectively adsorb specific cells.

[0004] To address these issues, Non-Patent Document 1 discloses a substrate on which a temperature-responsive polymer with an electric charge is immobilized. In this method, a temperature-responsive polymer (p(NIPAM-co-AA)) copolymerized with N-isopropylacrylamide (NIPAM) and a monomer having a carboxylic acid as an anionic charge is immobilized on a glass substrate, and cell separation is performed using temperature response. However, when performing cell separation using a cell separation substrate on which a copolymer with a lower critical solution temperature (LCST) such as p(NIPAM-co-AA) is immobilized, the temperature must be lowered to detach cells adsorbed to the substrate. Low-temperature conditions reduce cell motility, making cell detachment difficult and potentially reducing cell recovery. Furthermore, NIPAM-based polymers have a lower critical solution temperature (LCST), and are hydrophilic at low temperatures but hydrophobic at high temperatures. Therefore, high-temperature conditions are used to adsorb cells to the substrate, but the hydrophobic nature of the polymer makes them prone to nonspecific adsorption, potentially resulting in low cell purity after separation.

[0005] Furthermore, Patent Document 2 discloses a method for cell separation based on temperature response, in which a polymer copolymerized with N,N-dimethylaminoethyl methacrylate (DMAEMA) polymer as a temperature-responsive polymer and an anionic monomer for charge regulation is immobilized on a glass substrate. However, like the above-mentioned p(NIPAM-co-AA), this polymer has a lower critical solution temperature (LCST), which makes it difficult to detach cells, raising concerns about a resulting decrease in cell recovery rate. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 4475847 [Patent Document 2] Japanese Patent Application Publication No. 2017-014323 [Non-patent literature]

[0007] [Non-Patent Document 1] Colloids and Surfaces B,2020,185,p.110565 Summary of the Invention [Problem to be solved by the invention]

[0008] As described above, there is no known cell separation substrate that can selectively adsorb specific cells, that allows cells to be easily detached from the cell separation substrate, and that allows highly pure cells to be obtained.

[0009] An object of the present invention is to provide a cell separation substrate that can selectively adsorb specific cells, that allows cells to be easily detached from the cell separation substrate, and that allows highly pure cells to be obtained. [Means for solving the problem]

[0010] As a result of extensive research aimed at achieving the above-mentioned object, the inventors have found that the above-mentioned problems can be solved by a cell separation substrate having a copolymer fixed to its surface, which copolymer has an electric charge for selectively adsorbing specific cells, exhibits minimal changes in affinity with temperature, and has an upper critical solution temperature (UCST).

[0011] That is, the present invention provides at least the following [1] to

[15] . [1] A cell separation substrate having a copolymer immobilized on its surface, the copolymer having at least a ureido group-containing repeating sequence A, which is a repeating ureido group-containing structure represented by the following formula (1), and a charge-containing repeating sequence B, and having an upper critical solution temperature (UCST) in the range of 4 to 50°C:

[0012] [ka]

[0013] (In formula (1), R 1 represents a hydrogen atom or a methyl group, and A 1is absent, represents -(C=O)O- or -(C=O)NH-, and a represents an integer of 1 to 6. [2] The cell separation substrate according to the above [1], wherein the charged repeating sequence B is a charged repeating sequence B' which is a repetition of a charged structure represented by the following formula (2):

[0014] [ka]

[0015] (In formula (2), R 2 represents a hydrogen atom or a methyl group, and A 2 is absent, represents -(C=O)O- or -(C=O)NH-, b represents an integer of 0 to 6, and X is NH3 + , N(CH3)H2 + , N(CH3)2H + , N(CH3)3 + , S(CH3)2 + , COO - , SO3 - , C6H5SO3 - and PO3H - However, when b is 0, A 2 does not exist.) [3] X is NH3 + , N(CH3)H2 + , N(CH3)2H + , N(CH3)3 + and S(CH3)2 + The cell separation substrate according to the above [2], wherein the group is selected from the group consisting of: [4] R 1 is a methyl group, and A 1 is -(C=O)O-, a is 2, and R 2 is a methyl group, and A 2 is -(C=O)O-, b is 2, and X is N(CH3)2H + or N(CH3)3 + The cell separation substrate according to the above [3], [5] R1 is a hydrogen atom, and A 1 does not exist, a is 1, and R 2 is a hydrogen atom, and A 2 is absent, b is 1, and X is NH3 + The cell separation substrate according to the above [3], [6] X is the COO - , SO3 - , C6H5SO3 - and PO3H - The cell separation substrate according to the above [2], wherein the group is selected from the group consisting of: [7] R 1 is a methyl group, and A 1 is -(C=O)O-, a is 2, and R 2 is a hydrogen atom, and A 2 does not exist, b is 0, and X is COO - The cell separation substrate according to the above [6], [8] The cell separation substrate according to any one of [1] to [7] above, wherein the abundance ratio of the ureido group-containing repeating sequence A in all monomer units constituting the copolymer is 30%≦A<100% in molar ratio. [9] The cell separation substrate according to any one of the above [1] to [8], wherein the copolymer further comprises a phosphorylcholine group-containing repeating sequence C which is a repeat of a phosphorylcholine group-containing structure represented by the following formula (3):

[0016] [ka]

[0017] (In formula (3), R 3 represents a hydrogen atom or a methyl group, and A 3 is absent, represents -(C=O)O- or -(C=O)NH-, and c represents an integer of 1 to 6.

[10] R 3 is a methyl group, and A 3The cell separation substrate according to [9] above, where a is -(C=O)O- and c is 2.

[11] The cell separation substrate according to [9] or

[10] above, where the abundance ratio of the phosphorylcholine group-containing repeating sequence C in all monomer units constituting the copolymer is 0% < C ≦ 20% in molar ratio.

[12] The cell separation substrate according to any one of [1] to

[11] above, where the number average molecular weight of the copolymer is 1,000 to 5,000,000.

[13] The cell separation substrate according to any one of [1] to

[12] above, where the material of the substrate is glass, silicon or plastic.

[14] A method for separating a target cell from a cell group containing the target cell, the cell separation method being characterized by including the following [Step A], [Step B] and [Step C]. [Step A] A step of bringing a cell group containing the target cell into contact with the cell separation substrate according to any one of [1] to

[13] above, and adsorbing the target cell to the cell separation substrate [Step B] A step of washing the target cell adsorbed to the cell separation substrate [Step C] A step of detaching the target cell from the cell separation substrate by a temperature change

[15] In the above [Step C], the temperature change is to heat from a temperature below the upper critical solution temperature (UCST) of the copolymer immobilized on the surface of the cell separation substrate to a temperature above the upper critical solution temperature (UCST). The cell separation method according to

[14] above. [Advantages of the Invention]

[0018] According to the present invention, it is possible to provide a cell separation substrate that can selectively adsorb specific cells, easily detach cells from the cell separation substrate, and further obtain highly pure cells. [Modes for Carrying Out the Invention]

[0019] Preferred embodiments of the present invention will be described in detail below.

[0020] <Copolymer> The copolymer has at least a ureido group-containing repeating sequence A which is a repeating ureido group-containing structure represented by the following formula (1) and a charge-containing repeating sequence B such as a charge-containing repeating sequence B' which is a repeating charge-containing structure represented by the following formula (2), and has an upper critical solution temperature (UCST) in the range of 4 to 50°C.

[0021] [ka]

[0022] (In the above formula (1), R 1 represents a hydrogen atom or a methyl group, and A 1 is absent or represents -(C=O)O- or -(C=O)NH-, and a represents an integer of 1 to 6. 2 represents a hydrogen atom or a methyl group, and A 2 is absent, represents -(C=O)O- or -(C=O)NH-, b represents an integer of 0 to 6, and X is NH3 + , N(CH3)H2 + , N(CH3)2H + , N(CH3)3 + , S(CH3)2 + , COO - , SO3 - , C6H5SO3 - and PO3H - represents a group selected from In this specification, the term "repetition of a structure" refers to not only continuous repetition of the structure but also intermittent repetition.

[0023] In addition, R in the ureido group-containing structure represented by each formula (1) in the ureido group-containing repeating sequence A 1 , A 1and a, and R in the charge-containing structure represented by formula (2) in the charge-containing repeat sequence B. 2 , A 2 , b and X may be the same or different.

[0024] In the above formula (1), A 1 The absence of A in the above formula (1) 1 The upper carbon (C) and A 1 (CH2) at the bottom a This indicates that and are directly single bonded.

[0025] In the above formula (1), a represents an integer of 1 to 6, preferably 1 to 4, for example 1 or 2.

[0026] The ureido group-containing repeating sequence A is, for example, R 1 is a methyl group, and A 1 is -(C=O)O-, and a is 2; 1 is a hydrogen atom, and A 1 is -(C=O)O-, and a is 2; 1 is a methyl group, and A 1 is -(C=O)NH-, and a is 2; 1 is a hydrogen atom, and A 1 is -(C=O)NH-, and a is 2; 1 is a hydrogen atom, and A 1 Examples include a ureido group-containing repeating sequence A in which R does not exist and a is 1. 1 is a methyl group, and A 1 is -(C=O)O-, and a is 2; 1 is a hydrogen atom, and A 1 is -(C=O)O-, and a is 2; 1 is a hydrogen atom, and A 1 is -(C=O)NH-, and a is 2;1 is a hydrogen atom, and A 1 is preferably a ureido group-containing repeating sequence A in which R is absent and a is 1, 1 is a methyl group, and A 1 is -(C=O)O-, and a is 2; 1 is a hydrogen atom, and A 1 A ureido group-containing repeating sequence A in which a is not present and a is 1 is more preferred.

[0027] In the above formula (2), A 2 The absence of A in the above formula (2) 2 The upper carbon (C) and A 2 (CH2) at the bottom b This indicates that and are directly single bonded.

[0028] In the above formula (2), b represents an integer of 0 to 6, preferably 0 to 4, for example, 0, 1 or 2.

[0029] In the above formula (2), when X is cationic, the X is NH3 + , N(CH3)H2 + , N(CH3)2H + or N(CH3)3 + Preferably, NH3 + , N(CH3)H2 + or N(CH3)2H + In addition, when X is anionic, it is more preferable that X is COO - , SO3 - or C6H5SO3 - Preferably, COO - or SO3 - It is more preferable that:

[0030] The charge-containing repeating sequence B may be, for example, R 2 is a methyl group, and A 2 is -(C=O)O-, b is 2, and X is NH3 + A charge-containing repeating sequence B (AEMA), R2 is a methyl group, and A 2 is -(C=O)O-, b is 2, and X is N(CH3)2H + A charge-containing repeating sequence B (DMAEMA), R 2 is a methyl group, and A 2 is -(C=O)O-, b is 2, and X is N(CH3)3 + A charge-containing repeating sequence B (choline methacrylate), R 2 is a methyl group, and A 2 is -(C=O)O-, b is 2, and X is S(CH3)2 + A charge-containing repeating sequence B (a tertiary sulfonium methacrylate), R 2 is a hydrogen atom, and A 2 does not exist, b is 0, and X is COO - A charge-containing repeating sequence B (acrylic acid), R 2 is a methyl group, and A 2 is -(C=O)O-, b is 2, and X is COO - A charge-containing repeating sequence B (carboxyl ethyl methacrylate) in which R 2 is a methyl group, and A 2 is -(C=O)O-, b is 2, and X is SO3 - A charge-containing repeating sequence B (ethyl methacrylate sulfonate), R 2 is a methyl group, and A 2 is -(C=O)NH-, b is 2, and X is NH3 + A charge-containing repeating sequence B (aminoethyl methacrylamide), R 2 is a hydrogen atom, and A 2 is -(C=O)NH-, b is 2, and X is NH3 + A charge-containing repeating sequence B (aminoethylacrylamide), R 2 is a hydrogen atom, and A 2 is absent, b is 1, and X is NH3 + A charge-containing repeating sequence B (allylamine), R 2 is a hydrogen atom, and A 2 is absent, b is 0, and X is C6H5SO3 -Examples include the charge-containing repeating sequence B (styrene sulfonic acid), which is 2 is a methyl group, and A 2 is -(C=O)O-, b is 2, and X is NH3 + A charge-containing repeating sequence B (AEMA), R 2 is a methyl group, and A 2 is -(C=O)O-, b is 2, and X is N(CH3)2H + A charge-containing repeating sequence B (DMAEMA), R 2 is a methyl group, and A 2 is -(C=O)O-, b is 2, and X is N(CH3)3 + A charge-containing repeating sequence B (choline methacrylate), R 2 is a hydrogen atom, and A 2 does not exist, b is 0, and X is COO - A charge-containing repeating sequence B (acrylic acid), R 2 is a hydrogen atom, and A 2 is absent, b is 1, and X is NH3 + A charge-containing repeating sequence B (allylamine) or R 2 is a hydrogen atom, and A 2 is absent, b is 0, and X is C6H5SO3 - Preferred is a charge-containing repeat sequence B (styrene sulfonic acid) in which R 2 is a methyl group, and A 2 is -(C=O)O-, b is 2, and X is NH3 + A charge-containing repeating sequence B (AEMA), R 2 is a methyl group, and A 2 is -(C=O)O-, b is 2, and X is N(CH3)2H + A charge-containing repeating sequence B (DMAEMA), R 2 is a methyl group, and A 2 is -(C=O)O-, b is 2, and X is N(CH3)3 + A charge-containing repeating sequence B (choline methacrylate), R 2 is a hydrogen atom, and A 2 does not exist, b is 0, and X is COO- A charge-containing repeating sequence B (acrylic acid) or R 2 is a hydrogen atom, and A 2 is absent, b is 1, and X is NH3 + More preferred is a charge-containing repeating sequence B (allylamine) in which R 2 is a methyl group, and A 2 is -(C=O)O-, b is 2, and X is N(CH3)2H + A charge-containing repeating sequence B (DMAEMA), R 2 is a methyl group, and A 2 is -(C=O)O-, b is 2, and X is N(CH3)3 + A charge-containing repeating sequence B (choline methacrylate), R 2 is a hydrogen atom, and A 2 does not exist, b is 0, and X is COO - A charge-containing repeating sequence B (acrylic acid) or R 2 is a hydrogen atom, and A 2 is absent, b is 1, and X is NH3 + Particularly preferred is a charge-containing repeating sequence B (allylamine) which is:

[0031] The molar ratio of each repeating sequence in the copolymer can be appropriately determined so as to achieve the required performance, but in order to set the upper critical temperature in the range of 4 to 50°C, the abundance ratio of the ureido group-containing repeating sequence A in all monomer units constituting the copolymer is preferably in the range of 30%≦A<100%, more preferably 50%≦A<100%, and particularly preferably 50%≦A≦98%.

[0032] The copolymer may further have a phosphorylcholine group-containing repeating sequence C, which is a repeating phosphorylcholine group-containing structure represented by the following formula (3): Introduction of a phosphorylcholine group into the copolymer not only makes it easier to control the upper critical solution temperature (UCST) of the copolymer, but also improves hydrophilicity when used for cell separation, making it easier to suppress nonspecific adsorption of unwanted cells.

[0033] [Chemical formula]

[0034] (In the above formula (3), R 3 represents a hydrogen atom or a methyl group, and A 3 represents nothing, -(C=O)O- or -(C=O)NH-, and c represents an integer from 1 to 6.)

[0035] In the above formula (3), when A 3 does not exist, it means that in the above formula (3), the carbon (C) above A 3 and the (CH2) 3 below A c are directly bonded by a single bond.

[0036] In the above formula (3), c represents an integer from 1 to 6, preferably 1 to 4, for example, 1 or 2.

[0037] Examples of the above phosphorylcholine group-containing repeating sequence C include a phosphorylcholine group-containing repeating sequence C in which R 3 is a methyl group, A 3 is -(C=O)O-, and c is 2.

[0038] The content of the above phosphorylcholine group-containing repeating sequence C can be appropriately determined so that the required performance is exhibited. However, in order to make the upper critical temperature in the range of 4 to 50 °C, the abundance ratio of the above phosphorylcholine group-containing repeating sequence C in all monomer units constituting the above copolymer is preferably in the range of 0% < C ≦ 20% in molar ratio. More preferably, it is in the range of 0% < C ≦ 15%, and particularly preferably in the range of 0% < C ≦ 10%.

[0039] The copolymer may be a copolymer with other monomers. The type of other monomer can be appropriately selected to improve the efficiency of cell separation. To lower the upper critical solution temperature (UCST), hydrophilic monomers such as glycerol (meth)acrylate, (meth)acryloyloxyethyl phosphate, N-methylcarboxybetaine (meth)acrylate, N-methylsulfobetaine (meth)acrylate, aminoethyl (meth)acrylate, N,N'-dimethylacrylamide, S-methylsulfonium carboxylic acid (meth)acrylate, polyethylene glycol (meth)acrylate, polyethylene glycol monomethyl ether (meth)acrylate, 2-methoxyethyl (meth)acrylate, allyl alcohol acrylonitrile, acrolein, sodium vinyl sulfonate, N-vinylpyrrolidone, itaconic acid, and maleic acid can be used. Examples of monomers having a large effect of adjusting the upper critical temperature (UCST) include N-methylcarboxybetaine (meth)acrylate, N-methylsulfobetaine (meth)acrylate, aminoethyl (meth)acrylate, S-methylsulfonium carboxylic acid (meth)acrylate, polyethylene glycol (meth)acrylate, and polyethylene glycol monomethyl ether (meth)acrylate, and N-methylcarboxybetaine (meth)acrylate, N-methylsulfobetaine (meth)acrylate, aminoethyl (meth)acrylate, polyethylene glycol (meth)acrylate, and polyethylene glycol monomethyl ether (meth)acrylate are particularly preferred.To increase the upper critical solution temperature (UCST), hydrophobic monomers such as n-butyl (meth)acrylate, isobutyl (meth)acrylate, pentyl (meth)acrylate, hexyl (meth)acrylate, N-isopropyl (meth)acrylamide, vinyl acetate, styrene, chlorostyrene, vinylphenol, vinyl cinnamate, vinyl chloride, vinyl bromide, butadiene, vinylene carbonate, itaconate esters, fumarate esters, and maleate esters are used. Monomers with a significant UCST-regulating effect are preferred, with n-butyl (meth)acrylate, isobutyl (meth)acrylate, pentyl (meth)acrylate, hexyl (meth)acrylate, and N-isopropyl (meth)acrylamide being more preferred. Furthermore, monomers for binding ligands that recognize specific cells can also be used in cell separation. For example, (meth)acrylate propargyl, azidopropyl (meth)acrylate, (meth)acrylic acid succinimide, (meth)acrylic acid, aminoethyl (meth)acrylate, (meth)acrylic acid isocyanate, hydroxyethyl (meth)acrylate, etc. are preferred, and (meth)acrylate propargyl, azidopropyl (meth)acrylate, (meth)acrylic acid succinimide, or (meth)acrylic acid isocyanate are preferred due to their ease of binding to ligands, and (meth)acrylate propargyl, azidopropyl (meth)acrylate, or (meth)acrylic acid succinimide are particularly preferred. The amount of other monomers to be added is optional and can be selected appropriately, but in order to fully utilize the performance of the copolymer, the amount of the other monomers to be added is preferably 20 mol% or less, more preferably 15 mol% or less, and even more preferably 10 mol% or less of the total monomers.

[0040] The molecular weight of the copolymer can be appropriately determined by adjusting polymerization conditions, etc., so that the required performance can be achieved, but is typically about 1,000 to 5,000,000 in number average molecular weight. When preparing a cell separation substrate on which the copolymer is immobilized, a number average molecular weight of 2,000 to 2,000,000 is preferred, and 2,000 to 1,000,000 is more preferred, in order to improve cell separation efficiency. If the number average molecular weight of the copolymer is 1,000 or more, cell detachment in response to temperature becomes easier, and if it is 5,000,000 or less, the cells to be separated are more easily adsorbed to the cell separation substrate.

[0041] <Upper critical solution temperature (UCST) of copolymer> The upper critical solution temperature (UCST) is the boundary temperature between the temperature at which the copolymer insolubilizes in water or a medium to form an insoluble phase and the temperature at which the copolymer dissolves in water or a medium to form a dissolved phase. The copolymer is dissolved in water or a medium at a concentration of at least 1 mM, and the transmittance of visible light at 500 nm is measured in a quartz cell while the temperature is lowered. When the visible light transmittance of a clear solution in which the copolymer is completely dissolved is taken as 100%, the UCST can be determined as the temperature at which the transmittance begins to decrease upon lowering the temperature. The upper critical solution temperature (UCST) is not particularly limited, but should be suitable for cell culture, and is therefore in the range of 2 to 60°C, preferably 3 to 50°C, and more preferably 4 to 50°C.

[0042] <Method of producing copolymer 1> The copolymer can be produced, for example, by radical polymerization of a ureido monomer represented by the following formula (4) and a charged monomer represented by the following formula (5).

[0043] [ka]

[0044] (In the above formula (4), R 1 , A 1 and a are as defined above. 2 , A2 , b, and X are as defined above.

[0045] In the radical polymerization of the ureido monomer and the charged monomer, copolymerization with other monomers is also possible.

[0046] The radical polymerization of the ureido monomer and the charged monomer (and the other monomers) can be carried out by bulk polymerization, but a solvent can also be added for polymerization. The solvent is not particularly limited as long as it dissolves the ureido monomer and the charged monomer (and the other monomers), and any common solvent can be used. For example, the solvent can be selected from polar aprotic solvents such as acetone, dioxane, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), anisole, toluene, acetonitrile, and dimethylacetamide; polar protic solvents such as methanol, ethanol (EtOH), 2-propanol, and water; and mixtures thereof.

[0047] The radical polymerization of the ureido monomer and the charged monomer (and the other monomers) can be carried out by thermal polymerization or photopolymerization. The thermal polymerization can be carried out using, for example, a radical initiator. Examples of the thermal polymerization initiator include peroxide radical initiators (e.g., benzoyl peroxide, ammonium persulfate, etc.), azo radical initiators (e.g., azobisisobutyronitrile (AIBN), 2,2'-azobisdimethylvaleronitrile (ADVN)), 2,2'-azobiscyanovaleric acid (ACVA), azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-044), and water-soluble or oil-soluble redox radical initiators (composed of dimethylaniline and benzoyl peroxide). The amount of radical initiator used is typically 0.01 to 10 parts by mass per 100 parts by mass of the ureido monomer, the charged monomer (and the other monomers). The polymerization temperature and polymerization time can be appropriately selected and determined depending on the type of radical initiator, the presence or absence of other monomers, and their types, etc. For example, when radical polymerization of the ureido monomer and the charged monomer (and the other monomers) is carried out using AIBN, the polymerization temperature is preferably 40 to 90°C, and the polymerization time is preferably about 2 to 48 hours.

[0048] The photopolymerization can be carried out, for example, by irradiation with ultraviolet light (UV) at a wavelength of 254 nm or an electron beam (EB) at an acceleration voltage of 150 to 300 kV. The use of a photopolymerization initiator is optional, but is preferable from the viewpoint of reaction time. Examples of photopolymerization initiators include 2-hydroxy-2-methyl-1-phenyl-1-propanone and 1-hydroxy-cyclohexyl phenyl ketone, with 2-hydroxy-2-methyl-1-phenyl-1-propanone being preferred from the viewpoint of solubility.

[0049] In the radical polymerization of the ureido monomer and the charged monomer (and the other monomers), a chain transfer agent can be used, such as 2-mercaptoethanol, 1-mercapto-2-propanol, 3-mercapto-1-propanol, p-mercaptophenol, mercaptoacetic acid, 2-mercaptopropionic acid, 3-mercaptopropionic acid, and 2-mercaptonicotinic acid.

[0050] The radical polymerization of the ureido monomer and the charged monomer (and other monomers) can also be carried out by living radical polymerization, specifically atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer polymerization (RAFT), and nitroxide-mediated polymerization (NMP). For immobilization on the cell separation substrate of the present invention, reversible addition-fragmentation chain transfer polymerization (RAFT) and atom transfer radical polymerization (ATRP), which can directly modify the polymer on the substrate surface, are particularly preferred because they do not use metals and do not reduce enzyme activity.

[0051] As the RAFT polymerization method, known methods can be used, and for example, the methods described in WO99 / 31144, WO98 / 01478, U.S. Patent No. 6,153,705, etc. are effective. When radical polymerization of the ureido monomer and the charged monomer (and the other monomers) is carried out using RAFT polymerization, the polymerization can be carried out by adding a RAFT agent to ordinary radical polymerization. The RAFT agents include 4-cyanopentanoic acid dithiobenzoate, 2-cyano-2-propyl benzodithioate, benzyl benzodithioate, 2-phenyl-2-propyl benzodithioate, methyl 2-phenyl-2-(phenylcarbonothioylthio)acetate, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester, 4-cyano-4-(dodecylsulfanyl-thiocarbonyl)sulfanylpentanoic acid, 4-cyano-4-(dodecylsulfanyl-thiocarbonyl)sulfanylpentanol, 2-cyano-2-propyldodecyltrithiocarbonate, 2-(dodecylthiocarbonylthio)-2-methylpropionic acid, 4-cyano-4-(dodecylsulfanyl-thiocarbonyl)sulfanylpentanoic acid polyethylene glycol methyl ether ester, 2-(dodecyl ... The 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester, 4-cyano-4-(dodecylsulfanyl-thiocarbonyl)sulfanylpentanoic acid, 4-cyano-4-(dodecylsulfanyl-thiocarbonyl)sulfanylpentanol, or 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid-3-azido-1-propanol ester is preferred from the viewpoint of controlling the polymerization of the ureido monomer and the charged monomer (and the other monomers).

[0052] Known methods can be used for the ATRP method. The copolymer may be synthesized by the ATRP method and then modified onto a substrate, or the copolymer may be polymerized directly onto the surface of a substrate having an ATRP initiator. The catalyst used in the ATRP method is not particularly limited and can be selected from a wide range of catalysts commonly used in the ATRP method. For example, a transition metal complex can be used as the catalyst. The transition metal complex is not particularly limited and can be selected from a wide range of catalysts. For example, the transition metal salts and ligands shown below can be appropriately selected and combined. The ATRP initiator is not particularly limited, but examples thereof include 1-trimethoxysilyl-2-(p-chloromethylphenyl)ethane, 4-(chloromethyl)phenyltrimethoxysilane, 1-trichlorosilyl-2-(m-chloromethylphenyl)ethane, 1-trichlorosilyl-2-(p-chloromethylphenyl)ethane, 2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane, (3-(2-bromoisobutyryl)propyl)trimethoxysilane, 2-Bromo-2-methylpropanoic acid 3-(trichlorosilyl)propyl, 2-bromo-2-methylpropanoic acid 3-(trimethoxysilyl)propyl, 2-bromo-2-methyl-N-[3-(trimethoxysilyl)propyl]propanamide, 2-bromo-2-methyl-N-[3-(triethoxysilyl)propyl]propanamide, 2-bromo-2-methylpropionyl bromide, 2-t-butoxycarbonyl-2-bromopropane, 2-bromo-2-methylpropionic acid ethyl Examples of suitable silanes include methyl bromoisobutyrate, bromomethyl xylene, 1-bromoethylbenzene, 1-chloroethylbenzene, 2-hydroxyethyl 2-bromoisobutyrate, 2,2-dichloroacetophenone, methyl 2-chloropropionate, bromomethyl acetate, bromoethyl acetate, ethyl 2-bromoisobutyrate, bromoacetonitrile, 2-bromoisobutyryl bromide, and diethyl meso-2,5-dibromoadipate. However, from the viewpoint of controlling the polymerization of the ureido monomer and the charged monomer (and the other monomers), 1-trimethoxysilyl-2-(p-chloromethylphenyl)ethane, 4-(chloromethyl)phenyltrimethoxysilane, 1-trichlorosilyl-2-(p-chloromethylphenyl)ethane, 3-(trimethoxysilyl)propyl 2-bromo-2-methylpropanoate, 2-bromo-2-methyl-N-[3-(trimethoxysilyl)propyl]propanamide, chloromethyl xylene, or ethyl 2-bromoisobutyrate is preferred. As a simple method, a substance obtained by reacting 2-bromopropionyl bromide with dopamine or a substance obtained by reacting 2-bromopropionyl bromide with 3-aminopropyltrimethoxysilane or 3-aminopropyltriethoxysilane can be used as an ATRP initiator by modifying the surface of a substrate.

[0053] Examples of transition metal salts used in the ATRP method include CuCl, CuCl2, CuBr, CuBr2, TiCl2, TiCl3, TiCl4, TiBr4, FeCl2, FeCl3, FeBr2, FeBr3, CoCl2, CoBr2, NiCl2, NiBr2, MoCl3, MoCl5, and RuCl3.

[0054] The ligand for the transition metal salt is not particularly limited, and examples thereof include tris(2-(dimethylamino)ethyl)amine (Me6TREN), N,N,N,N-pentamethyldiethylenetriamine (PMDETA), 1,1,4,7,10,10-hexamethyltriethylenetetraamine (HMTETA), 1,4,8,11-tetramethyl-1,4,8,11-azacyclotetradecane (Me4Cyclam), 2,2-bipyridine, 4,4-dimethyl Examples of suitable transition metal salts include 2,2-dipyridyl, 4,4-di-t-butyl-2,2-dipyridyl, 4,4-dinonyl-2,2-dipyridyl, N-butyl-2-pyridylmethanimine, N-octyl-2-pyridylmethanimine, N-dodecyl-N-(2-pyridyl-methylene)amine, N-octadecyl-N-(2-pyridylmethylene)amine, tris(2-pyridylmethyl)amine, and N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine. Examples of suitable combinations of the transition metal salts and ligands include CuBr / 2,2-bipyridine, CuBr2 / 2,2-bipyridine, CuCl / Me6TREN, and CuCl2 / Me6TREN.

[0055] In addition to the transition metal salt and ligand, a reducing agent may be added as needed, such as ascorbic acid, sodium ascorbate, tin(II) 2-ethylhexanoate, or a monovalent copper salt.

[0056] When using the ATRP method, bulk polymerization is possible, but a solvent can also be added for polymerization. The solvent is not particularly limited as long as it dissolves the ureido monomer, the charged monomer, and the other monomers; any common solvent can be used. Examples include polar aprotic solvents such as acetone, dioxane, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), anisole, toluene, acetonitrile, and dimethylacetamide; polar protic solvents such as methanol, ethanol (EtOH), 2-propanol, and water; and mixtures thereof. The polymerization temperature is selected appropriately depending on the solvent, but is typically in the range of 25 to 120°C, preferably 25 to 70°C. At polymerization temperatures of 25°C or higher, polymerization proceeds more easily, while at temperatures of 120°C or lower, polymerization is more easily controlled. The polymerization time is also not particularly limited, but is typically in the range of 1 to 96 hours, preferably 1 to 48 hours. If the polymerization time is 1 hour or more, the polymerization is likely to proceed more sufficiently, and if it is 96 hours or less, there is less possibility of by-products being generated due to unnecessary termination reactions.

[0057] <Copolymer manufacturing method 2> Methods for producing the copolymer are already known, and for example, the method described in Japanese Patent No. 5800323 can be used. Specifically, a method can be used in which a primary amine-containing polymer having a repeating sequence in which the structure represented by the following formula (4) is repeated is dissolved in a solvent and reacted with a cyanate (MCNO).

[0058] [ka]

[0059] (In the above formula (4), R 4 represents a hydrogen atom or a methyl group, and A 4 is absent, represents -(C=O)O- or -(C=O)NH-, and d represents an integer of 1 to 6.

[0060] In the above formula (4), A4 does not exist means that in the above formula (4), A 4 The upper carbon (C) and A 4 (CH2) at the bottom d indicates that there is a direct single bond.

[0061] In the above formula (4), d represents an integer of 1 to 6, preferably 1 to 4, for example 1 or 2.

[0062] Examples of the primary amine-containing polymer include R 4 is a methyl group, and A 4 a primary amine-containing polymer in which R is —(C═O)O— and d is 2; 4 is a hydrogen atom, and A 4 a primary amine-containing polymer in which R is —(C═O)O— and d is 2; 4 is a methyl group, and A 4 a primary amine-containing polymer in which R is —(C═O)NH— and d is 2; 4 is a hydrogen atom, and A 4 a primary amine-containing polymer in which R is —(C═O)NH— and d is 2; 4 is a hydrogen atom, and A 4 Examples include primary amine-containing polymers in which R does not exist and d is 1. However, from the viewpoint of copolymerizability, 4 is a methyl group, and A 4 a primary amine-containing polymer in which R is —(C═O)O— and d is 2; 4 is a hydrogen atom, and A 4 a primary amine-containing polymer in which R is —(C═O)O— and d is 2; 4 is a hydrogen atom, and A 4 is —(C═O)NH— and d is 2; or a primary amine-containing polymer in which R 4 is a hydrogen atom, and A 4 is absent and d is 1, and primary amine-containing polymers in which R 4 is a methyl group, and A 4 is —(C═O)O— and d is 2; or a primary amine-containing polymer in which R 4 is a hydrogen atom, and A 4Primary amine-containing polymers in which is absent and d is 1 are more preferred.

[0063] The molecular weight of the primary amine-containing polymer can be appropriately determined by adjusting the polymerization conditions, etc., so that the required performance can be achieved, but is usually about 1,000 to 5,000,000 in number average molecular weight, and in order to increase the efficiency of cell separation in the cell separation substrate of the present invention, it is preferably 2,000 to 2,000,000, more preferably 2,000 to 1,000,000. When the molecular weight of the primary amine-containing polymer is 1,000 or more in number average molecular weight, temperature-responsive cell detachment becomes easier in the cell separation substrate of the present invention, and when it is 5,000,000 or less, the cells to be separated are more easily adsorbed to the cell separation substrate.

[0064] Examples of solvents for dissolving the primary amine-containing polymer include water, buffer solutions such as imidazole buffer solutions, organic solvents, and mixtures thereof. The organic solvent is not particularly limited as long as it dissolves the primary amine-containing polymer. Examples include alcohols such as methanol, ethanol, 1-propanol, 2-propanol, and 1-butanol; acetonitrile, formamide, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, and 1,4-dioxane. The concentration of the primary amine-containing polymer in the primary amine-containing polymer solution used in the reaction is not limited, but is typically 1 to 80% by weight, preferably 2 to 50% by weight, and more preferably 5 to 40% by weight. A concentration of the primary amine-containing polymer of 80% by weight or less is preferred because it prevents excessive increase in solution viscosity and facilitates maintaining reactivity with the cyanate salt.

[0065] Suitable examples of the cyanate (MCNO) to be reacted with the primary amine-containing polymer include alkali metal salts of cyanic acid, such as potassium cyanate and sodium cyanate. Potassium cyanate is preferred. The proportion of the cyanate used is not particularly limited, but is preferably 0.4 moles or more per mole of primary amino groups in the primary amine-containing polymer. When the proportion of the cyanate used is 0.4 moles or more, ureido groups can be introduced more sufficiently, making it easier to achieve the upper critical solution temperature (UCST).

[0066] The reaction between the primary amine-containing polymer and the cyanate (MCNO) is preferably carried out with stirring. The reaction temperature is not particularly limited, but is preferably maintained at 0 to 100°C, more preferably 30 to 60°C. At a reaction temperature of 0°C or higher, the reaction is more likely to proceed satisfactorily, while at a temperature of 100°C or lower, there is less risk of decomposition of the raw materials or undesired side reactions. The reaction time is also not particularly limited, but a solution of the copolymer can be obtained within 1 to 48 hours, preferably 1 to 25 hours. At a reaction time of 1 hour or longer, the reaction is more likely to proceed satisfactorily, while at a reaction time of 48 hours or shorter, there is less risk of decomposition of the raw materials or undesired side reactions.

[0067] The copolymer thus obtained can be used in the next step without further purification, or can be isolated and purified, preferably by reprecipitation, gel filtration chromatography, dialysis or the like.

[0068] The copolymer produced from the primary amine-containing polymer has a primary amino group as the charge-containing repeating sequence B, which can be converted to a secondary amine, tertiary amine, or quaternary amine by reacting with an alkyl halide, trifluoromethanesulfonyl alkyl, or methanesulfonyl alkyl, etc. Also, it can be converted to a carboxyl group by reacting with succinic anhydride.

[0069] <Base material> The material of the substrate is not particularly limited, but examples thereof include plastics, polysaccharides, metals, magnetic metals, silicon, glass, etc., and a combination of these materials may be used. Materials that are easy to use for cell separation are preferably silicon, glass, or plastic, which are resistant to water and solvents. Examples of plastics include acrylic polymers such as polymethyl methacrylate, various silicone rubbers such as polydimethylsiloxane, polystyrene, polyethylene terephthalate, and polycarbonate.

[0070] The shape of the substrate may be, for example, a container-like, plate-like, particle-like, or fibrous shape, or may be a porous shape with holes or grooves. Among these, container-like, plate-like, particle-like, fibrous, or magnetic particle-like shapes (magnetic particles) are preferred for ease of handling during cell separation, and container-like shapes such as petri dishes and flasks, and plate-like shapes such as silicon wafers and glass plates are particularly preferred.

[0071] The surface of the substrate may have at least one functional group selected from the group consisting of a hydroxyl group, an amino group, a carboxyl group, a tosyl group, an epoxy group, an N-hydroxysuccinimide group, a maleimide group, a thiol group, an azide group, a phenylazide group, a biotin residue, and an avidin residue.

[0072] <Method 1 for preparing cell separation substrate> The cell separation substrate of the present invention can be produced by the method of immobilizing a surface initiator on the substrate [step a] and polymerizing the above-mentioned copolymer from the surface initiator [step b].

[0073] When carrying out [Step a], a pretreatment step can usually be carried out on the substrate. The pretreatment step can be carried out by coating the surface of the substrate with an acidic solution. Examples of acidic solutions include sulfuric acid, hydrochloric acid, nitric acid, and hydrogen peroxide, which can be used alone or in combination of two or more. Among these, a mixed solution of equal amounts of sulfuric acid and hydrogen peroxide is particularly preferred. Coating with the acidic solution is not particularly limited as long as it can coat the surface of the substrate, and can be carried out by, for example, painting, spraying, dipping, etc. The treatment time with the acidic solution is preferably 1 to 48 hours, more preferably 3 to 24 hours. When the treatment time is 1 hour or more, surface modification can be carried out more sufficiently. When the treatment time is 48 hours or less, it is unlikely that further changes will occur on the material surface, and productivity will be further improved.

[0074] The surface initiator can be appropriately selected depending on the polymerization method used in the following [Step b], and examples thereof include the radical initiators, RAFT agents, ATRP initiators, etc. Other surface initiators include allyltrimethoxysilane, allyltriethoxysilane, (3-mercaptopropyl)trimethoxysilane, (3-mercaptopropyl)triethoxysilane, 3-(trimethoxysilyl)propyl (meth)acrylate, and 3-(triethoxysilyl)propyl (meth)acrylate.

[0075] Methods for immobilizing the surface initiator on a substrate include addition to the substrate, silane coupling reaction, click reaction, amidation, esterification, thioesterification, carbonation, Schiff base formation reaction, reductive amination, radical-mediated coupling reaction, Diels-Alder reaction, disulfidation, Michael addition reaction, ene-thiol reaction, aromatic boron compound-mediated coupling reaction, tertiary sulfonium formation reaction, and quaternary ammonium formation reaction. However, due to their high versatility, silane coupling reaction, click reaction, amidation, and esterification are preferred.

[0076] Other immobilization methods include reacting 2-bromopropionyl bromide with the functional groups of the substrate, reacting 2-bromopropionyl bromide with a surface that has been reacted with 3-aminopropyltrimethoxysilane or 3-aminopropyltriethoxysilane, or using a reaction product of dopamine and 2-bromopropionyl bromide. These methods can be used to introduce an ATRP initiator onto the surface of the substrate as a surface initiator. Furthermore, RAFT agents can be introduced onto the surface of the substrate as a surface initiator by reacting 4,4'-azobis(4-cyanopentanoic acid), a radical initiator having a carboxyl group, with the functional groups of the substrate, or by reacting 4-cyanopentanoic acid dithiobenzoate, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester, or 4-cyano-4-(dodecylsulfanyl-thiocarbonyl)sulfanylpentanoic acid.

[0077] The silane coupling reaction can be carried out by coating, spraying, dipping, vacuum deposition, ion plating, thermal CVD, or the like. Among these, coating or dipping is preferred as a simple method. The temperature for the silane coupling reaction is not particularly limited, but is typically 20 to 200°C, preferably 20 to 150°C, and more preferably 20 to 120°C. When the reaction temperature is 20°C or higher, the reaction proceeds more easily, while when the reaction temperature is 200°C or lower, undesired side reactions such as decomposition of the silane coupling agent are less likely to proceed. Furthermore, the time for the silane coupling reaction is typically 2 to 96 hours, preferably 2 to 72 hours, and more preferably 2 to 48 hours. When the reaction time is 2 hours or longer, the reaction proceeds more efficiently, while when the reaction time is 96 hours or shorter, undesired side reactions such as decomposition of the silane coupling agent are less likely to proceed.

[0078] The silane coupling reaction can also be carried out by adding a solvent for immobilization. The solvent is not particularly limited as long as it dissolves the surface initiator, and any common solvent can be used. For example, the solvent can be selected from polar aprotic solvents such as toluene, acetone, dioxane, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), anisole, toluene, xylene, acetonitrile, and dimethylacetamide; polar protic solvents such as methanol, ethanol (EtOH), 2-propanol, and water; and mixtures thereof.

[0079] The above [Step b] is a step of synthesizing the copolymer from the surface initiator immobilized on the surface of the base material in the above [Step a] by the grafting from method, and is the same as the above copolymer production method 1.

[0080] <Method 2 for preparing cell separation substrate> The cell separation substrate of the present invention can also be produced by the grafting to method, that is, by synthesizing the copolymer [step c] and immobilizing the copolymer on a substrate [step d].

[0081] The above [Step c] is the same as the above-mentioned method for producing the copolymer.

[0082] The method for immobilizing the copolymer on the substrate in the above [Step d] includes the same method as in the above [Step a].

[0083] <Cell separation method> By using the cell separation substrate of the present invention, separation of cells to be separated from a cell group containing cells to be separated can be carried out by a cell separation method comprising the following [Step A], [Step B], and [Step C].

[0084] [Process A] This is a step of contacting a cell population containing cells to be separated with the cell separation substrate, and allowing the cells to be separated to be adsorbed onto the cell separation substrate.

[0085] Examples of the cells include established cells for culture, fertilized eggs and egg cells of animals including humans, etc. Also included are stem cells such as sperm cells, ES cells, iPS cells, mesenchymal stem cells, hematopoietic stem cells, neural stem cells, and umbilical cord blood cells, as well as animal cells including humans or plant cells such as hepatocytes, nerve cells, cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells, and blood cells.

[0086] The cells to be separated refer to a group of cells obtained as a result of cell separation using the cell separation substrate of the present invention, and may be a single type of cell or a group of cells consisting of multiple types of cells.

[0087] The cell population containing the separated cells is a cell population containing preferably 0.1% or more separated cells, and there are no limitations on the type of cells contained.

[0088] The temperature at which the cell population including the cells to be separated is brought into contact with the cell separation substrate may be equal to or lower than the upper critical solution temperature (UCST) of the copolymer immobilized on the surface of the cell separation substrate, for example, 2 to 20° C., preferably 3 to 15° C., and more preferably 3 to 10° C. When the cell population including the cells to be separated is brought into contact with the cell separation substrate at a temperature of 2° C. or higher, there is less risk of freezing and damaging the cells.

[0089] Methods for contacting a cell group containing the cells to be separated with the cell separation substrate include centrifuging a plate, dish, or flask to press the cells against the cell separation substrate, and waiting for the cells to settle on the cell separation substrate.

[0090] The number of cells contained in the cell population containing the separated cells is usually 1.0 × 10 10 cells / mL or less, 1.0 × 10 9 cells / mL or less is preferable, and 1.0 × 10 8 A concentration of 1.0 x 10 cells / mL or less is particularly preferred. 10When the cell number is less than 100 cells / mL, the cells that come into contact with the cell separation substrate do not become saturated, and a sufficient separation effect is likely to be obtained.

[0091] The contact between the cell population containing the cells to be separated and the cell separation substrate is not particularly limited as long as it does not adversely affect the cells to be separated. A buffer or medium commonly used in this field can be used. Examples include phosphate buffer, Tris buffer, Good's buffer, glycine buffer, and borate buffer, and these may be used in combination. Phosphate buffer is preferred. Other examples include Dulbecco's Modified Eagle's MEM Medium (DMEM), α-MEM medium, Roswell Park Memorial Institute (RPMI) medium, F12 medium, TC199 medium, and GMEM medium. These may be mixed, or supplemented with fetal bovine serum (FBS), glutamine, or antibiotics as needed. However, serum-free media are preferred to avoid impairing the effect of the charge on the surface of the cell separation substrate.

[0092] [Process B] This is a step of washing the cells to be separated that have been adsorbed onto the cell separation substrate.

[0093] The washing temperature may be equal to or lower than the upper critical solution temperature (UCST) of the copolymer immobilized on the surface of the cell separation substrate, for example, 2 to 20° C., preferably 3 to 15° C., and more preferably 3 to 10° C. When washing is performed at a temperature of 2° C. or higher, there is less risk of freezing and damaging the cells.

[0094] The number of times of washing is usually 10 or less, preferably 7 or less, and more preferably 5 or less. When washing is performed 10 or less times, there is less risk of cells being damaged by shear stress during washing.

[0095] The buffer solution and medium used for the washing can be the same as those used in [Step A].

[0096] [Process C] This is a step in which the cells to be separated are detached from the cell separation substrate by a temperature change.

[0097] The temperature for the detachment may be equal to or higher than the upper critical solution temperature (UCST) of the copolymer immobilized on the surface of the cell separation substrate, for example, 20 to 50° C., preferably 25 to 40° C., and more preferably 30 to 40° C. When the detachment is performed at a temperature of 50° C. or lower, proteins contained in the cells are denatured, which reduces the risk of damaging the cells. [Example]

[0098] The present invention will be specifically described below based on examples, but the present invention is not limited to the following examples.

[0099] <Preparation of initiator-modified substrate>

[0100] [ka]

[0101] A glass substrate (10 mm × 10 mm) was dipped into a piranha solution (concentrated sulfuric acid / 30% hydrogen peroxide solution, 3 / 1 v / v) for 2 hours. After immersion, it was washed with ion-exchanged water and dried. The piranha-treated glass substrate was placed in toluene (8 mL), and 3-aminopropyltrimethoxysilane (50 μL) was added. The reaction was carried out at 30°C for 24 hours. After the reaction, the toluene was removed and the substrate was washed with ethanol. The substrate was then annealed at 120°C for 2 hours under a nitrogen atmosphere to obtain a glass substrate with amino groups on its surface. The glass substrate with amino groups on its surface was placed in THF (2 mL), and triethylamine (50 μL) and 2-bromopropionyl bromide (140 μL) were added. After reaction at room temperature for 3 hours, the substrate was washed with ethanol to obtain an initiator-modified substrate (hereinafter referred to as the "initiator-modified substrate").

[0102] <Example 1: Preparation of cell separation substrate A>

[0103] [ka]

[0104] (In the formula, co indicates a copolymer structure of three monomer units in the chemical formula.)

[0105] The initiator-modified substrate (10 mm x 10 mm) and the monomers ureidoethyl methacrylate (UMA) (230 mg, 1.35 mmol), N,N-dimethylaminoethyl methacrylate (DMAEMA) (11.8 mg, 0.075 mmol), 2-methacryloyloxyethyl phosphorylcholine (MPC) (22.1 mg, 0.075 mmol), 2-bromoisobutyrate (1.9 mg, 0.01 mmol), CuBr (8.82 mg, 0.036 mmol), and 2,2-bipyridine (11.4 mg, 0.072 mmol) were placed in a test tube and dissolved in 3 mL of ethanol / water (50 / 50 v / v). After bubbling with nitrogen gas, ascorbic acid (1.26 mg, 0.0072 mmol) was added to initiate the reaction. After 24 hours of reaction, the substrate was recovered from the reaction solution and washed to obtain cell separation substrate A. In addition, in order to analyze the polymer immobilized on cell separation substrate A, the reaction solution was reprecipitated with acetone to obtain free copolymer A' obtained by polymerization from ethyl 2-bromoisobutyrate. Yield of free copolymer A': 199 mg The composition ratio of free copolymer A' is x:y:z=89:6:5( 1 (calculated from H NMR) · 1 H-NMR (CD3OD+D2O, 400 MHz); 4.1 ppm (UMA, DMAEMA:COO-C H 2), 3.8-4.6 ppm (MPC:COO-C H 2,CH2-C H 2,PO-C H 2), 3.6 ppm (MPC:C H 2-N),3.4ppm(UMA, DMAEMA:CH2-C H 2), 3.2 ppm (N (C H3) 3), 2.8 ppm (DMAEMA: N(CH3)2H + ), 1.0-2.4 ppm (polymer main chain)

[0106] The number-average molecular weight of the free copolymer A' was determined by gel permeation chromatography using a multi-angle light scattering detector (MALS) manufactured by WYATT. The pump used was a Shimadzu LC-720AD, the detector (differential refractometer) was a Shimadzu RID10A, and the detector (UV) was an SPD-20A. The column used consisted of two columns, one TSKGel G3000PWXL and one TSKGel G5000PWXL (column size: 4.6 mm x 25 cm) manufactured by Tosoh Corporation. The developing solvent was distilled water / acetonitrile (5 / 5 v / v) containing 100 mM sodium nitrate. The measurement conditions were a flow rate of 0.6 mL / min, a column temperature of 40°C, a sample concentration of 2 mg / mL, and an injection volume of 100 μL. As a result of gel permeation chromatography measurement, the number average molecular weight of the free copolymer A' was 17,000.

[0107] The upper critical solution temperature (UCST) of the free copolymer A' was determined by measuring the transmittance using a UV-visible spectrophotometer, and the temperature at which the transmittance began to decrease was defined as the UCST. Free copolymer A' was dissolved in serum-free RPMI medium to a concentration of 2.5 mg / mL. The solution was placed in a quartz cell, and the solution temperature was changed from 70°C to 5°C. The transmittance (%) of the solution was measured using the UV-visible spectrophotometer. The transmittance (%) of the solution was calculated using the following formula. The UCST of free copolymer A' was found to be 27°C.

[0108]

number

[0109] <Example 2: Preparation of cell separation substrate B>

[0110] [ka]

[0111] (In the formula, co indicates a copolymer structure of three monomer units in the chemical formula.)

[0112] In a test tube, the initiator-modified substrate (10 mm x 10 mm) and the monomers ureidoethyl methacrylate (UMA) (230 mg, 1.35 mmol), choline methacrylate (CMA) (15.6 mg, 0.075 mmol), 2-methacryloyloxyethyl phosphorylcholine (MPC) (22.1 mg, 0.075 mmol), 2-ethyl 2-bromoisobutyrate (1.9 mg, 0.01 mmol), CuBr (8.82 mg, 0.036 mmol), and 2,2-bipyridine (11.4 mg, 0.072 mmol) were placed and dissolved in 3 mL of ethanol / water (50 / 50 v / v). After bubbling with nitrogen gas, ascorbic acid (1.26 mg, 0.0072 mmol) was added to initiate the reaction. After 24 hours of reaction, the resulting substrate was collected and washed to obtain cell separation substrate B. In addition, in order to analyze the polymer immobilized on the cell separation substrate, the reaction solution was reprecipitated with acetone to obtain free copolymer B' obtained by polymerization from ethyl 2-bromoisobutyrate. Yield of free copolymer B': 187 mg The composition ratio of free copolymer B' is x:y:z=88:6:6( 1 (calculated from H NMR)

[0113] The number average molecular weight of the free copolymer B' obtained was determined in the same manner as in Example 1. As a result of gel permeation chromatography measurement, the number average molecular weight of the free copolymer B' was found to be 20,000.

[0114] The upper critical solution temperature (UCST) of the free copolymer B' obtained was determined in the same manner as in Example 1. As a result of measurement using an ultraviolet-visible spectrophotometer, the upper critical solution temperature (UCST) was 25°C.

[0115] <Example 3: Synthesis of cell separation substrate C>

[0116] [ka]

[0117] (In the formula, co indicates a copolymer structure of three monomer units in the chemical formula.)

[0118] A test tube containing the initiator-modified substrate (10 mm x 10 mm) was charged with the monomers ureidoethyl methacrylate (UMA) (230 mg, 1.35 mmol), t-butyl acrylate (tBA) (9.6 mg, 0.075 mmol), 2-methacryloyloxyethyl phosphorylcholine (MPC) (22.1 mg, 0.075 mmol), 2-ethyl 2-bromoisobutyrate (1.9 mg, 0.01 mmol), CuBr (8.82 mg, 0.036 mmol), and 2,2-bipyridine (11.4 mg, 0.072 mmol), and dissolved in 3 mL of ethanol / water (50 / 50 v / v). After bubbling with nitrogen gas, ascorbic acid (1.26 mg, 0.0072 mmol) was added to initiate the reaction. After 24 hours of reaction, the resulting substrate was recovered and washed. Thereafter, the substrate was reacted with methanesulfonic acid to hydrolyze the t-butyl groups, thereby obtaining cell separation substrate C. In addition, in order to analyze the polymer immobilized on the cell separation substrate, the reaction solution was reprecipitated with acetone to obtain free copolymer C' obtained by polymerization from ethyl 2-bromoisobutyrate. Yield of free copolymer C': 157 mg The composition ratio of free copolymer C' is x:y:z=90:5:5( 1 (calculated from H NMR)

[0119] The number average molecular weight of the free copolymer C' obtained was determined in the same manner as in Example 1. As a result of gel permeation chromatography measurement, the number average molecular weight of the free copolymer C' was 16,000.

[0120] The upper critical solution temperature (UCST) of the free copolymer C' obtained was determined in the same manner as in Example 1. As a result of measurement using an ultraviolet-visible spectrophotometer, the upper critical solution temperature (UCST) was 23°C.

[0121] Example 4-1: Synthesis of copolymer D'

[0122] [ka]

[0123] (In the formula, co indicates a copolymer structure of two monomer units in the chemical formula.)

[0124] Polyallylamine (500 mg) with a number-average molecular weight of approximately 150,000 was placed in a four-neck flask, dissolved in 10 mL of ion-exchanged water, and heated to 50°C. Potassium cyanate (611 mg, 7.54 mmol) (0.83 mol per mol of amino groups in polyallylamine) was added and reacted for 24 hours. After the reaction was completed, the copolymer was purified by dialysis at the same temperature to obtain copolymer D'. Composition ratio (molar ratio) x:y = 81:19 ( 1 (calculated from H NMR)

[0125] The upper critical solution temperature (UCST) of the obtained copolymer D' was determined in the same manner as in Example 1. As a result of measurement using an ultraviolet-visible spectrophotometer, the upper critical solution temperature (UCST) was 30°C.

[0126] <Example 4-2: Synthesis of cell separation substrate D>

[0127] [ka]

[0128] (In the formula, co indicates a copolymer structure of three monomer units in the chemical formula.)

[0129] Copolymer D' (30 mg) and water-soluble carbodiimide (WSC) (2 mg) were placed in one of the cell culture surface-treated polystyrene 6-well plates (Corning Incorporated) with carboxylic acid on the surface, and the mixture was dissolved in 3 mL of water to initiate the reaction. After 3 hours of reaction, the polystyrene dish was washed to obtain cell separation substrate D.

[0130] <Test Example 1-1: Contact angle measurement> The contact angle of the prepared cell separation substrate A was measured using a contact angle meter DMo-702 (Kyowa Interface Science Co., Ltd.). Cell separation substrate A was placed on a stage, and measurements were performed by dropping 1 μL of ion-exchanged water at temperatures of 4°C and 37°C under a nitrogen atmosphere. The contact angle at each temperature was calculated by measuring the angle between the liquid surface and the solid surface.

[0131] <Test Examples 1-2 to 1-4> The contact angle was measured in the same manner as in Test Example 1-1, except that the cell separation substrate shown in Table 1 was used. The evaluation results are shown in Table 1.

[0132] <Test Example 2-1: Cell Separation Test> Cell culture substrate A was placed in a polystyrene 6-well plate as a substrate for cell separation, and a multi-cell mixture (1.0 × 10) of Jurkat cells and HL-60 cells pre-stained with Hoechest 33342 as the cells to be separated was mixed at a cell number ratio of 50:50. 5 Three mL of a solution of 1000 μg / mL of cellulose acetate (1000 μg / mL cells) was added at 4°C. The 6-well plate was centrifuged to precipitate the cells, then washed twice. A 1 mm x 1 mm area was observed under a fluorescent microscope and the number of separated cells on the cell separation substrate was counted (Count A). The plate was then heated to 37°C, washed, and the number of separated cells was counted again under a fluorescent microscope (Count B). The detachment rate was calculated using the formula below. Purity was calculated by analysis with a flow cytometer after the cell separation procedure. The evaluation results are shown in Table 2. Desorption rate = (Count A - Count B) / Count A x 100

[0133] <Test Examples 2-2 to 2-4> A cell separation test was carried out in the same manner as in Test Example 2-1, except that the cell separation substrate and separated cells shown in Table 2 were used. The evaluation results are shown in Table 2.

[0134] Comparative Example 1: Synthesis of Cell Separation Substrate E

[0135] [ka]

[0136] Initiator-modified substrates (10 mm x 10 mm) were placed in a test tube. The monomers, ureidoethyl methacrylate (UMA) (243 mg, 1.43 mmol), 2-methacryloyloxyethyl phosphorylcholine (MPC) (22.1 mg, 0.075 mmol), 2-bromoisobutyrate ethyl (1.9 mg, 0.01 mmol), CuBr (8.82 mg, 0.036 mmol), and 2,2-bipyridine (11.4 mg, 0.072 mmol), were dissolved in 3 mL of ethanol / water (50 / 50 v / v). After bubbling with nitrogen gas, ascorbic acid (1.26 mg, 0.0072 mmol) was added to initiate the reaction. After 24 hours of reaction, the resulting substrate was collected and washed to obtain cell separation substrate E. In addition, in order to analyze the polymer immobilized on the cell separation substrate, the reaction solution was reprecipitated with acetone to obtain free copolymer E' obtained by polymerization from ethyl 2-bromoisobutyrate. Yield of free copolymer E': 200 mg The composition ratio of free copolymer E' is x:y=94:6( 1 (calculated from H NMR)

[0137] The number average molecular weight of the free copolymer E' obtained was determined in the same manner as in Example 1. As a result of gel permeation chromatography measurement, the number average molecular weight of the free copolymer E' was found to be 20,000.

[0138] The upper critical solution temperature (UCST) of the free copolymer E' obtained was determined in the same manner as in Example 1. As a result of measurement using an ultraviolet-visible spectrophotometer, the upper critical solution temperature (UCST) was 25°C.

[0139] <Comparative Example 2: Synthesis of cell separation substrate F>

[0140] [ka]

[0141] The initiator-modified substrate (10 mm x 10 mm) was placed in a test tube. The monomers N-isopropylacrylamide (NIPAM) (162 mg, 1.43 mmol), t-butyl acrylate (tBA) (9.6 mg, 0.075 mmol), ethyl 2-bromoisobutyrate (1.9 mg, 0.01 mmol), CuBr (8.82 mg, 0.036 mmol), and 2,2-bipyridine (11.4 mg, 0.072 mmol) were dissolved in 3 mL of ethanol / water (80 / 20 v / v). After bubbling with nitrogen gas, ascorbic acid (1.26 mg, 0.0072 mmol) was added to initiate the reaction. After 24 hours of reaction, the resulting substrate was collected and washed. The substrate was then reacted with methanesulfonic acid to hydrolyze the t-butyl groups, yielding cell separation substrate F. In addition, in order to analyze the polymer immobilized on the cell separation substrate, the reaction solution was reprecipitated with acetone to obtain free copolymer F' obtained by polymerization from ethyl 2-bromoisobutyrate. Yield of free copolymer F': 200 mg The composition ratio of free copolymer F' is x:y=95:5( 1 (calculated from H NMR)

[0142] The number average molecular weight of the free copolymer F' obtained was determined in the same manner as in Example 1. As a result of gel permeation chromatography measurement, the number average molecular weight of the free copolymer F' was 12,000.

[0143] The lower critical solution temperature (LCST) of the obtained free copolymer F' was determined by measuring the transmittance using a UV-visible spectrophotometer, and the temperature at which the transmittance reached 50 was defined as the lower critical solution temperature (LCST). Free copolymer F' was dissolved in serum-free RPMI medium to a concentration of 2.5 mg / mL. The solution was placed in a quartz cell, and the solution temperature was changed between 70°C and 5°C, during which the transmittance (%) of the solution was measured using a UV-visible spectrophotometer. The transmittance (%) of the solution was calculated in the same manner as in Example 1. As a result, the lower critical solution temperature (LCST) of free copolymer F' was 31°C.

[0144] <Comparative Test Examples 1-1 to 1-2> The contact angle was measured in the same manner as in Test Example 1-1, except that the cell separation substrate shown in Table 1 was used. The evaluation results are shown in Table 1.

[0145] <Comparative Test Example 2-1> A cell separation test was carried out in the same manner as in Test Example 2-1, except that the cell separation substrate and separated cells shown in Table 2 were used. The evaluation results are shown in Table 2.

[0146] <Comparative Test Example 2-2> Cell culture substrate F was placed in a polystyrene 6-well plate as a substrate for cell separation, and a multi-cell mixture (1.0 × 10) of Jurkat cells and HUVEC cells pre-stained with Hoechest 33342 as the cells to be separated was mixed at a cell number ratio of 50:50. 5 Three mL of PBS (37.2% PBS / mL) was added at 37°C. The 6-well plate was centrifuged to precipitate the cells, then washed twice, and a 1 mm x 1 mm area was observed under a fluorescent microscope to count the number of separated cells on the cell separation substrate (Count A). The plate was then cooled to 4°C, washed, and the number of separated cells was counted again under a fluorescent microscope (Count B). The detachment rate and purity were calculated in the same manner as in Test Example 2-1. The evaluation results are shown in Table 2.

[0147] [Table 1]

[0148] [Table 2]

[0149] In the cell separation substrates A to E (Test Examples 1-1 to 1-4, Comparative Test Example 1-1) on which the copolymer having a ureido group-containing repeating sequence was immobilized, the change in contact angle between 4°C and 37°C was small, and hydrophilicity was maintained. On the other hand, in the cell separation substrate F (Comparative Test Example 1-2) on which a NIPAM-based polymer was immobilized, the change in contact angle between 4°C and 37°C was large, and it was found to be hydrophobic at 37°C. This demonstrates that the cell separation substrate of the present invention exhibits little change in affinity / affinity due to temperature changes and is always hydrophilic.

[0150] The cell separation substrate E, which is an uncharged substrate, had a low Count A value, i.e., the adsorption amount of separated cells was low. Furthermore, because it was uncharged, it was unable to adsorb only specific cells, resulting in low purity after separation (Comparative Test Example 2-1). Furthermore, with cell separation substrate F, on which a NIPAM-based polymer was immobilized, cells had to be detached at low temperatures, which reduced cell motility and resulted in a low detachment rate. Furthermore, the purity after separation was also low. This is likely because cell separation substrate F is hydrophobic at 37°C, and during cell adsorption, it adsorbed not only the target cells (HUVECs) but also non-target cells (Jurkat) through hydrophobic interactions. From these findings, we demonstrate that the use of a cell separation substrate of the present invention, in which a copolymer that has a charge for selectively adsorbing specific cells, exhibits minimal changes in affinity with temperature, and has an upper critical solution temperature (UCST) immobilized on the substrate surface, allows for selective adsorption of specific cells, facilitates cell detachment from the cell separation substrate, and yields highly pure cells. [Industrial Applicability]

[0151] The present invention provides a cell separation substrate that can selectively adsorb specific cells, facilitates detachment of cells from the cell separation substrate, and enables the production of highly pure cells.

Claims

1. A cell separation substrate having a copolymer immobilized on its surface, the copolymer having at least a ureido group-containing repeating sequence A, which is a repeat of a ureido group-containing structure represented by the following formula (1), and a charge-containing repeating sequence B, which is represented by the following formula (2), and having an upper critical solution temperature (UCST) in the range of 4 to 50°C. 【Chemistry 1】 (In formula (1), R １ represents a hydrogen atom or a methyl group, A １ represents -(C=O)O- or -(C=O)NH-, and a represents an integer of 1 to 6. 【Chemistry 2】 (In formula (2), R 2 represents a hydrogen atom or a methyl group, A 2 represents —(C═O)O— or —(C═O)NH—, b represents an integer of 1 to 6, and X represents a group selected from NH 3 + , N(CH 3 )H 2 + , N(CH 3 ) 2 H + , N(CH 3 ) 3 + and COO − .)

2. X is NH ３ ＋ , N(CH ３ ) H ２ ＋ , N(CH ３ ) ２ H ＋ and N(CH ３ ) ３ The cell separation substrate according to claim 1 , wherein the group is a group selected from the group consisting of aryl, arylsulfonyl ...

3. R １ is a methyl group, and A １ is —(C═O)O—, a is 2, and R ２ is a methyl group, and A ２ is -(C=O)O-, b is 2, and X is N(CH ３ ) ２ H ＋ or N(CH ３ ) ３ ＋ The cell separation substrate according to claim 2, wherein

4. The cell separation substrate according to claim 1, wherein X is COO-.

5. The cell separation substrate according to any one of claims 1 to 4, wherein the abundance ratio of the ureido group-containing repeating sequence A in all monomer units constituting the copolymer is 30%≦A<100% in molar ratio.

6. The cell separation substrate according to any one of claims 1 to 5, wherein the copolymer further comprises a phosphorylcholine group-containing repeating sequence C, which is a repeating phosphorylcholine group-containing structure represented by the following formula (3): 【Transformation 3】 (In formula (3), R ３ represents a hydrogen atom or a methyl group, A ３ is absent, represents —(C═O)O—, or —(C═O)NH—, and c represents an integer of 1 to 6.

7. R ３ is a methyl group, and A ３ The cell separation substrate according to claim 6, wherein: is —(C═O)O— and c is 2.

8. 8. The cell separation substrate according to claim 6, wherein the abundance ratio of the phosphorylcholine group-containing repeating sequence C in all monomer units constituting the copolymer is 0%<C≦20% in molar ratio.

9. The cell separation substrate according to any one of claims 1 to 8, wherein the number average molecular weight of the copolymer is 1,000 to 5,000,000.

10. The cell separation substrate according to any one of claims 1 to 9, wherein the material of the substrate is glass, silicon or plastic.

11. A method for separating cells to be separated from a cell group containing cells to be separated, the method comprising the following steps A, B, and C: [Process A] A step of contacting a cell population containing cells to be separated with the cell separation substrate according to any one of claims 1 to 10, thereby adsorbing the cells to be separated onto the cell separation substrate. [Process B] a step of washing the cells to be separated that have been adsorbed onto the cell separation substrate; [Process C] a step of detaching the cells to be separated from the cell separation substrate by changing the temperature.

12. The cell separation method according to claim 11, wherein the temperature change during step C is heating from a temperature below the upper critical solution temperature (UCST) of the copolymer immobilized on the surface of the cell separation substrate to a temperature above the upper critical solution temperature (UCST).

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