JP2023006594A - Cell separation substrate and cell separation method - Google Patents

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

In the field of regenerative medicine, a technique of extracting normal cells from a diseased patient, culturing them outside the body, and then transplanting them back into the patient is often used. However, it is difficult to extract only specific cells from the patient's body, blood, or bodily fluids. Therefore, a technique for isolating specific cells is required.
This cell separation method is also widely used in the field of pathological diagnosis or clinical examination. Known cell separation methods include a density gradient centrifugation method, a separation method using magnetic particles, a separation method using a substrate, and a method combining flow cytometry and a cell sorter. Among them, the method of separating specific cells using a base material has the advantage that the number of cells that can be treated is large and the operation of cell separation is simple.

As a technique for detaching cells adsorbed to a substrate, a method using a temperature-responsive polymer is known in recent years as a desorption method that does not use enzymes or the like and does not damage cells. For example, as a technique for recovering cultured cell structures from a cell culture vessel, Patent Document 1 discloses a cell culture support in which poly[N-isopropylacrylamide (NIPAM)], which is a temperature-responsive polymer, is introduced onto the surface by electron beam irradiation. A body material is disclosed. However, since the NIPAM polymer immobilized on this cell culture vessel does not contain an electric charge, there is a problem that specific cells cannot be selectively adsorbed.

In order to solve such problems, Non-Patent Document 1 discloses a substrate on which a temperature-responsive polymer having an electric charge is immobilized. A polymer (p(NIPAM-co-AA)) obtained by copolymerizing N-isopropylacrylamide (NIPAM) as a temperature-responsive polymer and a monomer having a carboxylic acid as an anionic charge was immobilized on a glass substrate, and cells were detected by temperature response. It is a method of performing separation. However, when cell separation is performed using a cell separation substrate on which a copolymer having a lower critical solution temperature (LCST) is immobilized, such as p(NIPAM-co-AA), cells adsorbed to the substrate are desorbed. When separating, it is necessary to incline to a low temperature. Since the motility of cells decreases under low temperature conditions, it becomes difficult to detach cells, and as a result, there is a concern that the recovery rate of cells decreases. Furthermore, the NIPAM-based polymer has a lower critical solution temperature (LCST) and exhibits hydrophilicity under low temperature conditions, but exhibits hydrophobicity under high temperature conditions. Therefore, when cells are adsorbed to a substrate, high-temperature conditions are used, but non-specific adsorption is likely to occur due to the hydrophobicity of the polymer, and there is a concern that the purity of the separated cells will be low.

Furthermore, in Patent Document 2, a polymer obtained by copolymerizing N,N-dimethylaminoethyl methacrylate (DMAEMA) polymer as a temperature-responsive polymer and an anionic monomer as a role of charge adjustment is fixed on a glass substrate, and cell separation is performed by temperature response. is disclosed. However, as with p(NIPAM-co-AA), the polymer has a lower critical solution temperature (LCST), which makes it difficult to detach cells, and as a result, there is a concern that the recovery rate of cells may decrease.

Japanese Patent No. 4475847 JP 2017-014323 A

Colloids and Surfaces B, 2020, 185, p. 110565

As described above, a cell separation substrate capable of selectively adsorbing specific cells, easily detaching cells from the cell separation substrate, and capable of obtaining cells of high purity is not known.

An object of the present invention is to provide a substrate for cell separation that can selectively adsorb specific cells, easily detach the cells from the substrate for cell separation, and provide cells of high purity.

The present inventors have conducted intensive studies to achieve the above-mentioned objects, and found that they have an electric charge for selectively adsorbing specific cells, have a small change in affinity due to temperature changes, and have an upper critical solution temperature The present inventors have found that a substrate for cell separation in which a copolymer having (UCST) is immobilized on the surface of the substrate can solve the above problems.

That is, the present invention provides at least the following [1] to [15].
[1]
The base material surface has at least a ureido group-containing repeating sequence A and a charge-containing repeating sequence B, which are repetitions of a ureido group-containing structure represented by the following formula (1), and has an upper critical solution temperature (UCST) of 4 to 50. A substrate for cell separation on which a copolymer having a temperature range of °C is immobilized.

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

(In formula (2), R ² represents a hydrogen atom or a methyl group, A ² is absent or represents -(C=O)O- or -(C=O)NH-, b is 0 to 6 and X is NH ₃ ⁺ , N(CH ₃ )H ₂ ⁺ , N(CH ₃ ) ₂ H ⁺ , N(CH ₃ ) ₃ ⁺ , S(CH ₃ ) ₂ ⁺ , COO ⁻ , SO ₃ ^- represents a group selected from C ₆ H ₅ SO ₃ ^- and PO ₃ H ^- , provided that when b is 0, A ² does not exist.)
[3]
[2] above, wherein X is a group selected from NH ₃ ⁺ , N(CH ₃ )H ₂ ⁺ , N(CH ₃ ) ₂ H ⁺ , N(CH ₃ ) ₃ ⁺ and S(CH ₃ ) ₂ ⁺ 3. The substrate for cell separation according to .
[4]
R ¹ is a methyl group, A ¹ is -(C=O)O-, a is 2, R ² is a methyl group, A ² is -(C=O)O-, The substrate for cell separation according to [3] above, wherein b is 2 and X is N(CH ₃ ) ₂ H ⁺ or N(CH ₃ ) ₃ ⁺ .
[5]
The above wherein R ¹ is a hydrogen atom, A ¹ is absent, a is 1, R ² is a hydrogen atom, A ² is absent, b is 1, and X is NH ₃ ⁺ The substrate for cell separation according to [3].
[6]
The substrate for cell separation according to [2] above, wherein X is a group selected from COO ^- , SO ₃ ^- , C ₆ H ₅ SO ₃ ^- and PO ₃ H ^- .
[7]
R ¹ is a methyl group, A ¹ is -(C=O)O-, a is 2, R ² is a hydrogen atom, A ² is absent, b is 0, X is COO ^- . The substrate for cell separation according to [6] above.
[8]
Any one of the above [1] to [7], wherein the abundance ratio of the ureido group-containing repeating sequence A in all the monomer units constituting the copolymer is 30% ≤ A < 100% in terms of molar ratio. Substrate for cell separation.
[9]
The substrate for cell separation according to any one of [1] to [8] above, wherein the copolymer further has a phosphorylcholine group-containing repeating sequence C that is a repetition of a phosphorylcholine group-containing structure represented by the following formula (3): .

(In formula (3), R ³ represents a hydrogen atom or a methyl group, A ³ is absent or represents -(C=O)O- or -(C=O)NH-, c is 1 to 6 represents an integer of
[10]
The substrate for cell separation according to [9] above, wherein R ³ is a methyl group, A ³ is —(C═O)O—, and c is 2.
[11]
For cell separation according to [9] or [10] above, 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 Base material.
[12]
The substrate for cell separation according to any one of [1] to [11] above, wherein the copolymer has a number average molecular weight of 1,000 to 5,000,000.
[13]
The substrate for cell separation according to any one of [1] to [12] above, wherein the material of the substrate is glass, silicon or plastic.
[14]
A cell separation method for separating cells to be separated from a cell group containing cells to be separated, comprising the following [step A], [step B] and [step C].
[Step A]
A step of contacting a cell group containing cells to be separated with the substrate for cell separation according to any one of [1] to [13] above to adsorb the cells to be separated to the substrate for cell separation [Step B]
A step of washing the cells to be separated adsorbed to the substrate for cell separation [Step C]
Detaching the cells to be separated from the cell separation substrate by temperature change [15]
During the above [Step C], the temperature changes 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, which is heating.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a cell separation substrate capable of selectively adsorbing specific cells, allowing the cells to be easily detached from the cell separation substrate, and yielding cells of high purity.

Preferred embodiments of the present invention are described in detail below.

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

(In the above formula (1), R ¹ represents a hydrogen atom or a methyl group, A ¹ does not exist or represents -(C=O)O- or -(C=O)NH-, a is 1 to represents an integer of 6. In the above formula (2), R ² represents a hydrogen atom or a methyl group, and A ² is absent or -(C=O)O- or -(C=O)NH- where b is an integer of 0 to 6, X is NH ₃ ⁺ , N(CH ₃ )H ₂ ⁺ , N(CH ₃ ) ₂ H ⁺ , N(CH ₃ ) ₃ ⁺ , S(CH ₃ ) ₂ ⁺ , COO ^- , SO ₃ ^- , C ₆ H ₅ SO ₃ ^- and PO ₃ H ^- .)
In this specification, "repeating a structure" refers not only to continuous repetition of the structure, but also to intermittent repetition.

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

In the above formula (1), the absence of A ¹ means that the upper carbon (C) of A ¹ and the lower (CH ₂ ) _a of A ¹ are directly single bonded in the above formula (1). is shown.

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

As the ureido group-containing repeating sequence A, for example, R ¹ is a methyl group, A ¹ is —(C═O)O—, and a is 2, and R ¹ is a hydrogen atom. and A ¹ is -(C=O)O-, and a is 2, the ureido group-containing repeating sequence A, R ¹ is a methyl group, and A ¹ is -(C=O)NH- , a is 2 ureido group-containing repeating sequence A, R ¹ is a hydrogen atom, A ¹ is -(C=O)NH-, a is 2 ureido group-containing repeating sequence A, R ¹ is A ureido group-containing repeating sequence A is a hydrogen atom, A ¹ is absent, and a is 1. From the viewpoint of copolymerizability, R ¹ is a methyl group, and A ¹ is -(C= O) a ureido group-containing repeating sequence A wherein a is 2, and a ureido group-containing repeating sequence where R ¹ is a hydrogen atom, A ¹ is -(C=O)O- and a is 2 Sequence A, R ¹ is a hydrogen atom, A ¹ is —(C═O)NH—, a is 2, the ureido group-containing repeating sequence A or R ¹ is a hydrogen atom, and A ¹ is not present A ureido group-containing repeating sequence A in which a is 1 is preferred, R ¹ is a methyl group, A ¹ is -(C=O) O-, and a is 2, or A ureido group-containing repeating sequence A in which R ¹ is a hydrogen atom, A ¹ is absent, and a is 1 is more preferable.

In the above formula (2), the absence of A ² means that the upper carbon (C) of A ² and the lower (CH ₂ ) _b of A ² are directly single bonded in the above formula (2). is shown.

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

In the above formula (2), when X is cationic, said X is NH ₃ ⁺ , N(CH ₃ )H ₂ ⁺ , N(CH ₃ ) ₂ H ⁺ or N(CH ₃ ) for ease of synthesis. It is preferably ₃ ⁺ , more preferably NH ₃ ⁺ , N(CH ₃ )H ₂ ⁺ or N(CH ₃ ) ₂ H ⁺ . When X is anionic, it is preferably COO ^- , SO ₃ ^- or C ₆ H ₅ SO ₃ ^- , more preferably COO ^- or SO ₃ ^- for ease of synthesis. .

As the charge-containing repeating sequence B, for example, R ² is a methyl group, A ² is -(C=O)O-, b is 2, and X is NH ₃ ⁺ . ₍ ^AEMA ) _, a charge ^- bearing repeat sequence ^B ( DMAEMA), a charge-bearing repeat sequence B (choline methacrylate), where R ² is a methyl group, A ² is -(C=O)O-, b is 2, and X is N(CH ₃ ) ₃ ^{+ )} , a charge ^- bearing repeat sequence ^B _{( tertiary} sulfonium methacrylate), a charge ^- bearing repeat sequence B (acrylic acid) in which R ² is a hydrogen atom, A ² is absent, b is 0, and X is COO-, R ² is a methyl group, and A ² is -(C=O)O-, b is 2, X is COO- ^, a charge-bearing repeat sequence B (carboxylethyl methacrylate), R ² is a methyl group, and A ² is -(C= O) a charge-bearing repeat sequence B (ethyl sulfonate methacrylate) where O-, b is ² and X is SO ^- , R ₂ is a methyl group and A ² is -(C=O)NH -, b is 2, X is NH ₃ ⁺ , a charge-bearing repeat sequence B (aminoethyl methacrylamide), R ² is a hydrogen atom, and A ² is -(C=O)NH- , a charge-bearing repeat sequence B (aminoethylacrylamide) where b is 2 and X is NH ₃ ⁺ , R ² is a hydrogen atom, A ² is absent, b is 1 and X is NH ₃ A charge ^- bearing repeat sequence B (allylamine) where R ² is a hydrogen atom, A ² is absent, b is 0, and X is C ₆ H ₅ SO ₃ ^— . styrene sulfonic acid), etc. From the viewpoint of copolymerizability, R ² is a methyl group, A ² is —(C═O)O—, b is 2, and X is NH ₃ ⁺ . A charge-bearing repeat sequence B (AEMA), where R ² is a methyl group, A ² is -(C=O)O-, b is 2, and X is N(CH ₃ ) ₂ H ⁺ Charge-bearing repeat sequence B (DMAEMA), R ² is a methyl group, A ² is -(C=O)O-, b is 2, X is N(CH ₃ ) ₃ ⁺ , charge-bearing repeat sequence B (choline methacrylate), R ² is a hydrogen atom, A ² is absent, b is 0, and X is COO- ^, charge-bearing repeat sequence B (acrylic acid ), a charge-bearing repeat sequence B (allylamine) wherein R ² is a hydrogen atom, A ² is absent, b is 1 and X is NH ₃ ⁺ or R ² is a hydrogen atom and A ² is A charge-bearing repeat sequence B (styrene sulfonic acid) is preferred, wherein B is absent, b is 0, X is C ₆ H ₅ SO ₃ ^— , R ² is a methyl group, and A ² is — (C═O ) O-, b is ₂ , X is NH ⁺ , a charge-bearing repeat sequence B (AEMA), R ² is a methyl group, A ² is -(C=O)O-, A charge-bearing repeat sequence B (DMAEMA) where b is 2 and X is N(CH ₃ ) ₂ H ⁺ , R ² is a methyl group, A ² is -(C=O)O-, b is 2 and X is N(CH ₃ ) ₃ ⁺ , R ² is a hydrogen atom, A ² is absent, b is 0 and X is COO a charge ^- bearing repeat sequence B (acrylic acid) wherein R ² is a hydrogen atom, A ² is absent, b is 1, and X is NH ₃ ⁺ (allylamine) More preferably, the charge ^- bearing ^repeat sequence ^B _{( DMAEMA} ), a charge-bearing repeat sequence B (choline methacrylate) where R ² is a methyl group, A ² is -(C=O)O-, b is 2, and X is N(CH ₃ ) ₃ ⁺ , R ² is a hydrogen atom, A ² is absent, b is 0, and X is COO- ^, a charge-containing repeat sequence B (acrylic acid) or R ² is a hydrogen atom, A ² is present and especially preferred is the charge-bearing repeat sequence B (allylamine) in which b is 1 and X is NH ₃ ⁺ .

The molar ratio of each repeating sequence in the above copolymer can be appropriately determined so as to exhibit the required performance. The abundance ratio of the ureido group-containing repeating sequence A in all monomer units is preferably in the range of 30%≦A<100% in terms of molar ratio. It is more preferably within the range of 50%≦A<100%, and particularly preferably within the range of 50%≦A≦98%.

The above copolymer may further have a phosphorylcholine group-containing repeating sequence C, which is a repetition of a phosphorylcholine group-containing structure represented by the following formula (3). By introducing a phosphorylcholine group into the copolymer, in addition to making it easier to control the upper critical solution temperature (UCST) of the copolymer, hydrophilicity is improved when used for cell separation, and unnecessary cells are non-existent. It becomes easier to suppress specific adsorption.

(In the above formula (3), R ³ represents a hydrogen atom or a methyl group, A ³ does not exist or represents -(C=O)O- or -(C=O)NH-, c is 1 to represents an integer of 6.)

In the above formula (3), the absence of A ³ means that the upper carbon (C) of A ³ and the lower (CH ₂ ) _c of A ³ are directly single bonded in the above formula (3). is shown.

In the above formula (3), c represents an integer of 1-6, preferably 1-4, for example 1 or 2.

Examples of the 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.

The content of the phosphorylcholine group-containing repeating sequence C can be appropriately determined so as to exhibit the required performance. It is preferable that the abundance ratio of the phosphorylcholine group-containing repeating sequence C in all monomer units is in the range of 0%<C≦20% in terms of molar ratio. It is more preferably within the range of 0%<C≦15%, and particularly preferably within the range of 0%<C≦10%.

The above copolymers may be copolymers with other monomers. Other types of monomers can be appropriately selected in order 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 ) acrylate, allyl alcohol acrylonitrile, acrolein, sodium vinyl sulfonate, N-vinylpyrrolidone, itaconic acid, maleic acid, etc., and a monomer having a large effect of adjusting the upper critical temperature (UCST) is N-methylcarboxybetaine (meth ) acrylate, N-methylsulfobetaine (meth)acrylate, aminoethyl (meth)acrylate, S-methylsulfonium carboxylic acid (meth)acrylate, polyethylene glycol (meth)acrylate or polyethylene glycol monomethyl ether (meth)acrylate are preferred, and N -Methylcarboxybetaine (meth)acrylate, N-methylsulfobetaine (meth)acrylate, aminoethyl (meth)acrylate, polyethylene glycol (meth)acrylate or polyethylene glycol monomethyl ether (meth)acrylate are particularly preferred. To raise 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)acrylate, ) acrylamide, vinyl acetate, styrene, chlorostyrene, vinyl phenol, vinyl cinnamate, vinyl chloride, vinyl bromide, butadiene, vinylene carbonate, itaconic acid ester, fumaric acid ester, maleic acid ester, etc., and the upper critical temperature (UCST ) is preferably n-butyl (meth)acrylate, isobutyl (meth)acrylate, pentyl (meth)acrylate, hexyl (meth)acrylate or N-isopropyl (meth)acrylamide, and n-butyl ( More preferred are meth)acrylates or N-isopropyl(meth)acrylamides. Furthermore, monomers for binding ligands that recognize specific cells in cell separation can also be used. For example, propargyl (meth)acrylate, azidopropyl (meth)acrylate, succinimide (meth)acrylate, (meth)acrylic acid, aminoethyl (meth)acrylate, isocyanate (meth)acrylate, hydroxyethyl (meth)acrylate, etc. is preferable, and propargyl (meth)acrylate, azidopropyl (meth)acrylate, succinimide (meth)acrylate or isocyanate (meth)acrylate is preferable from the viewpoint of ease of binding with the ligand, propargyl (meth)acrylate, azide Propyl (meth)acrylate or succinimide (meth)acrylate are particularly preferred. In addition, the amount of other monomers to be blended is arbitrary and can be selected as appropriate, but in order to fully bring out the performance of the copolymer, the other monomers to be blended should be 20 mol% or less in all the monomers. preferably 15 mol % or less, and even more preferably 10 mol % or less.

The molecular weight of the copolymer can be appropriately determined by adjusting the polymerization conditions and the like so that the required performance can be exhibited. When producing a cell separation substrate in which the above copolymer is immobilized, it is preferably 2,000 to 2,000,000, more preferably 2,000 to 1,000,000, in order to increase the efficiency of cell separation. preferable. When the molecular weight of the copolymer is 1,000 or more in terms of number average molecular weight, the detachment of cells due to temperature response becomes easier, and when it is 5,000,000 or less, cells to be separated are attached to the cell separation substrate. Easier to absorb.

<Upper critical solution temperature (UCST) of copolymer>
The upper critical solution temperature (UCST) is the temperature at which the copolymer becomes insoluble in water or medium and forms an insoluble phase, and the temperature at which the copolymer dissolves in water or medium and forms a dissolved phase. The boundary temperature of the copolymer is dissolved in water or a medium at a concentration of at least 1 mM, the transmittance of visible light at 500 nm is measured in a quartz cell while the temperature is lowered, and the copolymer is completely It can be determined as the temperature at which the visible light transmittance of the clear solution when dissolved is 100% and the transmittance starts to decrease when the temperature is lowered. The upper critical solution temperature (UCST) is not particularly limited, but it must be a temperature suitable for cell culture, so it is in the range of 2 to 60°C, preferably 3 to 50°C, more preferably 4 to 50°C.

<Copolymer production method 1>
The above copolymer can be produced, for example, by radically polymerizing a ureido monomer represented by the following formula (4) and a charged monomer represented by the following formula (5).

(In Formula (4) above, R ¹ , A ¹ and a have the same meanings as above. In Formula (5) above, R ² , A ² , b and X have the same meanings as above. )

Copolymerization with other monomers is also possible in the radical polymerization of the ureido monomer and the charged monomer.

Radical polymerization of the ureido monomer and the charged monomer (and other monomers) can be bulk polymerization, but it is also possible to add a solvent and subject the polymerization to polymerization. The solvent is not particularly limited as long as it dissolves the ureido monomer and the charged monomer (and the other monomers), and common solvents can be used. Polar aprotic solvents such as, for example, acetone, dioxane, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), tetrahydrofuran (THF), anisole, toluene, acetonitrile, dimethylacetamide, methanol, ethanol (EtOH), 2 - polar protic solvents such as propanol, water, and mixtures thereof.

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

The photopolymerization can be carried out, for example, by ultraviolet (UV) radiation with a wavelength of 254 nm or electron beam (EB) radiation with an acceleration voltage of 150 to 300 kV. At this time, the use of a photopolymerization initiator is optional, but it is preferable to use it in terms of reaction time. Examples of photopolymerization initiators include 2-hydroxy-2-methyl-1-phenyl-1-propanone and 1-hydroxy-cyclohexylphenyl ketone. -Phenyl-1-propanone is preferred.

A chain transfer agent can also be used in the radical polymerization of the ureido monomer and the charged monomer (and the other monomer). Examples of chain transfer agents include 2-mercaptoethanol, 1-mercapto-2-propanol, 3-mercapto-1-propanol, p-mercaptophenol, mercaptoacetic acid, 2-mercaptopropionic acid, 3-mercaptopropionic acid, 2-mercapto nicotinic acid and the like.

Radical polymerization of the ureido monomer and the charged monomer (and other monomers) can also be carried out by a living radical polymerization method, specifically an atom transfer radical polymerization method (ATRP method), reversible addition-fragmentation chain transfer. A polymerization method (RAFT polymerization method), a nitroxide-mediated polymerization method (NMP method), and the like can be used. In particular, in the immobilization application to the substrate for cell separation of the present invention, no metal is used and the enzyme activity is not lowered. Modifiable atom transfer radical polymerization (ATRP) is preferred.

A known method can be used as the RAFT polymerization method, and the methods described in, for example, WO99/31144, WO98/01478 and US Pat. No. 6,153,705 are effective. Radical polymerization of the ureido monomer and the charged monomer (and other monomers) using RAFT polymerization can be carried out by adding a RAFT agent to ordinary radical polymerization. Examples of the RAFT agent include 4-cyanopentanoic dithiobenzoate, 2-cyano-2-propylbenzodithioate, benzylbenzodithioate, 2-phenyl-2-propylbenzodithioate, methyl 2-phenyl-2-(phenyl- carbonothioylthio)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-(dodecylthiocarbonylthioylthio)-2-methylpropionic acid, 4-cyano-4-(dodecyl sulfanyl-thiocarbonyl)sulfanylpentanoic acid polyethylene glycol methyl ether ester, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid-3-azido-1-propanol ester, benzyl 1H-pyrrole-1-carbodithioate , 2-cyanopropan-2-yl-N-methyl-N-pyridin-4-yl carbodithioate, ethyl-2-ethyl propionate-2-ethylxanthate, etc., but the above ureido monomers and charged monomers (and above other monomer), 4-cyanopentanoic acid dithiobenzoate, 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 preferable.

A known method can be used as the ATRP method, and after the copolymer is synthesized by the ATRP method, the substrate may be modified, and the copolymer is directly applied from the surface of the substrate having the ATRP initiator. may be polymerized. The catalyst used in the ATRP method is not particularly limited, and can be widely selected from those commonly used in the ATRP method. A transition metal complex, for example, can be used as a catalyst. The transition metal complex is not particularly limited and can be selected widely. For example, the following transition metal salts and ligands can be appropriately selected and combined.
Examples of the ATRP initiator include, but are not limited to, 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, 3-(trichlorosilyl)propyl 2-bromo-2-methylpropanoate, 3-(trimethoxysilyl)propyl 2-bromo-2-methylpropanoate, 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, ethyl 2-bromo-2-methylpropionate, chloromethylxylene, 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, diethyl meso-2,5-dibromoadipate, etc., but the above ureido monomers 1-trimethoxysilyl-2-(p-chloromethylphenyl)ethane, 4-(chloromethyl)phenyltrimethoxysilane, 1-trichlorosilyl-2 from the viewpoint of polymerization control of and charged monomers (and other monomers above) -(p-chloromethylphenyl)ethane, 3-(trimethoxysilyl)propyl 2-bromo-2-methylpropanoate, 2-bromo-2-methyl-N-[3-(trimethoxysilyl)propyl]propanamide, Chloromethylxylene or ethyl 2-bromoisobutyrate are 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 is used. It can also be used as an ATRP initiator by modifying the substrate surface.

The transition metal salts in the ATRP method include CuCl, _CuCl2 , CuBr, CuBr2, _TiCl2 , _TiCl3 , _{TiCl4 , TiBr4} , _{FeCl2 ,} FeCl3, _FeBr2 , _FeBr3 , _CoCl2 , _CoBr2 . , NiCl ₂ , NiBr ₂ , MoCl ₃ , MoCl ₅ , RuCl ₃ and the like.

The ligand for the transition metal salt is also not particularly limited, but tris(2-(dimethylamino)ethyl)amine (Me ₆ TREN), N,N,N,N-pentamethyldiethylenetriamine (PMDETA), 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA), 1,4,8,11-tetramethyl 1,4,8,11-azacyclotetradecane (Me ₄ Cyclam), 2 ,2-bipyridine, 4,4-dimethyl-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, N,N,N,N-tetrakis(2-pyridylmethyl)-ethylenediamine and the like. Combinations of the transition metal salt and the ligand include, for example, CuBr/2,2-bipyridine, CuBr ₂ /2,2-bipyridine, CuCl/Me ₆ TREN, CuCl ₂ /Me ₆ TREN and the like.

Furthermore, in addition to the above transition metal salt and ligand, it is also possible to add a reducing agent, if necessary. Examples of the reducing agent include ascorbic acid, sodium ascorbate, tin(II) 2-ethylhexanoate, and monovalent copper salts.

When the ATRP method is used, bulk polymerization is possible, but a solvent may be added for polymerization. Moreover, the solvent is not particularly limited as long as it dissolves the ureido monomer, the charged monomer, and the other monomer, and general solvents can be used. Polar aprotic solvents such as, for example, acetone, dioxane, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), tetrahydrofuran (THF), anisole, toluene, acetonitrile, dimethylacetamide, methanol, ethanol (EtOH), 2 - polar protic solvents such as propanol, water, and mixtures thereof. The polymerization temperature is appropriately selected depending on the solvent, and is usually in the range of 25-120°C, preferably in the range of 25-70°C. When the polymerization temperature is 25° C. or higher, the polymerization proceeds more easily, while when it is 120° C. or lower, it is easier to control the polymerization. Furthermore, the polymerization time is not particularly limited, but it is usually in the range of 1 to 96 hours, preferably in the range of 1 to 48 hours. When the polymerization time is 1 hour or longer, the polymerization proceeds more easily, and when the polymerization time is 96 hours or shorter, the possibility of generating by-products due to unnecessary termination reactions is lower.

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

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

In the above formula (4), the absence of A ⁴ means that the upper carbon (C) of A ⁴ and the lower (CH ₂ ) _d of A ⁴ are directly single bonded in the above formula (4). showing.

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

Examples of the primary amine-containing polymer include a primary amine-containing polymer in which R ⁴ is a methyl group, A ⁴ is —(C═O)O—, and d is 2, R ⁴ is a hydrogen atom, and A A primary amine-containing polymer wherein ⁴ is -(C=O)O- and d is 2, R ⁴ is a methyl group, A ⁴ is -(C=O)NH- and d is 2 primary amine-containing polymer, wherein R ⁴ is a hydrogen atom, A ⁴ is -(C=O)NH-, and d is 2, R ⁴ is a hydrogen atom, and A ⁴ is absent; However, from the viewpoint of copolymerizability, R ⁴ is a methyl group, A ⁴ is —(C═O)O—, and d is 2. primary amine-containing polymer, R ⁴ is a hydrogen atom, A ⁴ is —(C═O)O—, and d is 2, primary amine-containing polymer, R ⁴ is a hydrogen atom, and A ⁴ is —( C═O)NH—, d is 2 or primary amine-containing polymers in which R ⁴ is a hydrogen atom, A ⁴ is absent and d is 1, and R ⁴ is methyl A primary amine-containing polymer wherein A ⁴ is -(C=O)O- and d is 2 or a primary amine wherein R ⁴ is a hydrogen atom, A ⁴ is absent and d is 1 Containing polymers are more preferred.

The molecular weight of the primary amine-containing polymer can be appropriately determined by adjusting the polymerization conditions and the like so that the required performance can be exhibited. , preferably 2,000 to 2,000,000, more preferably 2,000 to 1,000,000, in order to increase the efficiency of cell separation of the substrate for cell separation of the present invention. When the molecular weight of the primary amine-containing polymer is 1,000 or more in terms of number average molecular weight, detachment of cells by temperature response becomes easier in the substrate for cell separation of the present invention, and when it is 5,000,000 or less, It becomes easier for cells to be separated to be adsorbed to the cell separation substrate.

Solvents for dissolving the primary amine-containing polymer include, for example, water, buffers such as imidazole buffers, 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; Examples include dimethylsulfoxide, tetrahydrofuran, 1,4-dioxane and the like. The concentration of the primary amine-containing polymer in the primary amine-containing polymer solution used in the reaction is not limited, but is usually 1 to 80% by weight, preferably 2 to 50% by weight, more preferably 5 to 40% by weight. can. When the concentration of the primary amine-containing polymer is 80% by weight or less, the reactivity with the cyanate is easily maintained without excessively increasing the viscosity of the solution, which is preferable.

As the cyanate (MCNO) to be reacted with the primary amine-containing polymer, alkali metal salts of cyanic acid such as potassium cyanate and sodium cyanate can be suitably exemplified. Potassium cyanate is preferred. The ratio of the cyanate to be used is not particularly limited, but is preferably 0.4 mol or more per 1 mol of the primary amino group in the primary amine-containing polymer. When the proportion of cyanate used is 0.4 mol or more, ureido groups can be introduced more sufficiently, and the upper critical solution temperature (UCST) can be more easily achieved.

The reaction between the primary amine-containing polymer and cyanate (MCNO) is preferably carried out while stirring. Although the reaction temperature is not particularly limited, it is desirable to maintain it at preferably 0 to 100°C, more preferably 30 to 60°C. When the reaction temperature is 0° C. or higher, the reaction proceeds more easily, and when it is 100° C. or lower, the decomposition of the raw materials and the undesirable side reactions are less likely to occur. The reaction time is also not particularly limited, but usually 1 to 48 hours, preferably 1 to 25 hours, to obtain a solution of the copolymer. When the reaction time is 1 hour or more, the reaction tends to proceed sufficiently.

The obtained copolymer can be used as it is in the next step without being purified, or can be isolated and purified by treatment such as reprecipitation, gel filtration chromatography, dialysis, etc., preferably.

The above copolymer prepared from the above primary amine-containing polymer has a primary amino group as the charge-containing repeating sequence B, but can be reacted with an alkyl halide, trifluoromethanesulfonylalkyl, methanesulfonylalkyl, or the like to form a secondary amino group. It can be converted into amines, tertiary amines and quaternary amines. Also, it can be converted to a carboxyl group by reacting with succinic anhydride.

<Base material>
The material of the base material is not particularly limited, but plastics, polysaccharides, metals, magnetic metals, silicon, glass, etc. are used, and a plurality of these materials may be used, making it easy to use for cell separation. Preferred materials are silicon, glass, and 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.

Further, the shape of the substrate may be, for example, container-like, plate-like, particle-like, fiber-like, or porous with holes or grooves. Among them, container-shaped, plate-shaped, particle-shaped, fibrous or magnetic particles (magnetic particles) are preferable because of ease of handling during cell separation, and container-shaped such as petri dishes and flasks, silicon wafers and glass plates. Such a plate shape is particularly preferred.

The surface of the substrate is selected from the group consisting of hydroxyl group, amino group, carboxyl group, tosyl group, epoxy group, N-hydroxysuccinimide group, maleimide group, thiol group, azide group, phenylazide group, biotin residue and avidin residue. It may have at least one selected functional group on its surface.

<Method 1 for preparing cell separation substrate>
According to the present invention, a substrate for cell separation can be produced through [step a] of immobilizing a surface initiator on a substrate, and [step b] of polymerizing the copolymer from the surface initiator.

In carrying out [step a], a pretreatment step can generally be performed on the base material. The pretreatment step can be performed by coating the substrate surface with an acid solution. Acidic solutions include, for example, sulfuric acid, hydrochloric acid, nitric acid, hydrogen peroxide, etc. These may be used alone or in combination of two or more. Among these, a mixed solution obtained by mixing equal amounts of sulfuric acid and hydrogen peroxide is particularly preferred. The coating of the acid solution is not particularly limited as long as the method can coat the surface of the base material, and can be carried out, for example, by coating, spraying, dipping, or the like. 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, the surface modification can be performed more sufficiently. When the treatment time is 48 hours or less, it is difficult for the surface of the material to change further, and the productivity is further improved.

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

Methods for immobilizing the surface initiator on the substrate include addition to the substrate, silane coupling reaction, click reaction, amidation, esterification, thioesterification, carbonate formation, Schiff base formation reaction, reductive amination, Coupling reaction via radicals, Diels-Alder reaction, disulfidation, Michael addition reaction, ene-thiol reaction, coupling reaction via aromatic boron compound, tertiary sulfonium formation reaction, quaternary ammonium formation reaction, etc. However, silane coupling reaction, click reaction, amidation or esterification are preferred because of their high versatility.

Other immobilization methods include a method of reacting the functional group of the base material with 2-bromopropionyl bromide, and a method of reacting 3-aminopropyltrimethoxysilane or 3-aminopropyltriethoxysilane with 2-bromopropionyl bromide on the surface. and a method of using a reaction product of dopamine and 2-bromopropionyl bromide, by which an ATRP initiator can be introduced onto the surface of the substrate as a surface initiator. Furthermore, a method of reacting 4,4′-azobis(4-cyanopentanoic acid), which is a radical initiator having a carboxyl group, 4-cyanopentanoic acid dithiobenzoate, 4-cyano-4-(phenylcarbonothioyl Thio)pentanoic acid N-succinimidyl ester, 4-cyano-4-(dodecylsulfanyl-thiocarbonyl)sulfanylpentanoic acid, etc. are reacted with the functional groups of the base material to form a surface initiator on the surface of the base material. A RAFT agent can be introduced.

The silane coupling reaction can be carried out by coating, spraying, dipping, vacuum deposition, ion plating, thermal CVD, or the like. Among them, coating or dipping is preferable as a simple method. The temperature of the silane coupling reaction is not particularly limited, but is usually 20 to 200°C, preferably 20 to 150°C, more preferably 20 to 120°C. When the reaction temperature is 20° C. or higher, the reaction proceeds more easily, and when it is 200° C. or lower, undesirable side reactions such as decomposition of the silane coupling agent hardly proceed. Furthermore, the silane coupling reaction time is usually 2 to 96 hours, preferably 2 to 72 hours, more preferably 2 to 48 hours. When the reaction time is 2 hours or more, the reaction is likely to proceed sufficiently, and when it is 96 or less, undesirable side reactions such as decomposition of the silane coupling agent are less likely to proceed.

The silane coupling reaction can also be used for immobilization by adding a solvent. The solvent is not particularly limited as long as it dissolves the surface initiator, and common solvents can be used. Polar aprotic solvents such as toluene, acetone, dioxane, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), tetrahydrofuran (THF), anisole, toluene, xylene, acetonitrile, dimethylacetamide, methanol, ethanol ( EtOH), 2-propanol, polar protic solvent solvents such as water, mixtures thereof.

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 a Grafting from method, and is the same as the method 1 for producing the above copolymer. is.

<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, it can also be produced through [step c] of synthesizing the above copolymer and [step d] of immobilizing the above copolymer on a substrate.

The above [step c] is the same as the method for producing the above copolymer.

Examples of the method for immobilizing the copolymer on the substrate in the above [step d] include the same methods as in the above [step a].

<Cell separation method>
Separation of the cells to be separated from the cell group containing the cells to be separated by using the substrate for cell separation of the present invention is performed by a cell separation method including the following [Step A], [Step B] and [Step C]. be able to.

[Step A]
In this step, a cell group containing cells to be separated is brought into contact with the substrate for cell separation, and the cells to be separated are adsorbed to the substrate for cell separation.

Examples of the above-mentioned cells include established cells for culture, fertilized eggs and egg cells of animals including humans, and the like. In addition, stem cells such as sperm cells, ES cells, iPS cells, mesenchymal stem cells, hematopoietic stem cells, neural stem cells, umbilical cord blood cells, hepatocytes, nerve cells, cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells, blood cells, etc. Animal cells including human or plant cells can also be mentioned.

The above-mentioned cells to be separated refer to a group of cells obtained as a result of performing cell separation using the substrate for cell separation of the present invention. It can be a group.

The cell group containing the cells to be separated is a cell group containing preferably 0.1% or more of the cells to be separated, and the type of cells contained is not limited.

The temperature at which the cell group containing the cells to be separated is brought into contact with the cell separation substrate may be 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, more preferably 3 to 10°C. When the cell group containing the cells to be separated is brought into contact with the cell separation substrate at a temperature of 2° C. or higher, the cells are less likely to be frozen and damaged.

As a method of contacting the cell group containing the cells to be separated with the cell separation substrate, a method of pressing the cells against the cell separation substrate by centrifuging a plate, petri dish, or flask, A method of waiting for sedimentation on the substrate and the like can be mentioned.

The number of cells contained in the cell group containing the cells to be separated is usually 1.0×10 ¹⁰ cells/mL or less, preferably 1.0×10 ⁹ cells/mL or less, and 1.0×10 ⁸ cells/mL or less. Cells/mL or less is particularly preferred. When the cell count is 1.0×10 ¹⁰ cells/mL or less, sufficient separation effect can be easily obtained without saturating the cells in contact with the substrate for cell separation.

Contact between the cell group containing the cells to be separated and the substrate for cell separation is not particularly limited as long as it does not adversely affect the cells to be separated, but buffers and media that are commonly used in this field can be used. Examples thereof include phosphate buffer, Tris buffer, Good's buffer, glycine buffer, borate buffer and the like, and these may be mixed and used. Phosphate buffer is preferred. Dulbecco's Modified Eagle MEM medium (DMEM), α-MEM medium, Roswell Park Memorial Institute (RPMI) medium, F12 medium, TC199 medium, GMEM medium, etc., may be mixed, and if necessary bovine Supplements such as fetal serum (FBS), glutamine, and antibiotics may be added, but a serum-free medium is preferred so as not to impair the effect of the charge on the cell separation substrate surface.

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

The washing temperature may be 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. , more preferably 3 to 10°C. Washing at temperatures above 2°C is less likely to freeze and damage the cells.

The number of times of washing is usually 10 times or less, preferably 7 times or less, and more preferably 5 times or less. When washing is performed 10 times or less, there is less risk of damage to cells due to shear stress during washing.

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

[Step C]
This is a step of detaching the cells to be separated from the cell separation substrate by changing the temperature.

The desorption temperature may be at least 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. °C, more preferably 30-40°C. Desorption at a temperature of 50° C. or less denatures the proteins contained in the cells, which reduces the risk of damaging the cells.

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

<Preparation of initiator-modified substrate>

A glass substrate (10 mm×10 mm) was dipped in a piranha solution (concentrated sulfuric acid/30% hydrogen peroxide solution 3/1 v/v) and immersed for 2 hours. After immersion, it was washed with deionized water and dried. The piranha-treated glass substrate was placed in toluene (8 mL), 3-aminopropyltrimethoxysilane (50 μL) was added, and the mixture was reacted at 30° C. for 24 hours. After the reaction, toluene was removed and washed with ethanol. After that, annealing was performed at 120° C. for 2 hours in a nitrogen atmosphere to obtain a glass substrate having amino groups on its surface. A glass substrate having amino groups on its surface was placed in THF (2 mL), and triethylamine (50 μL) and 2-bromopropionyl bromide (140 μL) were added. After reacting at room temperature for 3 hours, the substrate was washed with ethanol to obtain an initiator-modified base material (hereinafter referred to as "initiator-modified base material").

<Example 1: Preparation of Cell Separation Substrate A>

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

In a test tube, an initiator-modified substrate (10 mm × 10 mm), ureidoethyl methacrylate (UMA) (230 mg, 1.35 mmol) and N,N-dimethylaminoethyl methacrylate (DMAEMA) (11.8 mg, 0.075 mmol) as monomers are placed in a test tube. , 2-methacryloyloxyethylphosphorylcholine (MPC) (22.1 mg, 0.075 mmol), ethyl 2-bromoisobutyrate (1.9 mg, 0.01 mmol), CuBr ₂ (8,82 mg, 0.036 mmol) and 2,2 - Bipyridine (11.4 mg, 0.072 mmol) was added 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 reacting for 24 hours, the substrate was recovered from the reaction solution and washed to obtain a cell separation substrate A.
In addition, in order to analyze the polymer immobilized on the cell separation substrate A, free copolymer A obtained by polymerizing ethyl 2-bromoisobutyrate by reprecipitating the reaction solution with acetone ' got.
- Yield of free copolymer A'; 199 mg
- Composition ratio of free copolymer A'; x: y: z = 89: 6: 5 (calculated from ¹ H NMR)
¹ H-NMR (CD ₃ OD+D ₂ O, 400 MHz);
4.1 ppm (UMA, DMAEMA: COO- CH ₂ ), 3.8-4, 6 ppm (MPC: COO- CH ₂ , CH ₂ -CH ₂ , PO- CH ₂ ), 3.6 ppm (MPC : CH ₂ —N), 3.4 ppm (UMA, DMAEMA: CH ₂ —CH ₂ ), 3.2 ppm (N( CH ₃ ) ₃ ), 2.8 ppm (DMAEMA: N(CH ₃ ) ₂ H ⁺ ), 1.0-2.4 ppm (polymer backbone)

The number average molecular weight of the resulting free copolymer A' was determined by gel permeation chromatography using a multi-angle light scattering detector (MALS) manufactured by WYATT as a detector. LC-720AD manufactured by Shimadzu Corporation was used as a pump, RID10A manufactured by Shimadzu Corporation as a detector (differential refractometer), and SPD-20A as a detector (UV). As the column, two columns of TSKGel G3000PWXL and TSKGel G5000PWXL (column size 4.6 mm×25 cm) manufactured by Tosoh Corporation were used. As a developing solvent, distilled water/acetonitrile (5/5 v/v) in which 100 mM sodium nitrate was dissolved was used. 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 free copolymer A' had a number average molecular weight of 17,000.

The upper critical solution temperature (UCST) of the obtained free copolymer A' was determined by measuring the transmittance with an ultraviolet-visible spectrophotometer, and the temperature at which the transmittance began to decrease was defined as the upper critical solution temperature (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, the solution temperature was changed in the range of 70° C. to 5° C., and the transmittance (%) of the solution was measured with an ultraviolet-visible spectrophotometer. The transmittance (%) of the solution was calculated from the following formula. As a result, the upper critical solution temperature (UCST) of free copolymer A' was 27°C.

<Example 2: Production of Cell Separation Substrate B>

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

In a test tube, an initiator-modified substrate (10 mm × 10 mm), ureidoethyl methacrylate (UMA) (230 mg, 1.35 mmol), choline methacrylate (CMA) (15.6 mg, 0.075 mmol), and 2-methacryloyloxyethyl as monomers are placed in a test tube. phosphorylcholine (MPC) (22.1 mg, 0.075 mmol), ethyl 2-bromoisobutyrate (1.9 mg, 0.01 mmol), CuBr ₂ (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 reacting for 24 hours, the obtained base material was collected and washed to obtain a base material B for cell separation.
In addition, in order to analyze the polymer immobilized on the cell separation substrate, free copolymer B' obtained by polymerizing ethyl 2-bromoisobutyrate by reprecipitating the reaction solution with acetone. got
- Yield of free copolymer B'; 187 mg
- Composition ratio of free copolymer B'; x: y: z = 88: 6: 6 (calculated from ¹ H NMR)

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

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

<Example 3: Synthesis of substrate C for cell separation>

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

In a test tube, an initiator-modified substrate (10 mm × 10 mm), ureidoethyl methacrylate (UMA) (230 mg, 1.35 mmol), t-butyl acrylate (tBA) (9.6 mg, 0.075 mmol), 2-methacryloyl as monomers Oxyethylphosphorylcholine (MPC) (22.1 mg, 0.075 mmol), ethyl 2-bromoisobutyrate (1.9 mg, 0.01 mmol), CuBr ₂ (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 reacting for 24 hours, the resulting substrate was collected and washed. After that, the cell separation substrate C was obtained by reacting with methanesulfonic acid to hydrolyze the t-butyl group.
In addition, in order to analyze the polymer immobilized on the cell separation substrate, free copolymer C′ obtained by polymerizing ethyl 2-bromoisobutyrate by reprecipitating the reaction solution with acetone. got
- Yield of free copolymer C'; 157 mg
- Composition ratio of free copolymer C'; x: y: z = 90: 5: 5 (calculated from ¹ H NMR)

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

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

<Example 4-1: Synthesis of copolymer D'>

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

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

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

<Example 4-2: Synthesis of substrate D for cell separation>

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

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 (manufactured by Corning) with carboxylic acid introduced on the surface, and added to 3 mL of water. dissolved to initiate the reaction. After reacting for 3 hours, the polystyrene dish was washed to obtain a substrate D for cell separation.

<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 (manufactured by Kyowa Interface Science Co., Ltd.). Cell separation substrate A was placed on the stage, and 1 μL of ion-exchanged water was added dropwise at temperatures of 4° C. and 37° C. under a nitrogen atmosphere for measurement. The contact angle at each temperature was calculated by measuring the angle between the liquid surface and the solid surface.

<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. Table 1 shows the evaluation results.

<Test Example 2-1: Cell separation test>
Cell culture substrate A was placed in a polystyrene 6-well plate as a cell separation substrate, and Jurkat cells and HL-60 cells previously stained with Hoechest 33342 as cells to be separated were mixed at a cell number of 50:50. 3 mL of multiple cell mixture (1.0×10 ⁵ cells/mL) was added at 4°C. After the cells were sedimented by centrifuging the 6-well plate, the cells were washed twice, an area of 1 mm × 1 mm was observed with a fluorescence microscope, and the number of cells to be separated on the cell separation substrate was counted (count A). After that, the temperature was raised to 37° C., the cells were washed, and the number of cells to be separated was again counted by observing with a fluorescence microscope (count B). The desorption rate was calculated from the following formula. Purity was calculated by flow cytometer analysis after cell separation. Table 2 shows the evaluation results.
Detachment rate = (count A - count B) / count A x 100

<Test Examples 2-2 to 2-4>
A cell separation test was performed 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. Table 2 shows the evaluation results.

<Comparative Example 1: Synthesis of Cell Separation Substrate E>

In a test tube, an initiator-modified substrate (10 mm × 10 mm), ureidoethyl methacrylate (UMA) (243 mg, 1.43 mmol), 2-methacryloyloxyethylphosphorylcholine (MPC) (22.1 mg, 0.075 mmol) and 2 as monomers - Ethyl bromoisobutyrate (1.9 mg, 0.01 mmol), CuBr ₂ (8,82 mg, 0.036 mmol) and 2,2-bipyridine (11.4 mg, 0.072 mmol) were added and ethanol/water (50/50 v/v) dissolved in 3 mL. After bubbling with nitrogen gas, ascorbic acid (1.26 mg, 0.0072 mmol) was added to initiate the reaction. After reacting for 24 hours, the resulting base material was collected and washed to obtain a cell separation base material E.
In addition, in order to analyze the polymer immobilized on the cell separation substrate, free copolymer E′ obtained by polymerizing ethyl 2-bromoisobutyrate by reprecipitating the reaction solution with acetone. got
- Yield of free copolymer E'; 200 mg
- Composition ratio of free copolymer E'; x: y = 94: 6 (calculated from ¹ H NMR)

The number average molecular weight of the resulting free copolymer E' was determined in the same manner as in Example 1. As a result of gel permeation chromatography measurement, the free copolymer E' had a number average molecular weight of 20,000.

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

<Comparative Example 2: Synthesis of Cell Separation Substrate F>

In a test tube, an initiator-modified substrate (10 mm × 10 mm), N-isopropylacrylamide (NIPAM) (162 mg, 1.43 mmol), t-butyl acrylate (tBA) (9.6 mg, 0.075 mmol) as monomers, 2-bromoiso Ethyl butyrate (1.9 mg, 0.01 mmol), CuBr ₂ (8,82 mg, 0.036 mmol) and 2,2-bipyridine (11.4 mg, 0.072 mmol) were charged and ethanol/water (80/20 v/ v) Dissolved in 3 mL. After bubbling with nitrogen gas, ascorbic acid (1.26 mg, 0.0072 mmol) was added to initiate the reaction. After reacting for 24 hours, the resulting substrate was collected and washed. After that, the cell separation substrate F was obtained by reacting with methanesulfonic acid to hydrolyze the t-butyl group.
In addition, in order to analyze the polymer immobilized on the cell separation substrate, free copolymer F' obtained by polymerizing ethyl 2-bromoisobutyrate by reprecipitating the reaction solution with acetone. got
- Yield of free copolymer F'; 200 mg
- Composition ratio of free copolymer F'; x: y = 95: 5 (calculated from ¹ H NMR)

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

The lower critical solution temperature (LCST) of the resulting free copolymer F' was determined by measuring the transmittance with an ultraviolet-visible spectrophotometer, and the temperature at which the transmittance was 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, the solution temperature was changed in the range of 70° C. to 5° C., and the transmittance (%) of the solution was measured with an ultraviolet-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 the free copolymer F' was 31°C.

<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. Table 1 shows the evaluation results.

<Comparative test example 2-1>
A cell separation test was performed 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. Table 2 shows the evaluation results.

<Comparative Test Example 2-2>
Cell culture substrate F as a cell separation substrate was placed in a polystyrene 6-well plate, and Jurkat cells and HUVEC cells previously stained with Hoechest 33342 as cells to be separated were mixed at a ratio of 50:50. 3 mL of the mixture (1.0×10 ⁵ cells/mL) was added at 37°C. After the cells were sedimented by centrifuging the 6-well plate, the cells were washed twice, an area of 1 mm × 1 mm was observed with a fluorescence microscope, and the number of cells to be separated on the cell separation substrate was counted (count A). After that, the temperature was lowered to 4° C., the cells were washed, and the number of cells to be separated was again counted by fluorescence microscope observation (count B). Desorption rate and purity were calculated in the same manner as in Test Example 2-1. Table 2 shows the evaluation results.

Cell separation substrates A to E (Test Examples 1-1 to 1-4, Comparative Test Example 1-1) on which the copolymer having the ureido group-containing repeating sequence was immobilized were 4° C. and 37° C. The change in contact angle is small at , maintaining hydrophilicity. On the other hand, the cell separation substrate F (Comparative Test Example 1-2) on which the NIPAM-based polymer was immobilized showed a large change in the contact angle between 4°C and 37°C, indicating hydrophobicity at 37°C. . From this, it was found that the substrate for cell separation of the present invention is always hydrophilic with little change in affinity due to temperature change.

Cell separation substrate E, which is a non-charged substrate, had a small value of count A, that is, a small amount of cells to be separated was adsorbed. Furthermore, since it does not have an electric charge, only specific cells cannot be adsorbed, resulting in low purity after separation (Comparative Test Example 2-1). In addition, in the cell separation substrate F to which the NIPAM-based polymer was immobilized, it was necessary to detach the cells under low temperature conditions, and as a result, the motility of the cells decreased, resulting in a decrease in the detachment rate. Furthermore, the purity after separation was also low. This is thought to be because the cell separation substrate F exhibits hydrophobicity at 37° C., and thus not only the target cells to be separated (HUVEC) but also non-target cells (Jurkat) were adsorbed by hydrophobic interaction during cell adsorption. be done. Based on the above, a copolymer having an electric charge for selectively adsorbing specific cells, a small change in affinity due to temperature change, and an upper critical solution temperature (UCST) was immobilized on the surface of the substrate. It was found that by using the cell separation substrate of the present invention, specific cells can be selectively adsorbed, the cells can be easily detached from the cell separation substrate, and highly pure cells can be obtained.

INDUSTRIAL APPLICABILITY The present invention provides a substrate for cell separation that can selectively adsorb specific cells, easily detach cells from the substrate for cell separation, and yield cells of high purity.

Claims (15)

The base material surface has at least a ureido group-containing repeating sequence A and a charge-containing repeating sequence B, which are repetitions of a ureido group-containing structure represented by the following formula (1), and has an upper critical solution temperature (UCST) of 4 to 50. A substrate for cell separation on which a copolymer having a temperature range of °C is immobilized.

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

(In formula (2), R ² represents a hydrogen atom or a methyl group, A ² is absent or represents -(C=O)O- or -(C=O)NH-, b is 0 to 6 and X is NH ₃ ⁺ , N(CH ₃ )H ₂ ⁺ , N(CH ₃ ) ₂ H ⁺ , N(CH ₃ ) ₃ ⁺ , S(CH ₃ ) ₂ ⁺ , COO ⁻ , SO ₃ ^- represents a group selected from C ₆ H ₅ SO ₃ ^- and PO ₃ H ^- , provided that when b is 0, A ² does not exist.) X is a group selected from _NH3 ⁺ , _N ( _CH3 )H2 ₊ , N( _CH3 ) ^{2H+ , N} ( _CH3 ) ₃₊ and S( _CH3 ) ₂₊ ^. Substrate for cell separation as described. R ¹ is a methyl group, A ¹ is -(C=O)O-, a is 2, R ² is a methyl group, A ² is -(C=O)O-, ^4. The substrate for cell separation according to claim 3, wherein b is ₂ and X is N( _CH3 )2H ⁺ or N( _CH3 ) ₃₊ . R ¹ is a hydrogen atom, A ¹ is absent, a is 1, R ² is a hydrogen atom, A ² is absent, b is 1, and X is NH ₃ ⁺ Item 4. The substrate for cell separation according to item 3. The substrate for cell separation according to claim 2, wherein X is a group selected from COO ^- , SO ₃ ^- , C ₆ H ₅ SO ₃ ^- and PO ₃ H ^- . R ¹ is a methyl group, A ¹ is -(C=O)O-, a is 2, R ² is a hydrogen atom, A ² is absent, b is 0, X The substrate for cell separation according to claim 6, wherein is COO ^- . The cell according to any one of claims 1 to 7, wherein the abundance ratio of the ureido group-containing repeating sequence A in all the monomer units constituting the copolymer is 30% ≤ A < 100% in terms of molar ratio. Substrate for separation. The substrate for cell separation according to any one of claims 1 to 8, wherein the copolymer further has a phosphorylcholine group-containing repeating sequence C which is a repetition of a phosphorylcholine group-containing structure represented by the following formula (3).

(In formula (3), R ³ represents a hydrogen atom or a methyl group, A ³ is absent or represents -(C=O)O- or -(C=O)NH-, c is 1 to 6 represents an integer of The substrate for cell separation according to claim 9, wherein R ³ is a methyl group, A ³ is -(C=O)O-, and c is 2. 11. The substrate for cell separation according to claim 9 or 10, wherein the abundance ratio of the phosphorylcholine group-containing repeating sequence C in all the monomer units constituting the copolymer is 0%<C≦20% in terms of molar ratio. The substrate for cell separation according to any one of claims 1 to 11, wherein the copolymer has a number average molecular weight of 1,000 to 5,000,000. The substrate for cell separation according to any one of claims 1 to 12, wherein the material of said substrate is glass, silicon or plastic. A cell separation method for separating cells to be separated from a cell group containing cells to be separated, comprising the following [step A], [step B] and [step C].
[Step A]
A step of contacting a cell group containing cells to be separated with the substrate for cell separation according to any one of claims 1 to 13 to adsorb the cells to be separated to the substrate for cell separation [Step B]
A step of washing the cells to be separated adsorbed to the substrate for cell separation [Step C]
A step of detaching the cells to be separated from the cell separation substrate by changing the temperature During the above [Step C], the temperature changes 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). 15. The cell separation method according to claim 14, which is heating.