Copolymer, substrate for cell separation, and cell separation method

Abstract

The present invention provides a copolymer which has a repeating unit A represented by formula (1) and a repeating unit B represented by formula (2), and in which the amount of the repeating unit A is 50 mol% or more relative to the total amount of the repeating unit A and the repeating unit B, and the lower critical solution temperature (LCST) is 3ºC to 40ºC (definitions of symbols in the formulae are as described in the description).

Description

Copolymer, cell separation substrate, and cell separation method

[0001] The present invention relates to a copolymer that is a temperature-responsive polymer, a cell separation substrate having a layer formed from the copolymer, and a cell separation method using the substrate.

[0002] In the field of regenerative medicine, a common technique involves extracting normal cells from diseased patients, culturing them outside the body, and then transplanting them back into the patient. However, it is difficult to extract only specific target cells from a patient's body, blood, or bodily fluids. Therefore, a method for isolating specific target cells is needed.

[0003] Cell separation methods are widely used in the fields of pathological diagnosis and clinical testing. Known cell separation methods include density gradient centrifugation, separation using magnetic particles, separation using substrates, and methods combining flow cytometry and cell sorting. Among these, methods using substrates to separate specific target cells offer advantages such as the ability to process a large number of cells and simple cell separation procedures. Furthermore, recent advancements in regenerative medicine have created a need for cell separation that is less irritating, more efficient, and of higher purity. Achieving cell separation with less irritation, higher efficiency, and higher purity is expected to improve diagnostic accuracy in pathological diagnosis and enhance therapeutic efficacy in regenerative medicine.

[0004] In cell separation methods using substrates, a method of separating cells from the substrate by enzymatic treatment is generally employed. However, it is known that methods using enzymes can damage cells. As a solution to this problem, Patent Document 1 discloses a cell culture support material in which poly[N-isopropylacrylamide], a temperature-responsive polymer, is introduced to the surface. It has been shown that a low-irritation cell separation method using such a temperature-responsive polymer and temperature changes can separate cells from the substrate with minimal irritation while suppressing damage to cells.

[0005] Patent Document 2 describes the use of a block copolymer consisting of blocks (A), (B), and (C) in the order (A)-(B)-(C) as a surface treatment agent for a substrate. Example 1 of Patent Document 2 describes a block copolymer consisting of an N-isopropylacrylamide polymer (block (A)), a butyl methacrylate polymer (block (B)), and an N,N-dimethylaminoethyl methacrylate polymer (block (C)).

[0006] Furthermore, Patent Document 2 states that in the block (C), the following formula is used:

[0007]

[0008] (In the above formula, R ３１ The disclosed material includes repeating units represented by ), where is a divalent hydrocarbon group (particularly alkylene) having 1 to 10 carbon atoms. By introducing a divalent hydrocarbon group (particularly alkylene) between the amino group and the main chain of the block copolymer in this way, the amino group is more easily exposed on the substrate surface, and it is expected that the recovery rate of target cells will increase.

[0009] Japanese Patent Publication No. 2003-038170 Japanese Patent Publication No. 2018-174919

[0010] The poly[N-isopropylacrylamide] described in Patent Document 1 exhibits hydrophilicity under low-temperature conditions and hydrophobicity under high-temperature conditions. Therefore, in cell separation using a cell separation substrate having a layer formed from the polymer, cells are adhered to the substrate at high temperatures, but due to the hydrophobicity of the polymer, nonspecific adhesion of cells is likely to occur, raising concerns that the number of target cells after separation will be small and the recovery efficiency will decrease. Furthermore, although cells are detached from the substrate at low temperatures, the polymer does not exhibit sufficient hydrophilicity, raising concerns that cell detachment will be insufficient and efficiency will decrease.

[0011] In the block copolymer described in Example 1 of Patent Document 2 mentioned above, the distance between the dimethylamino group in the N,N-dimethylaminoethyl methacrylate unit and the main chain skeleton of the block copolymer is short. Therefore, during cell separation, the dimethylamino group is embedded in the side chains of the block copolymer and does not come into contact with the substrate surface, raising concerns that it may not adhere to specific target cells and that purity may decrease.

[0012] As mentioned above, Patent Document 2 discloses the introduction of a divalent hydrocarbon group (particularly alkylene) between the main chain and the amino group of the block copolymer. However, because divalent hydrocarbon groups (particularly alkylene) are hydrophobic, when cells are adhered to a substrate, nonspecific adhesion of cells other than the target cells is likely to occur, raising concerns that the purity of the target cells after separation will decrease.

[0013] The present invention has been made in view of the above circumstances, and the problem that the present invention aims to solve is to provide a copolymer that can produce a cell separation substrate that enables separation of target cells under low-irritation conditions that reduce damage to target cells, and that can recover specific target cells with high efficiency and high purity.

[0014] As a result of diligent research by the inventors, we have found that the aforementioned problems can be solved by using a specific copolymer described later. Based on this finding, the present invention is as follows.

[0015] [1] A copolymer having repeating units A represented by the following formula (1) and repeating units B represented by the following formula (2), wherein the amount of repeating unit A relative to the total of repeating units A and B is 50 mol% or more, and the lower critical solution temperature (LCST) is 3°C to 40°C:

[0016]

[0017] (In the formula, R １ and R ２ Each of the following independently represents a hydrogen atom or a methyl group, n is a number from 0 to 12, when n is 0, X represents *-OH, and when n is a number greater than or equal to 1, X represents *-NH ２ , *-N(CH ３), H, *-N(CH ３ ), ２ , *-N ＋ (CH ３ ), ３ , *-S ＋ (CH ３ ), ２ , *-COOH, *-SO ３ , *-C ６ H ４ -SO ３ H, or *-PO ３ H ２ represents, and *-C ６ H ４ -* represents a phenylene group, and * represents the bonding position.).

[0018] [2] The copolymer according to [1], having a number average molecular weight of 1,000 to 5,000,000. [3] The copolymer according to [1] or [2], wherein both R １ and R ２ are methyl groups. [4] The copolymer according to any one of [1] to [3], wherein n is 0 and X is *-OH (where * represents the bonding position in the formula), or n is a number of 1 or more and X is *-N(CH ３ ), ２ or *-COOH (where * represents the bonding position in the formula).

[0019] [5] The copolymer according to any one of [1] to [4], further having a repeating unit C represented by the following formula (3):

[0020]

[0021] (In the formula, R ３ represents a hydrogen atom or a methyl group, Y represents a group represented by any one of the following formulas (4) to (8):

[0022]

[0023] , m represents an integer of 3 to 12, R ４ represents a hydrogen atom or a methyl group, R ５ represents a methyl group, a methoxy group, or an ethoxy group, and R ６ and R ７Each of these independently represents either a methoxy group or an ethoxy group, and * indicates the bond position.

[0024] [6] The copolymer according to [5], having a repeating unit A represented by the following formula (1a), a repeating unit B represented by the following formula (2a), and a repeating unit C represented by the following formula (3a):

[0025]

[0026] (In the formula, n-Bu represents a butyl group, and * represents a bond position.)

[0027] [7] The copolymer according to [5], having a repeating unit A represented by the following formula (1a), a repeating unit B represented by the following formula (2b), and a repeating unit C represented by the following formula (3a):

[0028]

[0029] (In the formula, n-Bu represents a butyl group, and * represents a bond position.)

[0030] [8] A cell separation substrate having a base material and a layer formed on the base material from a copolymer according to any one of [1] to [7]. [9] The cell separation substrate according to [8], wherein the material of the base material is glass, silicon, synthetic resin, or synthetic rubber.

[10] The cell separation substrate according to [8], wherein the thickness of the layer formed from the copolymer is 1 nm to 1000 nm.

[0031]

[11] A method for separating target cells from a group of cells containing target cells, comprising: step A, bringing a group of cells containing target cells into contact with a layer formed from a copolymer of any one of [8] to

[10] at a temperature higher than the lower critical solution temperature (LCST) of the copolymer to adhere the target cells to the layer; step B, washing the layer to which the target cells have adhered at a temperature higher than the lower critical solution temperature (LCST) of the copolymer; and step C, cooling the cell separation substrate having the washed layer to a temperature lower than the lower critical solution temperature (LCST) of the copolymer to detach and recover the target cells from the layer.

[0032] By using the copolymer of the present invention, it is possible to manufacture a cell separation substrate that enables the separation of target cells under low-irritation conditions with reduced damage to the target cells, and allows for the highly efficient and high-purity recovery of specific target cells.

[0033] In this specification, numerical ranges defined using "~" include the numbers at both ends (upper and lower limits) of "~". For example, "2~5" means 2 or more and 5 or less. Furthermore, each description in this specification can be combined with others unless it is clearly stated that they cannot be combined.

[0034] <Copolymer> The present invention provides a copolymer having repeating units A represented by the following formula (1) and repeating units B represented by the following formula (2), wherein the amount of repeating units A relative to the total of repeating units A and B is 50 mol% or more, and the lower critical solution temperature (LCST) is 3°C to 40°C.

[0035]

[0036] The symbols in equations (1) and (2) will be explained below in order. １ and R ２ Each is independently a hydrogen atom or a methyl group. From the viewpoint of copolymerization, R １ Preferably, it is a methyl group. From the viewpoint of copolymerization, R ２ The group is preferably a methyl group. １ and R ２ Preferably, both are methyl groups.

[0037] In formula (2), n represents a number from 0 to 12. This n is the oxyethylene group (-O-CH) in formula (2). ２ CH ２ n is a number (minus), and may be a decimal. In this specification, "n is 0" means that there is no oxyethylene group in formula (2). n is preferably a number from 1 to 10, more preferably a number from 4 to 10.

[0038] In equation (2), if n is 0, X represents *-OH, and if n is a number greater than or equal to 1, X represents *-NH.２ , *-N(CH ３ ) H, *-N (CH ３ ) ２ , *-N ＋ (CH ３ ) ３ , *-S ＋ (CH ３ ) ２ , *-COOH, *-SO ３ H, *-C ６ H ４ -SO ３ H, or *-PO ３ H ２ This indicates *-C ６ H ４ -* indicates a phenylene group, and * indicates the bond position.

[0039] In this specification, "*" represents a bond position, not a carbon atom, as described above. Therefore, "*-" in this specification represents a single bond. The phenylene group may be any of 1,2-phenylene, 1,3-phenylene, or 1,4-phenylene.

[0040] In formula (2), when n is 0, X is *-OH. In formula (2), when n is a number greater than or equal to 1, X is preferably *-N(CH ３ ) ２ or *-COOH. In formula (2), preferably n is 0 and X is *-OH, or n is a number of 1 or more and X is *-N(CH ３ ) ２ Or *-COOH, more preferably n is a number of 1 or more, and X is *-N(CH ３ ) ２ Alternatively, it is *-COOH.

[0041] When producing the copolymer of the present invention, only one monomer may be used to form the repeating unit A, or two or more monomers may be used in combination. Therefore, the multiple repeating units A may be the same or different. From the viewpoint of ease of copolymer production, the multiple repeating units A are preferably the same.

[0042] When producing the copolymer of the present invention, only one monomer may be used to form the repeating unit B, or two or more monomers may be used in combination. Therefore, the multiple repeating units B may be the same or different. From the viewpoint of ease of copolymer production, the multiple repeating units B are preferably the same.

[0043] Examples of repeating units B having an amino group or an ammonia group include the following: R ２ A is a hydrogen atom, n is 1, and X is *-NH ２ The repeating units B and R are ２ is a methyl group, n is 1, and X is *-NH ２ The repeating units B and R are ２ is a methyl group, n is 5, and X is *-NH ２ The repeating units B and R are ２ is a methyl group, n is 9, and X is *-NH ２ The repeating units B and R are ２ A is a hydrogen atom, n is 1, and X is *-N(CH ３ ) Repeating units B and R are H ２ is a methyl group, n is 1, and X is *-N(CH ３ ) Repeating units B and R are H ２ is a methyl group, n is 5, and X is *-N(CH ３ ) Repeating units B and R are H ２ The group is a methyl group, n is 9, and X is *-N(CH ３ ) Repeating units B and R are H ２ A is a hydrogen atom, n is 1, and X is *-N(CH ３ ) ２ The repeating units B and R are ２ is a methyl group, n is 1, and X is *-N(CH ３ ) ２ The repeating units B and R are ２ is a methyl group, n is 5, and X is *-N(CH ３ ) ２ The repeating units B and R are２ is a methyl group, n is 9, and X is *-N(CH ３ ). ２ The repeating unit B, where R ２ is a hydrogen atom, n is 1, and X is *-N ＋ (CH ３ ). ３ The repeating unit B, where R ２ is a methyl group, n is 1, and X is *-N ＋ (CH ３ ). ３ The repeating unit B, where R ２ is a methyl group, n is 5, and X is *-N ＋ (CH ３ ). ３ The repeating unit B, and R ２ is a methyl group, n is 9, and X is *-N ＋ (CH ３ ). ３ The repeating unit B.

[0044] Preferred repeating units B having an amino group or an ammonium group include the following: R ２ is a methyl group, n is 1, and X is *-NH ２ . ２ The repeating unit B, where R ２ is a methyl group, n is 5, and X is *-NH ２ . ２ The repeating unit B, where R ２ is a methyl group, n is 9, and X is *-NH ３ . ２ The repeating unit B, where R ３ is a methyl group, n is 1, and X is *-N(CH ３ ). ３ H ２ The repeating unit B, where R ３ is a methyl group, n is 5, and X is *-N(CH ２The repeating units B and R are ２ is a methyl group, n is 5, and X is *-N(CH ３ ) ２ The repeating units B and R are ２ The group is a methyl group, n is 9, and X is *-N(CH ３ ) ２ The repeating units B and R are ２ is a methyl group, n is 1, and X is *-N ＋ (CH ３ ) ３ The repeating units B and R are ２ The group is a methyl group, n is 5, and X is *-N ＋ (CH ３ ) ３ The repeating units B and R ２ The group is a methyl group, n is 9, and X is *-N ＋ (CH ３ ) ３ The repeating unit B is...

[0045] The following are more preferred as repeating units B having an amino group or an ammonia group: R ２ is a methyl group, n is 1, and X is *-NH ２ The repeating units B and R are ２ is a methyl group, n is 5, and X is *-NH ２ The repeating units B and R are ２ is a methyl group, n is 9, and X is *-NH ２ The repeating units B and R are ２ is a methyl group, n is 1, and X is *-N(CH ３ ) ２ The repeating units B and R are ２ is a methyl group, n is 5, and X is *-N(CH ３ ) ２ The repeating units B and R are ２ The group is a methyl group, n is 9, and X is *-N(CH ３ ) ２ The repeating units B and R are ２is a methyl group, n is 1, and X is *-N ＋ (CH ３ ) ３ The repeating units B and R are ２ The group is a methyl group, n is 5, and X is *-N ＋ (CH ３ ) ３ The repeating units B and R ２ The group is a methyl group, n is 9, and X is *-N ＋ (CH ３ ) ３ The repeating unit B is...

[0046] Examples of repeating units B having an acidic functional group include the following: R ２ Repeating units B and R where n is a hydrogen atom, n is 0, and X is *-OH ２ Repeating units B and R where n is a hydrogen atom, n is 5, and X is *-COOH. ２ Repeating units B and R where n is a hydrogen atom, n is 9, and X is *-COOH. ２ Repeating units B and R where n is a methyl group, n is 0, and X is *-OH ２ Repeating units B and R where n is a methyl group, n is 5, and X is *-COOH ２ Repeating units B and R where n is a methyl group, n is 9, and X is *-COOH ２ The group is a methyl group, n is 1, and X is *-SO ３ H is the repeating unit B, R ２ The group is a methyl group, n is 5, and X is *-SO ３ H is the repeating unit B, R ２ The group is a methyl group, n is 9, and X is *-SO ３ H is the repeating unit B, R ２ is a methyl group, n is 1, and X is *-C ６ H ４ -SO ３ H is the repeating unit B, R ２ is a methyl group, n is 5, and X is *-C ６ H４ -SO ３ H is the repeating unit B, R ２ is a methyl group, n is 9, and X is *-C ６ H ４ -SO ３ H is the repeating unit B, R ２ is a methyl group, n is 1, and X is *-PO ３ H ２ The repeating units B and R are ２ is a methyl group, n is 5, and X is *-PO ３ H ２ The repeating units B and R ２ is a methyl group, n is 9, and X is *-PO ３ H ２ The repeating unit B is...

[0047] The following are preferred as repeating units B having an acidic functional group: R ２ Repeating units B and R where n is a hydrogen atom, n is 0, and X is *-OH ２ Repeating units B and R where n is a hydrogen atom, n is 5, and X is *-COOH. ２ Repeating units B and R where n is a hydrogen atom, n is 9, and X is *-COOH. ２ Repeating units B and R where n is a methyl group, n is 0, and X is *-OH ２ Repeating units B and R where n is a methyl group, n is 5, and X is *-COOH ２ Repeating units B and R where n is a methyl group, n is 9, and X is *-COOH ２ The group is a methyl group, n is 1, and X is *-SO ３ H is the repeating unit B, R ２ The group is a methyl group, n is 5, and X is *-SO ３ H is the repeating unit B, R ２ The group is a methyl group, n is 9, and X is *-SO ３ H is the repeating unit B, R ２ is a methyl group, n is 1, and X is *-C６ H ４ -SO ３ H is the repeating unit B, R ２ is a methyl group, n is 5, and X is *-C ６ H ４ -SO ３ H is a repeating unit B, and R ２ is a methyl group, n is 9, and X is *-C ６ H ４ -SO ３ H is the repeating unit B.

[0048] The following are more preferred as the repeating unit B having an acidic functional group: R ２ Repeating units B and R where n is a hydrogen atom, n is 0, and X is *-OH ２ Repeating units B and R where n is a hydrogen atom, n is 5, and X is *-COOH. ２ Repeating units B and R where n is a hydrogen atom, n is 9, and X is *-COOH. ２ Repeating units B and R where n is a methyl group, n is 0, and X is *-OH ２ Repeating units B and R where n is a methyl group, n is 5, and X is *-COOH ２ Repeating units B and R where n is a methyl group, n is 9, and X is *-COOH ２ The group is a methyl group, n is 1, and X is *-SO ３ H is the repeating unit B, R ２ The group is a methyl group, n is 5, and X is *-SO ３ H is a repeating unit B, and R ２ The group is a methyl group, n is 9, and X is *-SO ３ H is the repeating unit B.

[0049] For cell adhesion and detachment, the lower critical solution temperature (LCST) of the copolymer of the present invention is 3°C to 40°C. In this specification, “lower critical solution temperature (LCST)” means the boundary temperature between the temperature at which the polymer dissolves in water or culture medium and the temperature at which the copolymer does not dissolve in water or culture medium. The value of the “lower critical solution temperature (LCST)” of the copolymer of the present invention is measured as follows: First, the copolymer is dissolved in serum-free RPMI 1640 medium at a concentration of 2.5 mg / mL, and the transmittance of visible light at 500 nm is measured in a quartz cell while increasing the temperature. When the transmittance of the visible light of the solution when the copolymer is completely dissolved is taken as 100%, the temperature at which the transmittance becomes 50% when the solution is heated is defined as the “lower critical solution temperature (LCST)”. For cell adhesion and detachment, the lower critical solution temperature (LCST) of the copolymer of the present invention is preferably 3°C to 39°C, more preferably 6°C to 35°C, and even more preferably 10°C to 35°C.

[0050] To achieve a lower critical solution temperature (LCST) of the copolymer of the present invention of 3°C to 40°C, the amount of repeating unit A relative to the total of repeating unit A and repeating unit B is 50 mol% or more. To achieve a preferred lower critical solution temperature (LCST), the amount of repeating unit A is preferably 50 mol% to 98 mol%, and more preferably 55 mol% to 95 mol%.

[0051] The number-average molecular weight of the copolymer of the present invention can be appropriately adjusted by adjusting polymerization conditions, etc., to achieve the required performance. The number-average molecular weight is preferably 1,000 to 5,000,000, more preferably 2,000 to 2,000,000, even more preferably 2,000 to 1,000,000, and particularly preferably 10,000 to 600,000 for cell adhesion and detachment. When the number-average molecular weight is 1,000 or more, cells are more easily detached in response to temperature, and when the number-average molecular weight is 5,000,000 or less, the solubility of the copolymer increases, making it easier to use and facilitating better adhesion of target cells to a cell separation substrate.

[0052] The copolymer of the present invention may further have repeating units C represented by the following formula (3).

[0053]

[0054] For example, the copolymer of the present invention having repeating units C in which Y is a group represented by the following formula (4) has improved affinity to organic substrates (e.g., polystyrene substrates), and peeling of the layer formed from the copolymer from the organic substrate is suppressed.

[0055] The following explains the symbols in equation (3) in order. In equation (3), R ３ R represents a hydrogen atom or a methyl group. From the viewpoint of ease of copolymer synthesis, ３ The group is preferably a methyl group.

[0056] In formula (3), Y represents a group represented by any of the following formulas (4) to (8). In this specification, the "group represented by formula (4)" may be abbreviated as "group (4)". Groups, compounds, monomers, and copolymers represented by other formulas may also be abbreviated in the same way.

[0057]

[0058] In formula (4), m represents an integer from 3 to 12. That is, group (4) is an alkyl group having 3 to 12 carbon atoms. The alkyl group may be linear or branched. From the viewpoint of ease of obtaining monomers, m is preferably an integer from 3 to 8, and more preferably an integer from 4 to 6.

[0059] In formula (5), R ４ R represents a hydrogen atom or a methyl group. From the viewpoint of ease of copolymer synthesis, ４ Preferably, it is a hydrogen atom.

[0060] In formula (8), R ５ R represents a methyl group, a methoxy group, or an ethoxy group, and R ６ and R ７ Each of these independently represents either a methoxy group or an ethoxy group. In formulas (3) to (8), * indicates the bond position.

[0061] Y is preferably group (4) or group (8), more preferably group (4). Explanation of m in group (4), R in group (8) ４ The explanation is as stated above.

[0062] When producing the copolymer of the present invention, only one monomer may be used to form the repeating unit C, or two or more monomers may be used in combination. Therefore, the multiple repeating units C may be the same or different. From the viewpoint of ease of copolymer production, the multiple repeating units C are preferably the same.

[0063] Examples of repeating units C include: R ３ The repeating unit C, R is a methyl group, Y is a group (4), and m is 4. ３ is a methyl group, Y is a group (5), and R ４ The repeating units C and R are hydrogen atoms. ３ is a methyl group, Y is a group (5), and R ４ The repeating units C and R are methyl groups. ３ The repeating unit C, R is a methyl group and Y is a group (6). ３ The repeating unit C, R is a methyl group and Y is a group (7). ３ is a methyl group, Y is a group (8), and R ５ is a methyl group, and R ６ and R ７ Repeating units C and R, both of which are methoxy groups ３ is a methyl group, Y is a group (8), and R ５ ~R ７ Repeating units C and R, both of which are methoxy groups ３ is a methyl group, Y is a group (8), and R ５ is a methyl group, and R ６ and R ７ The repeating unit C and R are both ethoxy groups. ３ is a methyl group, Y is a group (8), and R ５ ~R ７ A repeating unit C in which both are ethoxy groups.

[0064] When the copolymer of the present invention has repeating units C, the amount of repeating units C is preferably 0.1 mol% to 20 mol%, more preferably 0.1 mol% to 15 mol%, and even more preferably 1 mol% to 10 mol%, relative to the total of repeating units A, B, and C, from the viewpoint of improving the adhesion of the copolymer to the substrate and maintaining the temperature responsiveness of the copolymer.

[0065] The copolymer of the present invention may have other repeating units different from repeating unit A, repeating unit B, or repeating unit C. The monomers that form the other repeating units (hereinafter referred to as "other monomers") will be described in order below.

[0066] To raise the lower critical solution temperature (LCST) of the copolymer of the present invention, hydrophilic monomers can be used as other monomers. Examples of hydrophilic monomers that are other monomers include 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 (excluding diethylene glycol monomethyl ether (meth)acrylate), 2-methoxyethyl (meth)acrylate, allyl alcohol acrylonitrile, acrolein, sodium vinylsulfonate, N-vinylpyrrolidone, itaconic acid, maleic acid, and the like. Among these, N-methylcarboxybetaine (meth)acrylate, N-methylsulfobetaine (meth)acrylate, aminoethyl (meth)acrylate, and S-methylsulfonium carboxylic acid (meth)acrylate, polyethylene glycol (meth)acrylate, or polyethylene glycol monomethyl ether (meth)acrylate (excluding diethylene 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 (excluding diethylene glycol monomethyl ether (meth)acrylate) are more preferred.

[0067] To lower the lower critical solution temperature (LCST) of the copolymer of the present invention, hydrophobic monomers can be used as other monomers. Examples of hydrophobic monomers include N-isopropyl(meth)acrylamide, vinyl acetate, styrene, chlorostyrene, vinylphenol, vinyl cinnamate, vinyl chloride, vinyl bromide, butadiene, vinylene carbonate, itaconic acid ester, fumarate ester, maleate ester, and the like. Among these, N-isopropyl(meth)acrylamide is preferred.

[0068] Other monomers can be used to bind ligands that recognize specific cells. Examples of such monomers include propargyl (meth)acrylate, azidopropyl (meth)acrylate, succinimidyl (meth)acrylate, (meth)acrylic acid, aminoethyl (meth)acrylate, 2-isocyanatoethyl (meth)acrylate, and hydroxyethyl (meth)acrylate. Among these, propargyl (meth)acrylate, azidopropyl (meth)acrylate, succinimidyl (meth)acrylate, and 2-isocyanatoethyl (meth)acrylate are preferred from the viewpoint of excellent ligand binding, and propargyl (meth)acrylate, azidopropyl (meth)acrylate, and succinimidyl (meth)acrylate are more preferred.

[0069] When producing the copolymer of the present invention, only one other monomer may be used to form the other repeating unit C, or two or more monomers may be used in combination. Therefore, the multiple other repeating units may be the same or different from one another.

[0070] The amount of other repeating units in the copolymer of the present invention is preferably 20 mol% or less, more preferably 15 mol% or less, and even more preferably 10 mol% or less, relative to the total number of repeating units (for example, if repeating unit C and other repeating units are present, the sum of repeating unit A, repeating unit B, repeating unit C, and other repeating units). It is particularly preferable that the copolymer of the present invention does not have other repeating units. That is, the total number of repeating units in the copolymer of the present invention is particularly preferably composed of repeating unit A and repeating unit B, or repeating unit A, repeating unit B, and repeating unit C, and most preferably composed of repeating unit A, repeating unit B, and repeating unit C.

[0071] The copolymer of the present invention may be a random copolymer, a block copolymer, or a copolymer comprising a random copolymer portion and a block copolymer portion. The copolymer of the present invention is preferably a random copolymer.

[0072] A preferred copolymer of the present invention is a copolymer having a repeating unit A represented by the following formula (1a) as a skeletal structure, a repeating unit B represented by any of the following formulas (2a) to (2f) as a skeletal structure, and a repeating unit C represented by the following formula (3a) or (3b) as a skeletal structure. In this specification, "skeletal structure" means a structural formula in which carbon atoms other than carbon atoms in the functional group are not indicated by the atomic symbol C, and the carbon atoms not indicated by the atomic symbol C are located at the vertices (angles) and ends of line segments, and the carbon atoms are bonded to enough hydrogen atoms to give them four bonds.

[0073]

[0074]

[0075]

[0076] (In the formulas, n-Bu represents a butyl group, and * indicates a bond position.) The entire repeating unit of the copolymer more preferably consists of repeating unit A represented by formula (1a), repeating unit B represented by any of formulas (2a) to (2f), and repeating unit C represented by formula (3a) or (3b). The amounts of repeating unit A, repeating unit C, lower critical solution temperature (LCST), and number-average molecular weight of the copolymer are described above.

[0077] Note that in the above formula (1a), R １ R is a methyl group. In the above formula (2a), R ２ is a methyl group, n is 9, and X is *-N(CH ３ ) ２ Therefore, in the above equation (2b), R ２ R is a methyl group, n is 9, and X is *-COOH, and in the above formula (2c), R ２ R is a methyl group, n is 0, and X is *-OH, and in formula (2d) above, ２ R is a methyl group, n is 5, and X is *-COOH, and in the above formula (2e), R ２ is a methyl group, n is 1, and X is *-N(CH ３ ) ２ And in the above equation (2f), R ２ is a methyl group, n is 5, and X is *-N(CH ３ ) ２ Therefore, in the above equation (3a), R ３ is a methyl group, and Y is a butyl group, and in formula (3b) above, R ２ is a methyl group, Y is a group represented by the above formula (5), and R ４ It is a hydrogen atom.

[0078] Specific examples of the preferred copolymers described above include: (i) a copolymer having a repeating unit A represented by formula (1a), a repeating unit B represented by formula (2c), and a repeating unit C represented by formula (3a); (ii) a copolymer having a repeating unit A represented by formula (1a), a repeating unit B represented by formula (2d), and a repeating unit C represented by formula (3a); (iii) a copolymer having a repeating unit A represented by formula (1a), a repeating unit B represented by formula (2b), and a repeating unit C represented by formula (3a); (iv) a copolymer having a repeating unit A represented by formula (1a), a repeating unit B represented by formula (2e), and a repeating unit C represented by formula (3a); (v) a copolymer having a repeating unit A represented by formula (1a), a repeating unit B represented by formula (2f), and a repeating unit C represented by formula (3a). (vi) A copolymer having repeating unit A represented by formula (1a), repeating unit B represented by formula (2a), and repeating unit C represented by formula (3a); (vii) A copolymer having repeating unit A represented by formula (1a), repeating unit B represented by formula (2b), and repeating unit C represented by formula (3b); and (viiii) A copolymer having repeating unit A represented by formula (1a), repeating unit B represented by formula (2a), and repeating unit C represented by formula (3b). The total repeating units of the copolymer are more preferably made up of repeating unit A, repeating unit B, and repeating unit C. The amounts of repeating unit A, repeating unit C, lower critical solution temperature (LCST), and number-average molecular weight of the copolymer are described above.

[0079] A more preferred copolymer of the present invention is a copolymer having a repeating unit A represented by the following formula (1a), a repeating unit B represented by the following formula (2a), and a repeating unit C represented by the following formula (3a):

[0080]

[0081] (In the formulas, n-Bu represents a butyl group (i.e., a normal butyl group), and * indicates the bond position.) The entire repeating unit of the copolymer more preferably consists of repeating unit A represented by formula (1a), repeating unit B represented by formula (2a), and repeating unit C represented by formula (3a). The amounts of repeating unit A, repeating unit C, lower critical solution temperature (LCST), and number-average molecular weight of the copolymer are described above.

[0082] Another more preferred copolymer of the present invention is a copolymer having a repeating unit A represented by the following formula (1a), a repeating unit B represented by the following formula (2b), and a repeating unit C represented by the following formula (3a):

[0083]

[0084] (In the formulas, n-Bu represents a butyl group (i.e., a normal butyl group), and * indicates the bond position.) The entire repeating unit of the copolymer more preferably consists of repeating unit A represented by formula (1a), repeating unit B represented by formula (2b), and repeating unit C represented by formula (3a). The amounts of repeating unit A, repeating unit C, lower critical solution temperature (LCST), and number-average molecular weight of the copolymer are described above.

[0085] <Method for Producing Copolymers> The copolymer of the present invention can be produced, for example, by radical polymerization of monomers that form repeating unit A and monomers that form repeating unit B, and, if necessary, monomers that form repeating unit C and / or other monomers. Commercial monomers may be used as the monomers, or monomers produced by known methods may be used.

[0086] As for the radical polymerization described above, bulk polymerization without a solvent is possible, but radical polymerization may also be carried out using a solvent. The solvent is not particularly limited as long as it dissolves the monomer, but examples include aprotic polar solvents such as acetone, dioxane, N,N-dimethylformamide (abbreviated as "DMF"), dimethyl sulfoxide (abbreviated as "DMSO"), tetrahydrofuran (abbreviated as "THF"), anisole, toluene, acetonitrile, and dimethylacetamide, and protic polar solvents such as methanol, ethanol, 2-propanol (abbreviated as "IPA"), and water. Only one solvent may be used, or two or more solvents may be used in combination.

[0087] When a solvent is used for radical polymerization, the total monomer concentration is not particularly limited, but is preferably 1% to 80% by weight, more preferably 2% to 50% by weight, and even more preferably 5% to 40% by weight.

[0088] The radical polymerization may be either thermal polymerization or photopolymerization. Examples of thermal polymerization initiators include peroxide-based radical initiators (e.g., benzoyl peroxide, ammonium persulfate, etc.), azo-based radical initiators (e.g., azobisisobutyronitrile (abbreviated as "AIBN"), 2,2'-azobis-dimethylvaleronitrile (abbreviated as "ADVN"), etc.), 2,2'-azobiscyanovaleric acid (abbreviated as "ACVA"), azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (abbreviated as "VA-044")), and water-soluble or oil-soluble redox-based radical initiators (e.g., initiators consisting of dimethylaniline and benzoyl peroxide). The amount of thermal polymerization initiator used is preferably 0.01 to 10% by weight of the total amount of monomers. The temperature and time of thermal polymerization can be appropriately selected depending on the type of thermal polymerization initiator, the type of monomer, etc. For example, when using AIBN, the thermal polymerization temperature is 40 to 90°C, and the time is approximately 2 to 48 hours.

[0089] The photopolymerization can be carried out, for example, by irradiation with ultraviolet light (UV) with a wavelength of 254 nm or electron beam (EB) with an accelerating voltage of 150 to 300 kV. In this case, a photopolymerization initiator may be used. Examples of photopolymerization initiators include 2-hydroxy-2-methyl-1-phenyl-1-propanone and 1-hydroxycyclohexylphenyl ketone. Among these, 2-hydroxy-2-methyl-1-phenyl-1-propanone is preferred from the viewpoint of solubility and other factors.

[0090] In the radical polymerization described above, a chain transfer agent may also be used. Examples of the chain transfer agent include 2-mercaptoethanol, 1-mercapto-2-propanol, 3-mercapto-1-propanol, p-mercaptophenol, mercaptoacetic acid, 2-mercaptopropionic acid, 3-mercaptopropionic acid, and 2-mercaptonicotinic acid.

[0091] The radical polymerization may also be living radical polymerization. Examples of living radical polymerization methods include reversible addition-cleavage chain transfer polymerization (hereinafter sometimes referred to as "RAFT polymerization"), atom transfer radical polymerization (hereinafter sometimes referred to as "ATRP"), and polymerization via nitroxide. Among these, RAFT polymerization and ATRP are preferred from the viewpoint of ease of polymerization.

[0092] Known methods can be used for RAFT polymerization, including, for example, the methods described in WO99 / 31144, WO98 / 01478, and U.S. Patent No. 6,153,705. RAFT polymerization can be carried out by using a RAFT agent in addition to a conventional radical polymerization initiator (thermal polymerization initiator).

[0093] Examples of RAFT agents include 4-cyanopentanoate dithiobenzoate, 2-cyano-2-propylbenzodithioate, benzylbenzodithioate, 2-phenyl-2-propylbenzodithioate, methyl 2-phenyl-2-(phenyl-carbonothiothio)acetate, 4-cyano-4-(phenylcarbonothiothio)pentanoate N-succinimidyl ester, 4-cyano-4-(dodecylsulfanyl-thiocarbonyl)sulfanylpentanoic acid, 4-cyano-4-(dodecylsulfanyl-thiocarbonyl)sulfanylpentanol, and 2-cyano-2 Examples include propyl dodecyl trithiocarbonate, 2-(dodecylthiocarbonylthio)-2-methylpropionic acid, 4-cyano-4-(dodecylsulfanylthiocarbonyl)sulfanypentanoate polyethylene glycol methyl ether ester, 2-(dodecylthiocarbonothio)-2-methylpropionic acid-3-azido-1-propanol ester, benzyl 1H-pyrrole-1-carbodhithioate, 2-cyanopropan-2-yl-N-methyl-N-pyridine-4-ylcarbodhithioate, and ethyl-2-ethylsanthate propionate. Among these, from the viewpoint of polymerization control, 4-cyanopentanoate dithiobenzoate, 4-cyano-4-(phenylcarbonothioylthio)pentanoate N-succinimidyl ester, 4-cyano-4-(dodecylsulfanyl-thiocarbonyl)sulfanylpentanoic acid, 4-cyano-4-(dodecylsulfanyl-thiocarbonyl)sulfanylpentanol, and 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid-3-azido-1-propanol ester are preferred.

[0094] The ATRP initiator is not particularly limited, but examples include 1-trimethoxysilyl-2-(p-chloromethylphenyl)ethane, 4-(chloromethyl)phenyltrimethoxysilane, (3-chloropropyl)trimethoxysilane, 1-trichlorosilyl-2-(m-chloromethylphenyl)ethane, 1-trichlorosilyl-2-(p-chloromethylphenyl)ethane, 2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane, (3-(2-bromoisobutyryl)propyl)trimethoxysilane, 2-bromo-2-methylpropanoate 3-(trichlorosilyl)propyl, 2-bromo-2-methylpropanoate 3-(trimethoxysilyl)propyl, 2-bromo-2-methyl-N-[ Examples include 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, and diethylmeso-2,5-dibromoadipate. Among these, from the viewpoint of polymerization control, 1-trimethoxysilyl-2-(p-chloromethylphenyl)ethane, 4-(chloromethyl)phenyltrimethoxysilane, (3-chloropropyl)trimethoxysilane, 1-trichlorosilyl-2-(p-chloromethylphenyl)ethane, 3-(trimethoxysilyl)propyl 2-bromo-2-methylpropanoate, 2-bromo-2-methyl-N-[3-(trimethoxysilyl)propyl]propanamide, chloromethylxylene, and ethyl 2-bromoisobutyrate are preferred.

[0095] Furthermore, by using 2-bromopropionyl bromide and dopamine, 3-aminopropyltrimethoxysilane, or 3-aminopropyltriethoxysilane to bond a group formed from 2-bromopropionyl bromide to the surface of a substrate, this group can also be used as an ATRP initiator.

[0096] The catalyst used in the ATRP method is not particularly limited. For example, a transition metal complex can be used as the catalyst. The transition metal complex is not particularly limited, and for example, a transition metal complex formed from a transition metal salt and a ligand, as described later, can be used.

[0097] The transition metal salt is not particularly limited, but for example, CuCl, CuCl ２ , CuBr, CuBr ２ TiCl ２ TiCl ３ TiCl ４ TiBr ４ FeCl ２ FeCl ３ , FeBr ２ , FeBr ３ CoCl ２ CoBr ２ NiCl ２ NiBr ２ MoCl ３ MoCl ５ RuCl ３ These are some examples.

[0098] The ligand is not particularly limited, but for example, tris(2-(dimethylamino)ethyl)amine (abbreviated as "Me ６ TREN), N,N,N,N-pentamethyldiethylenetriamine (abbreviated as PMDETA), 1,1,4,7,10,10-hexamethyltriethylenetetraamine (abbreviated as HMTETA), 1,4,8,11-tetramethyl-1,4,8,11-azacyclotetradecane (abbreviated as Me ４Examples of transition metal salts include 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-pyridylmethanymine, N-octyl-2-pyridylmethanymine, N-dodecyl-N-(2-pyridylmethylene)amine, N-octadecyl-N-(2-pyridylmethylene)amine, tris(2-pyridylmethyl)amine, N,N,N,N-tetrakis(2-pyridylmethyl)-ethylenediamine, etc. Examples of combinations of the transition metal salt and ligand include CuBr / 2,2-bipyridine, CuBr ２ / 2,2-bipyridine, CuCl / Me ６ TREN, CuCl ２ / Me ６ TREN is one example.

[0099] In the ATRP method, a reducing agent may be used as needed. Examples of reducing agents include ascorbic acid, sodium ascorbate, tin(II) 2-ethylhexanoate, and monovalent copper salts.

[0100] Although bulk polymerization without solvents is possible using the ATRP method, polymerization may also be carried out using a solvent. The solvent is not particularly limited as long as it dissolves the monomer, but examples include aprotic polar solvents such as acetone, dioxane, N,N-dimethylformamide (abbreviated as "DMF"), dimethyl sulfoxide (abbreviated as "DMSO"), tetrahydrofuran (abbreviated as "THF"), anisole, toluene, acetonitrile, and dimethylacetamide, and protic polar solvents such as methanol, ethanol, 2-propanol (abbreviated as "IPA"), and water. Only one solvent may be used, or two or more solvents may be used in combination.

[0101] The polymerization temperature of the ATRP method can be appropriately selected depending on the type of ATRP initiator, the type of monomer, etc. The temperature is preferably 25°C to 120°C, more preferably 25°C to 70°C. The polymerization time of the ATRP method is not particularly limited, but is preferably 1 hour to 96 hours, more preferably 1 hour to 48 hours.

[0102] <Substrate for Cell Separation> The present invention provides a substrate for cell separation having a substrate and a layer formed from the copolymer of the present invention on the substrate. In this specification, the "layer formed from the copolymer" may be abbreviated as the "polymer layer".

[0103] There are no particular limitations on the material of the substrate. From the viewpoint of suitability for cell culture, the material of the substrate is preferably glass, silicon, synthetic resin, or synthetic rubber. Examples of synthetic resins or synthetic rubbers include acrylic polymers such as polymethyl methacrylate, silicone resins or silicone rubbers such as polydimethylsiloxane, polystyrene, polyethylene terephthalate, and polycarbonate.

[0104] There are no particular limitations on the shape of the substrate, but examples include container-shaped, plate-shaped, granular, and fibrous forms. Substrates with holes or grooves can be used. In particular, due to the ease of handling during cell separation, the substrate is preferably a container-shaped substrate (e.g., a petri dish, a flask) or a plate-shaped substrate (e.g., a silicon wafer, a glass plate).

[0105] The substrate may have functional groups on its surface. Examples of such functional groups include hydroxyl groups, amino groups, carboxyl groups, tosyl groups, epoxy groups, succinimidyl groups, maleimidyl groups, sulfanyl groups, azide groups, azidophenyl groups, biotin residues, avidin residues, and the like.

[0106] The thickness of the copolymer layer is preferably 1 nm to 1000 nm, more preferably 1 nm to 500 nm, and even more preferably 1 nm to 300 nm, from the viewpoint of adhesion between the copolymer layer and the substrate and the efficiency of detachment of cells attached to the substrate from the substrate. The thickness can be measured by spectroscopic ellipsometry.

[0107] <Method for manufacturing a cell separation substrate 1> The cell separation substrate of the present invention can be manufactured, for example, by coating a substrate with a solution of the copolymer of the present invention and drying it.

[0108] The solvent for preparing a solution of the copolymer of the present invention is not particularly limited as long as the copolymer is soluble in it, but examples include aprotic polar solvents such as acetone, dioxane, N,N-dimethylformamide (abbreviated as "DMF"), dimethyl sulfoxide (abbreviated as "DMSO"), tetrahydrofuran (abbreviated as "THF"), anisole, toluene, acetonitrile, and dimethylacetamide, and protic polar solvents such as methanol, ethanol, 2-propanol (abbreviated as "IPA"), and water. One solvent may be used alone, or two or more solvents may be used in combination.

[0109] The concentration of the copolymer of the present invention in the solution is preferably 1 mg / mL to 100 mg / mL, more preferably 1 mg / mL to 10 mg / mL, in order to efficiently produce the copolymer layer.

[0110] There are no particular limitations on the method for coating a substrate with a solution of the copolymer of the present invention, but examples include spin coating, dip coating, bar coating, spray coating, roll coating, air knife coating, blade coating, and the like.

[0111] The drying temperature after coating a substrate with a solution of the copolymer of the present invention is not particularly limited and can be set appropriately depending on the solvent used in the solution. When using an organic substrate (e.g., a polystyrene substrate), the drying temperature is preferably 10°C to 70°C, more preferably 15°C to 50°C. When using an inorganic substrate (e.g., a glass substrate), the drying temperature is preferably 10°C to 150°C, more preferably 15°C to 50°C.

[0112] When using a copolymer having repeating unit C containing groups (5) to (7) and an organic substrate, it is preferable to perform UV treatment on the copolymer layer after drying. By performing UV treatment, the copolymer layer can be firmly adhered to the organic substrate.

[0113] The wavelength of the light irradiated in the UV treatment is preferably 200 nm to 400 nm, more preferably 254 nm to 365 nm.

[0114] When using a copolymer having repeating units C including group (8) and a glass substrate, it is preferable to apply a solution containing at least one selected from the group consisting of water, methanol, and ethanol, and the copolymer to the glass substrate and heat dry it in order to firmly bond the copolymer layer and the glass substrate by performing a silane coupling reaction. The heat drying temperature is preferably 10°C to 150°C, more preferably 15°C to 50°C, and the time is preferably 2 hours to 96 hours, more preferably 2 hours to 72 hours.

[0115] <Method for manufacturing a cell separation substrate 2> The cell separation substrate of the present invention can be manufactured, for example, by forming polymerization initiation points on the surface of the substrate and polymerizing monomers from those initiation points to form the copolymer of the present invention.

[0116] For example, when using a glass substrate, a glass substrate having hydroxyl groups on its surface can be obtained by treating the glass substrate with a piranha solution (a mixture of concentrated sulfuric acid and hydrogen peroxide). Next, a glass substrate having amino groups on its surface can be obtained by treating the glass substrate with a silane coupling agent having amino groups (for example, 3-aminopropyltrimethoxysilane). Next, a glass substrate having bromo groups (Br) on its surface can be obtained by treating the glass substrate with, for example, 2-bromopropionyl bromide. Then, a glass substrate having a copolymer layer in which the copolymer of the present invention is bonded to the glass surface can be obtained by performing polymerization in a monomer solution containing the glass substrate, for example, by the ATRP method, starting from the bromo groups on the surface of the glass substrate. The polymerization (especially the ATRP method) is described above.

[0117] <Method for Separating Target Cells> The present invention provides a method for separating target cells from a group of cells containing target cells. The method of the present invention comprises the following steps: Step A: Contacting a group of cells containing target cells with a layer formed from the copolymer of the present invention of the cell separation substrate of the present invention at a temperature higher than the lower critical solution temperature (LCST) of the copolymer to adhere the target cells to the layer; Step B: Washing the layer to which the target cells have adhered at a temperature higher than the lower critical solution temperature (LCST) of the copolymer; and Step C: Cooling the cell separation substrate having the washed layer to a temperature lower than the lower critical solution temperature (LCST) of the copolymer to detach and recover the target cells from the layer.

[0118] (Step A) In Step A, a group of cells including the target cells and a layer formed from the copolymer of the present invention of the cell separation substrate of the present invention are brought into contact at a temperature higher than the lower critical solution temperature (LCST) of the copolymer (hereinafter sometimes simply referred to as "LCST") (hereinafter sometimes referred to as "contact temperature").

[0119] The lower limit of the contact temperature is preferably "LCST + 1"°C, more preferably "LCST + 2"°C, in order to ensure sufficient adhesion of the target cells to the copolymer layer. In other words, the contact temperature is preferably "LCST + 1"°C or higher, more preferably "LCST + 2"°C or higher. On the other hand, the upper limit of the contact temperature is preferably 40°C, more preferably 37°C, in order to avoid damage to the target cells. In other words, the contact temperature is preferably 40°C or lower, more preferably 37°C or lower. Naturally, the lower limit of the contact temperature is less than or equal to the upper limit of the contact temperature.

[0120] Examples of target cells included in the cell population include established cells for culture, fertilized eggs, and egg cells. The target cells may be either animal cells or plant cells. Other examples of target cells include sperm cells, ES cells, iPS cells, mesenchymal stem cells, hematopoietic stem cells, neural stem cells, umbilical cord blood cells and other stem cells, hepatocytes, nerve cells, cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells, and hematopoietic cells.

[0121] There are no particular limitations on the state of the target cells; for example, they can be dispersed, in sheet form, as spheroids, or as organoids.

[0122] The method of the present invention may isolate one type of target cell from the cell population, or it may isolate two or more types of target cells.

[0123] The amount of target cells in the cell group is preferably 0.1% or more of the total number of cells in the cell group. There are no particular limitations on the type of cell group.

[0124] The target cells are preferably cells with a positive charge on their surface or cells with a negative charge on their surface. Here, "cells with a positive charge on their surface" means cells with a surface zeta potential of 3 mV or more, and "cells with a negative charge on their surface" means cells with a surface zeta potential of -3 mV or less. Examples of cells with a positive charge on their surface include HUVEC cells. Examples of cells with a negative charge on their surface include HL-60 cells.

[0125] If the target cell is a cell with a positive charge on its surface, it is preferable that n is 0 and X is *-OH, or that n is 1 or more and X is *-COOH, *-SO ３ H, *-C ６ H ４ -SO ３ H, or *-PO ３ H ２ The cell separation substrate of the present invention is used, which has a copolymer layer, more preferably n is 0 and X is *-OH, or n is a number of 1 or more and X is *-COOH, and even more preferably n is a number of 1 or more and X is *-COOH. By using the cell separation substrate of the present invention having the above copolymer layer, target cells (cells having a positive charge on their surface) can be efficiently separated.

[0126] In another embodiment of the present invention, when the target cells are cells having a positive charge on their surface, a cell separation substrate of the present invention is used to efficiently separate the target cells, preferably a layer formed from a copolymer having a repeating unit A represented by formula (1a), a repeating unit B represented by formula (2b), formula (2c), or formula (2d), and a repeating unit C represented by formula (3a) or formula (3b), more preferably a copolymer having a repeating unit A represented by formula (1a), a repeating unit B represented by formula (2c), and a repeating unit C represented by formula (3a), a copolymer having a repeating unit A represented by formula (1a), a repeating unit B represented by formula (2d), and a repeating unit C represented by formula (3a), or a layer formed from a copolymer having a repeating unit A represented by formula (1a), a repeating unit B represented by formula (2b), and a repeating unit C represented by formula (3b). The entire repeating unit of the copolymer preferably consists of repeating unit A, repeating unit B, and repeating unit C. The amounts of repeating unit A, repeating unit C, lower critical solution temperature (LCST), and number-average molecular weight of the copolymer are described above.

[0127] If the target cells are cells with a positive charge on their surface, it is preferable to separate the target cells (cells with a positive charge on their surface) from a group of cells that includes both target cells and cells without a positive charge on their surface.

[0128] If the target cells are cells having a negative charge on their surface, preferably n is a number of 1 or more, and X is *-NH ２ , *-N(CH ３ ) H, *-N (CH ３ ) ２ , *-N ＋ (CH ３ ) ３ , or *-S ＋ (CH ３ ) ２A copolymer layer wherein n is 1 or more, and X is *-N(CH ３ ) ２ The present invention uses a cell separation substrate having a copolymer layer. By using the cell separation substrate of the present invention having the copolymer layer, target cells (cells with a negative charge on their surface) can be efficiently separated.

[0129] In another embodiment of the present invention, when the target cells are cells having a negative charge on their surface, a cell separation substrate of the present invention is used to efficiently separate the target cells, preferably a layer formed from a copolymer having a repeating unit A represented by formula (1a), a repeating unit B represented by formula (2a), formula (2e), or formula (2f), and a repeating unit C represented by formula (3a) or formula (3b), more preferably a copolymer having a repeating unit A represented by formula (1a), a repeating unit B represented by formula (2e), and a repeating unit C represented by formula (3a), a copolymer having a repeating unit A represented by formula (1a), a repeating unit B represented by formula (2f), and a repeating unit C represented by formula (3a), or a layer formed from a copolymer having a repeating unit A represented by formula (1a), a repeating unit B represented by formula (2a), and a repeating unit C represented by formula (3b). The entire repeating unit of the copolymer preferably consists of repeating unit A, repeating unit B, and repeating unit C. The amounts of repeating unit A, repeating unit C, lower critical solution temperature (LCST), and number-average molecular weight of the copolymer are described above.

[0130] If the target cells are cells with a negative charge on their surface, it is preferable to separate the target cells from a cell population that includes both target cells (cells with a negative charge on their surface) and cells without a negative charge on their surface.

[0131] Contact between the cell population containing the target cells and the copolymer layer is preferably brought into contact with the copolymer layer by bringing the cell population into contact with a culture medium or buffer containing the cell population. There are no particular limitations on the culture medium or buffer, and any culture medium or buffer commonly used in this field can be used.

[0132] Examples of culture media include Dulbecco's modified Eagle MEM medium (DMEM), α-MEM medium, Roswell Park Memorial Institute (RPMI) medium, F12 medium, TC199 medium, and GMEM medium. One type of culture medium may be used, or two or more may be used in combination. Additionally, supplements such as fetal bovine serum (FBS), glutamine, and antibiotics may be added to the culture medium as needed. A serum-free culture medium is preferred.

[0133] Examples of buffer solutions include phosphate buffer, Tris buffer, Good's buffer, glycine buffer, and borate buffer. One buffer solution may be used alone, or two or more may be used in combination. The buffer solution is preferably a phosphate buffer.

[0134] When using a culture medium or buffer containing a cell population, the total number of cells in the cell population per 1 mL of the culture medium or buffer is preferably 1.0 × 10⁶. １０ cells / mL or less, more preferably 1.0 × 10 ９ cells / mL or less, more preferably 1.0 × 10 ８ The cell count is less than or equal to cells / mL. The total cell count is 1.0 × 10⁻⁶. １０ When the cells / mL level is below a certain value, the cells can make sufficient contact with the copolymer layer, making it easier to separate the target cells.

[0135] To ensure sufficient adhesion of the target cells to the copolymer layer, it is preferable to allow the cell population and the cell separation substrate of the present invention to stand at the contact temperature after contact. The standing time is preferably 5 minutes to 6 hours, more preferably 15 minutes to 3 hours. In addition, to promote adhesion, centrifugal force may be applied to the cell separation substrate (e.g., flask, test tube) containing the cell population to press the cells against the substrate.

[0136] (Step B) In Step B, the copolymer layer to which the target cells are attached is washed at a temperature higher than that of LCST (hereinafter sometimes referred to as the "washing temperature").

[0137] The lower limit of the washing temperature is preferably "LCST + 1"°C, more preferably "LCST + 2"°C, in order to remove other cells without detaching the target cells. In other words, the washing temperature is preferably "LCST + 1"°C or higher, more preferably "LCST + 2"°C or higher. On the other hand, the upper limit of the washing temperature is preferably 40°C, more preferably 37°C, in order to avoid damage to the target cells. In other words, the washing temperature is preferably 40°C or lower, more preferably 37°C or lower. Naturally, the lower limit of the washing temperature is less than or equal to the upper limit of the washing temperature.

[0138] Washing is preferably performed by replacing the culture medium or buffer. For example, if a culture medium or buffer containing a cell population is used in step A, first remove the culture medium or buffer and add a new culture medium or buffer heated to the washing temperature. If no culture medium or buffer is used in step A, add a culture medium or buffer heated to the washing temperature to the cell population on the cell separation substrate, then remove the culture medium or buffer and add a new culture medium or buffer heated to the washing temperature.

[0139] When the removal of culture medium or buffer and the addition of new culture medium or buffer are counted as one wash, the number of washes is preferably 10 or less, more preferably 7 or less, and even more preferably 5 or less, in order to avoid damage to the target cells. The culture medium or buffer used for washing is described above.

[0140] (Step C) In Step C, the cell separation substrate having the washed copolymer layer is cooled to a temperature lower than that of LCST (hereinafter sometimes referred to as the "desorption temperature"), and the target cells are desorbed from the copolymer layer and recovered.

[0141] The upper limit of the desorption temperature is preferably "LCST-1"°C, more preferably "LCST-2"°C, in order to sufficiently desorb the target cells. In other words, the desorption temperature is preferably "LCST-1"°C or lower, more preferably "LCST-2"°C or lower. Note that "-" in the above formula means minus. On the other hand, the lower limit of the desorption temperature is preferably 2°C, more preferably 4°C, in order to avoid freezing of the cells. In other words, the desorption temperature is preferably 2°C or higher, more preferably 4°C or higher. Of course, the lower limit of the desorption temperature is less than or equal to the upper limit of the desorption temperature.

[0142] The detached target cells can be recovered by known methods. There are no particular limitations on the recovery method; the target cells can be recovered using methods commonly used in fields that handle cells.

[0143] The present invention will be described in detail below based on examples, but the present invention is not limited to the following examples.

[0144] Synthesis Example 1: Synthesis of monomer (m1)

[0145]

[0146] Compound (c1) (5.69 g, 21.4 mmol), triethylamine (8.67 g, 85.6 mmol), and 4-dimethylaminopyridine (0.524 g, 4.28 mmol) were added to a flask, and toluene (15 mL) was added to dissolve compound (c1). After cooling the resulting mixture to 0°C, methacrylic anhydride (6.60 g, 42.8 mmol) was added, and the reaction was carried out at 0°C for 1 hour. Then, methanol (1.50 mL) was added to stop the reaction. Dichloromethane was added to the reaction mixture, and the resulting dichloromethane solution was washed three times by liquid-liquid extraction using saturated sodium bicarbonate solution. The washed dichloromethane solution was dehydrated with sodium sulfate, the sodium sulfate was removed by filtration, and the solvent of the resulting filtrate was removed by vacuum distillation to obtain monomer (m1) (yield: 3.35 g). １ H-NMR (CDCl ３ , 400MHz) δ 6.2ppm (CH ２ -C(CH ３)-COO), 5.6ppm (CH ２ -C(CH ３ )-COO), 4.4ppm (COO-CH ２ ), 3.8-3.5ppm (O-CH ２ -CH ２ -O), 2.4 ppm (CH ２ -COOH), 1.9ppm (CH ２ -C(CH ３ ) - COO)

[0147] In addition, １ For H-NMR measurements, a JEOL JNM-ECS400 was used. The same procedure was followed for subsequent measurements.

[0148] Synthesis Example 2: Synthesis of monomer (m2)

[0149]

[0150] Compound (C2) (9.46 g, 21.4 mmol), triethylamine (8.67 g, 85.6 mmol), and 4-dimethylaminopyridine (0.524 g, 4.28 mmol) were added to a flask, and toluene (15 mL) was added to dissolve compound (C2). After cooling the resulting mixture to 0°C, methacrylic anhydride (6.60 g, 42.8 mmol) was added, and the reaction was carried out at 0°C for 1 hour. Then, methanol (1.50 mL) was added to stop the reaction. Dichloromethane was added to the reaction mixture, and the resulting dichloromethane solution was washed three times by liquid-liquid extraction using saturated sodium bicarbonate solution. The washed dichloromethane solution was dehydrated with sodium sulfate, the sodium sulfate was removed by filtration, and the solvent of the resulting filtrate was removed under reduced pressure to obtain monomer (M2) (yield: 5.61 g). １ H-NMR (CDCl ３ , 400MHz) δ 6.2ppm (CH ２ -C(CH ３ )-COO), 5.6ppm (CH ２ -C(CH ３ )-COO), 4.4ppm (COO-CH ２ ), 3.8-3.5ppm (O-CH ２ -CH ２ -O), 2.4 ppm (CH ２-COOH), 1.9ppm (CH ２ -C(CH ３ ) - COO)

[0151] Synthesis Example 3: Synthesis of monomers (m3)

[0152]

[0153] Compound (C3) (5.67 g, 21.4 mmol), triethylamine (8.67 g, 85.6 mmol), and 4-dimethylaminopyridine (0.524 g, 4.28 mmol) were added to a flask, and toluene (15 mL) was added to dissolve compound (C3). After cooling the resulting mixture to 0°C, methacrylic anhydride (6.60 g, 42.8 mmol) was added, and the reaction was carried out at 0°C for 1 hour. Then, methanol (1.50 mL) was added to stop the reaction. Dichloromethane was added to the reaction mixture, and the resulting dichloromethane solution was washed three times by liquid-liquid extraction using saturated sodium bicarbonate solution. The washed dichloromethane solution was dehydrated with sodium sulfate, the sodium sulfate was removed by filtration, and the solvent of the resulting filtrate was removed under reduced pressure to obtain monomer (M3) (yield: 3.78 g). １ H-NMR (CDCl ３ , 400MHz) δ 6.2ppm (CH ２ -C(CH ３ )-COO), 5.6ppm (CH ２ -C(CH ３ )-COO), 4.4ppm (COO-CH ２ ), 3.8-3.5ppm (O-CH ２ -CH ２ -O), 2.5ppm (CH ２ -N(CH ３ ) ２ ), 2.3ppm (N(CH ３ ) ２ ), 1.9ppm (CH ２ -C(CH ３ ) - COO)

[0154] Synthesis Example 4: Synthesis of monomer (m4)

[0155]

[0156] Compound (C4) (9.44 g, 21.4 mmol), triethylamine (8.67 g, 85.6 mmol), and 4-dimethylaminopyridine (0.524 g, 4.28 mmol) were added to a flask, and toluene (15 mL) was added to dissolve compound (C4). After cooling the resulting mixture to 0°C, methacrylic anhydride (6.60 g, 42.8 mmol) was added, and the reaction was carried out at 0°C for 1 hour. Then, methanol (1.50 mL) was added to stop the reaction. Dichloromethane was added to the reaction mixture, and the resulting dichloromethane solution was washed three times by liquid-liquid extraction using saturated sodium bicarbonate solution. The washed dichloromethane solution was dehydrated with sodium sulfate, the sodium sulfate was removed by filtration, and the solvent of the resulting filtrate was removed under reduced pressure to obtain monomer (M4) (yield: 6.02 g). １ H-NMR (CDCl ３ , 400MHz) δ 6.2ppm (CH ２ -C(CH ３ )-COO), 5.6ppm (CH ２ -C(CH ３ )-COO), 4.4ppm (COO-CH ２ ), 3.8-3.5ppm (O-CH ２ -CH ２ -O), 2.5ppm (CH ２ -N(CH ３ ) ２ ), 2.3ppm (N(CH ３ ) ２ ), 1.9ppm (CH ２ -C(CH ３ ) - COO)

[0157] Synthesis Example 5: Synthesis of monomer (m5)

[0158]

[0159] Compound (C5) (10.7 g, 21.4 mmol), triethylamine (8.67 g, 85.6 mmol), and 4-dimethylaminopyridine (0.524 g, 4.28 mmol) were added to a flask, and toluene (15 mL) was added to dissolve compound (C5). After cooling the resulting mixture to 0°C, methacrylic anhydride (6.60 g, 42.8 mmol) was added, and the reaction was carried out at 0°C for 1 hour. Then, methanol (1.50 mL) was added to stop the reaction. Dichloromethane was added to the reaction mixture, and the resulting dichloromethane solution was washed three times by liquid-liquid extraction using saturated sodium bicarbonate solution. The washed dichloromethane solution was dehydrated with sodium sulfate, the sodium sulfate was removed by filtration, and the solvent of the resulting filtrate was removed under reduced pressure to obtain monomer (M5) (yield: 6.94 g). １ H-NMR (CDCl ３ , 400MHz) δ 6.2ppm (CH ２ -C(CH ３ )-COO), 5.6ppm (CH ２ -C(CH ３ )-COO), 4.4ppm (COO-CH ２ ), 3.8-3.5ppm (O-CH ２ -CH ２ -O), 2.4 ppm (CH ２ -COO-t-Bu), 1.9ppm (CH ２ -C(CH ３ )-COO), 1.7ppm (COO-t-Bu)

[0160] <Example 1: Synthesis of Copolymer> Example 1-1: Synthesis of Copolymer (P1)

[0161]

[0162] Monomer (m6) (=diethylene glycol monomethyl ether methacrylate) (5.13 g, 27.3 mmol), monomer (m7) (=methacrylic acid) (0.839 g, 9.75 mmol), and monomer (m8) (=butyl methacrylate) (0.277 g, 1.95 mmol) were added to a flask, and then IPA (46.7 mL) and deionized water (3.3 mL) were added to dissolve the monomers. After 15 minutes of nitrogen bubbling, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (24.4 mg, 0.0604 mmol) and AIBN (9.90 mg, 0.0604 mmol) were added. After filling with nitrogen gas for 30 minutes, the reaction mixture was heated to 65°C and polymerization was carried out at that temperature for 20 hours. Subsequently, the reaction mixture was dialyzed with ethanol, and the resulting purified product was dried to obtain copolymer (P1) having repeating unit A represented by formula (1a), repeating unit B represented by formula (2c), and repeating unit C represented by formula (3a) (molar ratio of repeating unit A:repeating unit B:repeating unit C = 70:24:6, yield = 3.88 g). １ H-NMR (CD) ３ OD, 400MHz) δ 4.0-4.3ppm (COO-CH ２ ), 3.8-3.5ppm (O-CH ２ -CH ２ -O), 3.4ppm (O-CH ３ ), 0.8-2.2 ppm (polymer main chain, repeating unit C O-CH ２ -CH ２ -CH ２ -CH ３ )

[0163] The number-average molecular weight of copolymer (P1) was 81,000, and the polydispersity (weight-average molecular weight / number-average molecular weight) was 1.25. The lower critical solution temperature (LCST) of copolymer (P1) was 31°C.

[0164] The number-average molecular weight, weight-average molecular weight, and polydispersity of the copolymer were determined by gel permeation chromatography using a differential refractometer as the detector. A Shimadzu LC-720AD pump was used, a Shimadzu RID10A differential refractometer was used, and a Shimadzu SPD-20A UV detector was used. An Agilent Technologies PLgel MIXED-D column (column size: 7.5 mm × 30 cm) was used. DMF containing 50 mM lithium bromide was used as the developing solvent. Measurement conditions were: flow rate: 0.6 mL / min, column temperature: 40°C, sample concentration: 2 mg / mL, and injection volume: 70 μL. Polyethylene glycol was used as the standard. The same applies below.

[0165] The lower critical solution temperature (LCST) of the copolymer was determined by measuring the transmittance using a UV-Vis spectrophotometer, and the temperature at which the transmittance reached 50% was defined as the LCST. Specifically, the copolymer was dissolved in serum-free RPMI 1640 medium at a concentration of 2.5 mg / mL. The resulting solution was placed in a quartz cell, and the solution temperature was varied from 4°C to 70°C. The transmittance (%) of the solution during this range was measured using a UV-Vis spectrophotometer. The transmittance (%) of the solution is given by the following formula: Transmittance (%) = 10⁻¹⁰ （－吸光度） This was calculated from the following. The same applies below.

[0166] Examples 1-2: Synthesis of copolymer (P2)

[0167]

[0168] Into a flask, monomer (m6) (= diethylene glycol monomethyl ether methacrylate) (6.38 g, 33.9 mmol), monomer (m1) (1.04 g, 3.12 mmol), and monomer (m8) (= butyl methacrylate) (0.277 g, 1.95 mmol) were added, and then IPA (46.7 mL) and ion-exchanged water (3.3 mL) were added to dissolve the monomers. After nitrogen bubbling for 15 minutes, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (14.6 mg, 0.0361 mmol), and AIBN (5.90 mg, 0.0361 mmol) were added. After nitrogen gas enclosure for 30 minutes, the reaction mixture was heated to 65 °C and polymerization was carried out at that temperature for 20 hours. Then, the reaction mixture was dialyzed with ethanol, and the obtained purified product was dried to obtain a copolymer (P2) having a repeating unit A represented by formula (1a), a repeating unit B represented by formula (2d), and a repeating unit C represented by formula (3a) (molar ratio of repeating unit A: repeating unit B: repeating unit C = 87:8:5, yield = 5.81 g). １ H-NMR (CD ３ OD, 400 MHz) δ 4.0 - 4.3 ppm (COO-CH ２ ), 3.8 - 3.5 ppm (O-CH ２ -CH ２ -O), 3.4 ppm (O-CH ３ ), 0.8 - 2.2 ppm (copolymer main chain, O-CH of repeating unit C ２ -CH ２ -CH ２ -CH ３ )

[0169] The number average molecular weight of the copolymer (P2) was 183,000 and the polydispersity was 1.28. The lower critical solution temperature (LCST) of the copolymer (P2) was 22 °C.

[0170] Example 1-3: Synthesis of copolymer (P3)

[0171]

[0172] Into a flask, monomer (m6) (= diethylene glycol monomethyl ether methacrylate) (4.91 g, 26.1 mmol), monomer (m2) (5.57 g, 10.9 mmol), and monomer (m8) (= butyl methacrylate) (0.277 g, 1.95 mmol) were added. Then, IPA (46.7 mL) and ion-exchanged water (3.3 mL) were added to dissolve the monomers. After nitrogen bubbling for 15 minutes, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (72.4 mg, 0.179 mmol) and AIBN (29.4 mg, 0.179 mmol) were added. After nitrogen gas encapsulation for 30 minutes, the reaction mixture was heated to 65 °C and polymerization was carried out at that temperature for 20 hours. Then, the reaction mixture was dialyzed with ethanol, and the obtained purified product was dried to obtain a copolymer (P3) having a repeating unit A represented by formula (1a), a repeating unit B represented by formula (2b), and a repeating unit C represented by formula (3a) (molar ratio of repeating unit A:repeating unit B:repeating unit C = 67:28:5, yield = 7.11 g). １ H-NMR (CD ３ OD, 400 MHz) δ 4.0 - 4.3 ppm (COO-CH<00004​​​​​​​​​​​​​​​​​​​​​​​​​​Monomer (m6) (=diethylene glycol monomethyl ether methacrylate) (6.82 g, 36.3 mmol), monomer (m9) (=2-(dimethylamino)ethyl methacrylate) (0.122 g, 0.780 mmol), and monomer (m8) (=butyl methacrylate) (0.277 g, 1.95 mmol) were added to a flask, and then IPA (46.7 mL) and deionized water (3.3 mL) were added to dissolve the monomers. After 15 minutes of nitrogen bubbling, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (25.6 mg, 0.0634 mmol) and AIBN (10.4 mg, 0.0634 mmol) were added. After 30 minutes of nitrogen gas sealing, the reaction mixture was heated to 65°C and polymerization was carried out at that temperature for 20 hours. Subsequently, the reaction mixture was dialyzed with ethanol, and the resulting purified product was dried to obtain a copolymer (P4) having repeating unit A represented by formula (1a), repeating unit B represented by formula (2e), and repeating unit C represented by formula (3a) (molar ratio of repeating unit A:repeating unit B:repeating unit C = 93:3:4, yield = 5.78 g). １ H-NMR (CD) ３ OD, 400MHz) δ 4.0-4.3ppm (COO-CH ２ ), 3.8-3.5ppm (O-CH ２ -CH ２ -O), 3.4ppm (O-CH ３ ), 2.5-3.0ppm (N(CH ３ ) ２ ), 0.8-2.2 ppm (polymer main chain, repeating unit C O-CH ２ -CH ２ -CH ２ -CH ３ )

[0177] The number-average molecular weight of copolymer (P4) was 104,100, and the polydispersity was 1.22. The lower critical solution temperature (LCST) of copolymer (P4) was 15°C.

[0178] Examples 1-5: Synthesis of copolymer (P5)

[0179]

[0180] Monomer (m6) (=diethylene glycol monomethyl ether methacrylate) (5.87 g, 31.2 mmol), monomer (m3) (2.60 g, 7.80 mmol), and monomer (m8) (=butyl methacrylate) (0.277 g, 1.95 mmol) were added to a flask, and then IPA (46.7 mL) and deionized water (3.3 mL) were added to dissolve the monomers. After 15 minutes of nitrogen bubbling, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (73.2 mg, 0.181 mmol) and AIBN (29.7 mg, 0.181 mmol) were added. After 30 minutes of nitrogen gas sealing, the reaction mixture was heated to 65°C and polymerization was carried out at that temperature for 20 hours. Subsequently, the reaction mixture was dialyzed with ethanol, and the resulting purified product was dried to obtain a copolymer (P5) having repeating unit A represented by formula (1a), repeating unit B represented by formula (2f), and repeating unit C represented by formula (3a) (molar ratio of repeating unit A:repeating unit B:repeating unit C = 80:15:5, yield = 4.39 g). １ H-NMR (CD) ３ OD, 400MHz) δ 4.0-4.3ppm (COO-CH ２ ), 3.8-3.5ppm (O-CH ２ -CH ２ -O), 3.4ppm (O-CH ３ ), 2.5-3.0ppm (N(CH ３ ) ２ ), 0.8-2.2 ppm (polymer main chain, repeating unit C O-CH ２ -CH ２ -CH ２ -CH ３ )

[0181] The number-average molecular weight of copolymer (P5) was 36,400, and the polydispersity was 1.26. The lower critical solution temperature (LCST) of copolymer (P5) was 24°C.

[0182] Examples 1-6: Synthesis of copolymer (P6)

[0183]

[0184] Monomer (m6) (=diethylene glycol monomethyl ether methacrylate) (4.11 g, 21.8 mmol), monomer (m4) (7.74 g, 15.2 mmol), and monomer (m8) (=butyl methacrylate) (0.277 g, 1.95 mmol) were added to a flask, and then IPA (46.7 mL) and deionized water (3.3 mL) were added to dissolve the monomers. After 15 minutes of nitrogen bubbling, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (76.9 mg, 0.190 mmol) and AIBN (31.2 mg, 0.190 mmol) were added. After filling with nitrogen gas for 30 minutes, the reaction mixture was heated to 65°C and polymerization was carried out at that temperature for 20 hours. Subsequently, the reaction mixture was dialyzed with ethanol, and the resulting purified product was dried to obtain copolymer (P6) having repeating unit A represented by formula (1a), repeating unit B represented by formula (2a), and repeating unit C represented by formula (3a) (molar ratio of repeating unit A:repeating unit B:repeating unit C = 56:38:6, yield 6.04 g). １ H-NMR (CD) ３ OD, 400MHz) δ 4.0-4.3ppm (COO-CH ２ ), 3.8-3.5ppm (O-CH ２ -CH ２ -O), 3.4ppm (O-CH ３ ), 2.5-3.0ppm (N(CH ３ ) ２ ), 0.8-2.2 ppm (polymer main chain, repeating unit C O-CH ２ -CH ２ -CH ２ -CH ３ )

[0185] The number-average molecular weight of copolymer (P6) was 52,000, and the polydispersity was 1.21. The lower critical solution temperature (LCST) of copolymer (P6) was 35°C.

[0186] Examples 1-7: Synthesis of copolymer (P7)

[0187]

[0188] Monomer (m6) (=diethylene glycol monomethyl ether methacrylate) (6.53 g, 34.7 mmol), monomer (m2) (6.53 g, 2.34 mmol), and monomer (m10) (=4-benzoylphenyl methacrylate) (0.518 g, 1.95 mmol) were added to a flask, and then IPA (46.7 mL) and deionized water (3.3 mL) were added to dissolve the monomers. After 15 minutes of nitrogen bubbling, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (65.7 mg, 0.163 mmol) and AIBN (26.7 mg, 0.163 mmol) were added. After filling with nitrogen gas for 30 minutes, the reaction mixture was heated to 65°C and polymerization was carried out at that temperature for 20 hours. Subsequently, the reaction mixture was dialyzed with ethanol, and the resulting purified product was dried to obtain a copolymer (P7) having repeating unit A represented by formula (1a), repeating unit B represented by formula (2b), and repeating unit C represented by formula (3b) (molar ratio of repeating unit A:repeating unit B:repeating unit C = 89:6:5, yield = 5.11 g). １ H-NMR (CD) ３ (OD, 400MHz) δ 7.0–7.8 ppm (benzene ring with repeating unit C), 4.0–4.3 ppm (COO–CH ２ ), 3.8-3.5ppm (O-CH ２ -CH ２ -O), 3.4ppm (O-CH ３ ), 0.8-2.2ppm (copolymer main chain)

[0189] The number-average molecular weight of copolymer (P7) was 40,600, and the polydispersity was 1.20. The lower critical solution temperature (LCST) of copolymer (P7) was 18°C.

[0190] Examples 1-8: Synthesis of copolymer (P8)

[0191]

[0192] Monomer (m6) (=diethylene glycol monomethyl ether methacrylate) (6.75 g, 35.9 mmol), monomer (m4) (0.596 g, 1.17 mmol), and monomer (m10) (=4-benzoylphenyl methacrylate) (0.518 g, 1.95 mmol) were added to a flask, and then IPA (46.7 mL) and deionized water (3.3 mL) were added to dissolve the monomers. After 15 minutes of nitrogen bubbling, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (43.7 mg, 0.108 mmol) and AIBN (17.7 mg, 0.108 mmol) were added. After filling with nitrogen gas for 30 minutes, the reaction mixture was heated to 65°C and polymerization was carried out at that temperature for 20 hours. Subsequently, the reaction mixture was dialyzed with ethanol, and the resulting purified product was dried to obtain a copolymer (P8) having repeating unit A represented by formula (1a), repeating unit B represented by formula (2a), and repeating unit C represented by formula (3b) (molar ratio of repeating unit A:repeating unit B:repeating unit C = 92:3:5, yield = 5.04 g). １ H-NMR (CD) ３ (OD, 400MHz) δ 7.0–7.8 ppm (benzene ring with repeating unit C), 4.0–4.3 ppm (COO–CH ２ ), 3.8-3.5ppm (O-CH ２ -CH ２ -O), 3.4ppm (O-CH ３ ), 2.5-3.0ppm (N(CH ３ ) ２ ), 0.8-2.2ppm (copolymer main chain)

[0193] The number-average molecular weight of copolymer (P8) was 61,000, and the polydispersity was 1.21. The lower critical solution temperature (LCST) of copolymer (P8) was 16°C.

[0194] Examples 1-9: Synthesis of copolymer (P9)

[0195]

[0196] In a flask, monomer (m6) (=diethylene glycol monomethyl ether methacrylate) (6.23 g, 33.2 mmol), monomer (m5) (1.10 g, 1.95 mmol), monomer (m8) (=butyl methacrylate) (0.554 g, 3.80 mmol), ethyl 2-bromoisobutyrate (17.6 mg, 0.125 mmol), copper(II) bromide (0.0882 mg, 0.360 mmol), and 2,2-bipyridine (114 mg, 0.720 mmol) were added. Then, 50 mL of a mixed solvent of ethanol / water (volume ratio = 50 / 50) was added to dissolve the monomers. After filling with nitrogen gas for 30 minutes, L-ascorbic acid (18.0 mg, 0.100 mmol) was added. The reaction mixture was heated to 30°C, and polymerization was carried out at that temperature for 20 hours to obtain a copolymer (P9') having repeating unit A represented by formula (1a), repeating unit B' represented by formula (iib), and repeating unit C represented by formula (3a).

[0197] Subsequently, methanesulfonic acid (961 mg, 10 mmol) was added to the reaction mixture containing copolymer (P9'), and the resulting reaction mixture was heated at 30°C for 3 hours to hydrolyze the repeating units represented by formula (iib). The reaction mixture was then dialyzed with ethanol, and the resulting purified product was dried to obtain copolymer (P9) having repeating units A represented by formula (1a), repeating units B represented by formula (2b), and repeating units C represented by formula (3a) (molar ratio of repeating units A: repeating units B: repeating units C = 85:5:10, yield = 4.98 g). １ H-NMR (CD) ３ OD, 400MHz) δ 4.0-4.3ppm (COO-CH ２ ), 3.8-3.5ppm (O-CH ２ -CH ２ -O), 3.4ppm (O-CH ３ ), 0.8-2.2 ppm (polymer main chain, repeating unit C O-CH ２ -CH ２ -CH ２ -CH ３ )

[0198] The number average molecular weight of the copolymer (P9) was 54,200, and the polydispersity was 2.28. The lower critical solution temperature (LCST) of the copolymer (P9) was 18°C.

[0199] Example 1-10: Synthesis of Copolymer (P10)

[0200]

[0201] In a flask, monomer (m6) (= diethylene glycol monomethyl ether methacrylate) (6.60 g, 35.1 mmol), monomer (m2) (0.995 g, 1.95 mmol), and monomer (m8) (= butyl methacrylate) (0.277 g, 1.95 mmol) were added. Then, IPA (46.7 mL) and ion-exchanged water (3.3 mL) were added to dissolve the monomers. After nitrogen bubbling for 15 minutes, AIBN (2.10 mg, 0.0131 mmol) was added. After nitrogen gas encapsulation for 30 minutes, the reaction mixture was heated to 65°C and polymerization was carried out at that temperature for 20 hours. Then, the reaction mixture was dialyzed with ethanol and dried to obtain a copolymer (P10) having repeating unit A represented by formula (1a), repeating unit B represented by formula (2b), and repeating unit C represented by formula (3a) (molar ratio of repeating unit A: repeating unit B: repeating unit C = 89:6:5, yield = 5.11 g). The １ H-NMR (CD ３ OD, 400 MHz) δ 4.0 - 4.3 ppm (COO-CH ２ ), 3.8 - 3.5 ppm (O-CH ２ -CH ２ -O), 3.4 ppm (O-CH ３ ), 0.8 - 2.2 ppm (copolymer main chain, O-CH of repeating unit C ２ -CH ２ -CH ２ -CH ３ ) <​​​​<Example 2: Preparation of cell separation substrate> Example 2-1: Preparation of cell separation substrate (S1) having a layer formed from copolymer (P1) The copolymer (P1) was dissolved in ethanol to a concentration of 3 mg / mL. The obtained solution was added to a polystyrene 6-well plate at a volume of 2 mL / well. Then, the polystyrene 6-well plate was gently shaken several times to remove excess solution, and the plate was dried at room temperature for 17 hours to obtain a cell separation substrate (S1) having a layer formed from copolymer (P1).

[0204] Examples 2-2 to 2-6: Preparation of cell separation substrates (S2) having a layer formed from copolymer (P2) to cell separation substrates (S6) having a layer formed from copolymer (P6) Except that one of copolymers (P2) to copolymer (P6) was used instead of copolymer (P1), cell separation substrates (S2) having a layer formed from copolymer (P2) to cell separation substrates (S6) having a layer formed from copolymer (P6) were prepared in the same manner as in Example 2-1.

[0205] Example 2-7: Preparation of a cell separation substrate (S7) having a layer formed from copolymer (P7) The copolymer (P7) was dissolved in ethanol to a concentration of 3 mg / mL. The obtained solution was added to a 6-well polystyrene plate at a volume of 2 mL / well. The 6-well polystyrene plate was then gently shaken several times to remove excess solution, and dried at room temperature for 17 hours. The dried 6-well polystyrene plate was irradiated with 254 nm light to obtain a cell separation substrate (S7) having a layer formed from copolymer (P7).

[0206] Example 2-8: Preparation of a cell separation substrate (S8) having a layer formed from copolymer (P8) A cell separation substrate (S8) having a layer formed from copolymer (P8) was prepared in the same manner as in Example 2-7, except that copolymer (P8) was used instead of copolymer (P7).

[0207] Examples 2-9 and 2-10: Preparation of cell separation substrate (S9) having a layer formed from copolymer (P9) and cell separation substrate (S10) having a layer formed from copolymer (P10) A cell separation substrate (S9) having a layer formed from copolymer (P9) and a cell separation substrate (S10) having a layer formed from copolymer (P10) were prepared in the same manner as in Example 2-1, except that copolymer (P9) or copolymer (P10) was used instead of copolymer (P1).

[0208] Example 2-11: Preparation of a cell separation substrate (S11) having a layer formed from a copolymer (P11)

[0209]

[0210] A glass substrate (10 mm long x 10 mm wide x 0.15 mm thick) was dipped in a piranha solution (volume ratio of concentrated sulfuric acid / 30% by weight hydrogen peroxide = 3 / 1) and immersed for 2 hours to obtain a glass substrate having hydroxyl groups on its surface.

[0211] The glass substrate was removed from the piranha solution, washed with deionized water, and dried. The dried glass substrate was placed in toluene (8 mL), to which 3-aminopropyltrimethoxysilane (50 μL) was added, and the mixture was reacted at 30°C for 24 hours. After the reaction, the glass substrate was removed from the toluene and washed with ethanol. Subsequently, annealing was performed at 120°C for 2 hours under a nitrogen atmosphere to obtain a glass substrate having amino groups on its surface.

[0212] The glass substrate was placed in THF (2 mL), and triethylamine (50 μL) and 2-bromopropionyl bromide (140 μL) were added. The mixture was maintained at room temperature for 3 hours. After that, the glass substrate was removed from the THF and washed with ethanol to obtain a glass substrate having bromo groups (Br) on its surface.

[0213] The glass substrate was placed in a test tube. Next, monomer (m6) (=diethylene glycol monomethyl ether methacrylate) (7.05 g, 37.5 mmol), monomer (m5) (1.10 g, 1.95 mmol), monomer (m8) (=butyl methacrylate) (0.277 g, 1.95 mmol), ethyl 2-bromoisobutyrate (10.9 mg, 0.560 mmol), copper(II) bromide (0.0882 mg, 0.360 mmol), and 2,2-bipyridine (114 mg, 0.720 mmol) were added to the test tube. Then, 50 mL of a mixed solvent of ethanol / water (volume ratio = 50 / 50) was added to dissolve the monomers. After filling with nitrogen gas for 30 minutes, L-ascorbic acid (18.0 mg, 0.100 mmol) was added. The reaction mixture containing the glass substrate was heated to 30°C, and polymerization was carried out at that temperature for 20 hours, starting from the bromo groups on the surface of the glass substrate. This produced a glass substrate having a copolymer layer on the glass surface in which a copolymer (P11') having repeating units A represented by formula (1a), repeating units B' represented by formula (iib), and repeating units C represented by formula (3a) was bonded.

[0214] Methanesulfonic acid (961 mg, 10 mmol) was added to the reaction mixture containing the glass substrate, and the reaction mixture containing the glass substrate and methanesulfonic acid was heated at 30°C for 3 hours to hydrolyze the repeating units represented by formula (iib), thereby producing a cell separation substrate (S11) having a copolymer layer on its surface in which a copolymer (P11) having repeating units A represented by formula (1a), repeating units B represented by formula (2b), and repeating units C represented by formula (3a) was bonded.

[0215] After removing the cell separation substrate (S11) from the reaction mixture, the reaction mixture was dialyzed with ethanol, and the resulting purified product was dried to obtain a copolymer (P11) not bound to the glass substrate, which was then analyzed (molar ratio of repeating unit A:repeating unit B:repeating unit C = 90:5:5, yield: 4.21 g). １ H-NMR (CD) ３ OD, 400MHz) δ 4.0-4.3ppm (COO-CH２ ), 3.8-3.5ppm (O-CH ２ -CH ２ -O), 3.4ppm (O-CH ３ ), 0.8-2.2 ppm (polymer main chain, repeating unit C O-CH ２ -CH ２ -CH ２ -CH ３ )

[0216] The number-average molecular weight of the unbonded copolymer (P11) was 101,300, and its polydispersity was 2.49. The lower critical solution temperature (LCST) of the unbonded copolymer (P11) was 20°C.

[0217] <Comparative Example 1: Synthesis of Copolymer> Comparative Example 1-1: Synthesis of copolymer (CP1)

[0218]

[0219] Monomer (m11) (= N-isopropylacrylamide, abbreviated as "NIPAM") (6.35 g, 56.2 mmol) and monomer (m2) (3.18 g, 6.24 mmol) were added to a flask, followed by the addition of IPA (46.7 mL) and deionized water (3.3 mL) to dissolve the monomers. After 15 minutes of nitrogen bubbling, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (44.1 mg, 0.109 mmol) and AIBN (17.9 mg, 0.109 mmol) were added. After 30 minutes of nitrogen gas sealing, the reaction mixture was heated to 65°C and polymerization was carried out at that temperature for 20 hours. Subsequently, the reaction mixture was dialyzed with ethanol, and the resulting purified product was dried to obtain a copolymer (CP1) having repeating units represented by formula (r1) (hereinafter referred to as "NIPAM units") and repeating units B represented by formula (2b) (molar ratio of NIPAM units to repeating units B = 89:11, yield = 4.15 g). １ H-NMR (CD) ３ OD, 400MHz) δ 4.0-4.3ppm (COO-CH ２ ), 3.8-3.5ppm (O-CH ２ -CH ２-O), 3.4ppm (O-CH ３ ), 0.8-2.2 ppm (polymer main chain, NIPAM units CH-(CH ３ ) ２ )

[0220] The number-average molecular weight of copolymer (CP1) was 60,500, and the polydispersity was 1.28. The lower critical solution temperature (LCST) of copolymer (CP1) was 21°C.

[0221] Comparative Example 1-2: Synthesis of copolymer (CP2)

[0222]

[0223] Monomer (m6) (=diethylene glycol monomethyl ether methacrylate) (6.97 g, 37.1 mmol) and monomer (m8) (=butyl methacrylate) (0.277 g, 1.95 mmol) were added to a flask, followed by the addition of IPA (46.7 mL) and deionized water (3.3 mL) to dissolve the monomers. After 15 minutes of nitrogen bubbling, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (63.6 mg, 0.158 mmol) and AIBN (25.8 mg, 0.158 mmol) were added. After 30 minutes of nitrogen gas sealing, the reaction mixture was heated to 65°C and polymerization was carried out at that temperature for 20 hours. Subsequently, the reaction mixture was dialyzed with ethanol, and the resulting purified product was dried to obtain a copolymer (CP2) having repeating units A represented by formula (1a) and repeating units C represented by formula (3a) (molar ratio of repeating units A to repeating units C = 94:6, yield = 4.82 g). １ H-NMR (CD) ３ OD, 400MHz) δ 4.0-4.3ppm (COO-CH ２ ), 3.8-3.5ppm (O-CH ２ -CH ２ -O), 3.4ppm (O-CH ３ ), 0.8-2.2 ppm (polymer main chain, repeating unit C O-CH ２ -CH ２ -CH ２ -CH ３ )

[0224] The number-average molecular weight of the copolymer (CP2) was 41,900, and the polydispersity was 1.30. The lower critical solution temperature (LCST) of the copolymer (CP2) was 13°C.

[0225] Comparative Example 1-3: Synthesis of copolymer (CP3)

[0226]

[0227] Monomer (m6) (=diethylene glycol monomethyl ether methacrylate) (2.27 g, 12.1 mmol), monomer (m2) (3.18 g, 6.24 mmol), and monomer (m8) (=butyl methacrylate) (0.277 g, 1.95 mmol) were added to a flask, and then IPA (46.7 mL) and deionized water (3.3 mL) were added to dissolve the monomers. After 15 minutes of nitrogen bubbling, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (74.9 mg, 0.185 mmol) and AIBN (30.4 mg, 0.185 mmol) were added. After filling with nitrogen gas for 30 minutes, the reaction mixture was heated to 65°C and polymerization was carried out at that temperature for 20 hours. Subsequently, the reaction mixture was dialyzed with ethanol, and the resulting purified product was dried to obtain a copolymer (CP3) having repeating unit A represented by formula (1a), repeating unit B represented by formula (2b), and repeating unit C represented by formula (3a) (molar ratio of repeating unit A:repeating unit B:repeating unit C = 31:64:5, yield = 3.65 g). １ H-NMR (CD) ３ OD, 400MHz) δ 4.0-4.3ppm (COO-CH ２ ), 3.8-3.5ppm (O-CH ２ -CH ２ -O), 3.4ppm (O-CH ３ ), 0.8-2.2 ppm (polymer main chain, repeating unit C O-CH ２ -CH ２ -CH ２ -CH ３ )

[0228] The number-average molecular weight of the copolymer (CP3) was 53,400, and the polydispersity was 1.28. The lower critical solution temperature (LCST) of the copolymer (CP3) was 53°C.

[0229] <Comparative Example 2: Preparation of Cell Separation Substrates> Comparative Examples 2-1 to 2-3: Preparation of Cell Separation Substrates (CS1) Having a Layer Formed from Copolymer (CP1) to Cell Separation Substrates (CS3) Having a Layer Formed from Copolymer (CP3) Cell separation substrates (CS1) Having a layer formed from copolymer (CP1) to cell separation substrates (CS3) Having a layer formed from copolymer (CP3) were prepared in the same manner as in Example 2-1, except that one of copolymers (CP1) to copolymer (CP3) was used instead of copolymer (P1).

[0230] <Test Example 1: Measurement of the Thickness of the Copolymer Layer of the Cell Separation Substrate> The thickness of the copolymer layer of the cell separation substrate obtained as described above was measured by spectroscopic ellipsometry using an alpha 2.0 manufactured by J. A. Wollam. The results are shown in Table 1. In Table 1, "cell separation substrate (S1)," etc., are written as "(S1)," etc., and "copolymer (P1)," etc., are written as "(P1)," etc.

[0231]

[0232] <Test Example 2: Measurement of the Contact Angle of the Copolymer Layer of the Cell Separation Substrate with Water> The contact angle of the copolymer layer of the cell separation substrate obtained as described above was measured with water using a "Contact Angle Meter DMo-702" manufactured by Kyowa Interface Science Co., Ltd. Specifically, the cell separation substrate was placed on a stage with the copolymer layer facing upwards, and 1 μL of deionized water was dropped at temperatures of 4°C and 37°C under a nitrogen atmosphere, and the contact angle at each temperature was measured. The results are shown in Table 2. In Table 2, "Cell Separation Substrate (S1)", etc. are written as "(S1)", etc., and "Copolymer (P1)", etc. are written as "(P1)", etc.

[0233]

[0234] <Example 3: Method for Isolating Target Cells> Example 3-1: Method for Isolating Target Cells Using Cell Separation Substrate (S1) Jurkat cells (cells without surface charge) and HUVEC cells (cells with surface charge) pre-stained with Hoechest 33342 as target cells were mixed in a cell number ratio of 50:50, and the resulting cell mixture was suspended in serum-free RPMI 1640 medium to obtain a cell suspension (1.0 × 10⁻⁶). ５ Cells / mL (3 mL) was added to the cell separation substrate (S1) at 37°C.

[0235] A cell separation substrate (S1) containing a cell suspension was left to stand in an incubator at a temperature of 37°C for 1 hour to allow the cells to adhere to the cell separation substrate (Step A).

[0236] The culture medium was removed from the cell suspension, and then serum-free RPMI1640 medium heated to 37°C was added. This washing procedure was performed twice to remove cells that did not adhere to the cell separation substrate (Step B). A 1 mm × 1 mm area was observed with a fluorescence microscope, and the number of target cells on the cell separation substrate (S1) was counted (cell count 1).

[0237] Subsequently, the cell separation substrate to which the target cells were attached was cooled to 4°C to detach the target cells from the cell separation substrate (Step C), and the culture medium containing the target cells was collected, thereby recovering the cell population containing the target cells (Step D). The number of target cells remaining on the cell separation substrate (S1) after recovery in Step D was counted again by fluorescence microscopy (Cell Count 2).

[0238] Using cell counts 1 and 2 obtained as described above, the elimination rate was calculated using the following formula: Elimination rate (%) = 100 × (cell count 1 - cell count 2) / cell count 1. The results are shown in Table 3. In Table 3, "substrate for cell separation (S1)", etc. are written as "(S1)", etc., and "copolymer (P1)", etc. are written as "(P1)", etc.

[0239] By analyzing with a flow cytometer, the total number of cells (cell count 3) and the number of target cells (cell count 4) in the cell population recovered in step D were counted. Using the obtained cell counts 3 and 4, the purity was calculated using the following formula: Purity (%) = 100 × cell count 4 / cell count 3. The results are shown in Table 3.

[0240] The separated cell population, including non-separated cells, was stained with trypan blue, and the number of viable cells and the total number of cells were counted using a cell counter. Using the obtained number of viable cells and total number of cells, the survival rate was calculated using the following formula: Survival rate (%) = 100 × number of viable cells / total number of cells. The results are shown in Table 3.

[0241] Examples 3-2, 3-3, and 3-7: Methods for separating target cells using any of the cell separation substrates (S2), (S3), and (S7). The separation of target cells was carried out in the same manner as in Example 3-1, except that one of the cell separation substrates (S2), (S3), and (S7) was used instead of the cell separation substrate (S1), and the elimination rate, purity, and viability were calculated. The results are shown in Table 3.

[0242] Examples 3-4 to 3-6 and 3-8: Method for separating target cells using any of the cell separation substrates (S4) to (S6) and (S8). The separation of target cells was carried out in the same manner as in Example 3-1, except that any of the cell separation substrates (S4) to (S6) and (S8) were used instead of the cell separation substrate (S1), and HL-60 cells (cells with a negative charge on the surface) were used as the target cells instead of HUVEC cells (cells with a positive charge on the surface). The detachment rate, purity, and viability were calculated. The results are shown in Table 3.

[0243] <Comparative Example 3: Method for Isolating Target Cells> Comparative Example 3-1: Method for Isolating Target Cells by Enzyme Treatment HUVEC cells (target cells) were suspended in DMEM medium containing 10% by weight of bovine serum albumin (FBS), and the resulting cell suspension was placed in a 6-well polystyrene plate (1.0 × 10⁶). ５Cells / mL (3 mL) was added at 4°C. After centrifugation of the 6-well plate to settle the HUVEC cells, they were cultured at 37°C for 12 hours. Subsequently, the culture medium was removed from the cell suspension, and then serum-free RPMI 1640 medium heated to 37°C was added. This washing procedure was performed twice, and then a 1 mm × 1 mm area was observed with a fluorescence microscope to count the number of target cells on the 6-well plate (cell count 1). Subsequently, 0.25 wt% trypsin solution (1 mL) was added to the cell suspension and incubated at 37°C for 5 minutes. The supernatant was removed, and the number of target cells remaining on the 6-well plate was counted again by fluorescence microscopy (cell count 2). The elimination rate and viability were calculated in the same manner as in Example 3-1. The results are shown in Table 3.

[0244] <Comparative Examples 3-2 to 3-4: Method for Isolating Target Cells Using Any of the Cell Separation Substrates (CS1) to (CS3)> Target cells were separated in the same manner as in Example 3-1, except that one of the cell separation substrates (CS1) to (CS3) was used instead of the cell separation substrate (S1), and the elimination rate, purity, and viability were calculated. The results are shown in Table 3.

[0245]

[0246] As shown in Table 3, the method of detaching target cells adhered to a polystyrene 6-well plate by enzymatic treatment resulted in a low survival rate (Comparative Example 3-1). On the other hand, the method of detaching target cells by temperature change using cell separation substrates (S1) to (S8) did not result in a decrease in survival rate, and target cells could be separated with low irritation (Examples 3-1 to 3-8).

[0247] As shown in Table 2, the cell separation substrate (CS1) having a layer formed from a copolymer (CP1) containing NIPAM units had a large contact angle with water at 37°C and exhibited hydrophobicity at 37°C (Test Example 2-12). On the other hand, the cell separation substrates (S1) to (S8) having a layer formed from a copolymer (P1) containing repeating units A represented by formula (1a) had a small contact angle with water at 37°C and exhibited hydrophilicity at 37°C (Test Examples 2-1 to 2-8).

[0248] As a result, as shown in Table 3, in the method of detaching target cells using the cell separation substrate (CS1), nonspecific adhesion occurred at 37°C, resulting in a decrease in purity. Furthermore, since the copolymer layer of the substrate did not exhibit sufficient hydrophilicity even at 4°C, the target cells remained in the copolymer layer, reducing the detachment rate, and thus the recovery efficiency of the target cells decreased in the method using this substrate (Comparative Example 3-2). On the other hand, in the method using the cell separation substrates (S1) to (S8), the copolymer layer exhibited high hydrophilicity at 37°C, suppressing nonspecific adhesion and resulting in improved purity. Furthermore, since the copolymer layer of the substrate remained hydrophilic even at 4°C, the target cells did not remain in the copolymer layer, improving the detachment rate, and the target cells could be recovered with high efficiency in the method using this substrate (Examples 3-1 to 3-8).

[0249] As shown in Table 3, in the method using a cell separation substrate (CS2) having a layer formed from a copolymer (CP2) without repeating units B, the amount of target cells adhered was small, resulting in low purity (Comparative Example 3-3). On the other hand, in the method using cell separation substrates (S1) to (S6) having a layer formed from a copolymer with repeating units B, as n in formula (2) increases, the substituents of the repeating units B become more easily exposed from the polymer side chains while maintaining hydrophilicity, allowing for selective adhesion of target cells and improving purity (Examples 3-1 to 3-6).

[0250] As shown in Table 3, in the method using a cell separation substrate (CS3) having a layer formed from a copolymer (CP3) with a lower critical solution temperature (LCST) exceeding 50°C due to a high content of repeating unit B, adhesion of target cells was difficult in step A, which was performed at 37°C. Furthermore, in the method using the cell separation substrate (CS3), the target cells could not be sufficiently detached, resulting in a reduced detachment rate (Comparative Examples 3-4).

[0251] From the above, it was found that by using the cell separation substrate of the present invention, which has a layer formed from the copolymer of the present invention, it is possible to perform low-irritation separation with reduced damage to target cells, and furthermore, target cells can be recovered with high efficiency and high purity.

[0252] The copolymer and cell separation substrate of the present invention are useful for separating target cells from a group of cells that include the target cells.

[0253] This application is based on Japanese Patent Application No. 2025-10828, the contents of which are fully incorporated herein.

Claims

1. A copolymer having a repeating unit A represented by the following formula (1) and a repeating unit B represented by the following formula (2), wherein the amount of the repeating unit A relative to the total of the repeating unit A and the repeating unit B is 50 mol% or more, and the lower critical solution temperature (LCST) is 3°C to 40°C: (In the formula, R １ and R ２ each independently represent a hydrogen atom or a methyl group, n represents a number from 0 to 12, when n is 0, X represents *-OH, when n is a number of 1 or more, X represents *-NH ２ , *-N(CH ３ )H, *-N(CH ３ ) ２ , *-N ＋ (CH ３ ) ３ , *-S ＋ (CH ３ ) ２ , *-COOH, *-SO ３ H, *-C ６ H ４ -SO ３ H, or *-PO ３ H ２ represents, *-C ６ H ４ - * represents a phenylene group, and * represents the bonding position.).

2. The copolymer according to claim 1, wherein the number-average molecular weight is 1,000 to 5,000,000.

3. R １ and R ２ The copolymer according to claim 1, wherein both are methyl groups.

4. n is 0 and X is *-OH (where * indicates a bond position), or n is a number of 1 or more and X is *-N(CH ３ ) ２ The copolymer according to claim 1, or *-COOH (wherein * indicates a bond position).

5. The copolymer according to claim 1, further comprising a repeating unit C represented by the following formula (3): (In the formula, R ３ represents a hydrogen atom or a methyl group, and Y is represented by the following formulas (4) to (8): The base is represented by one of the following, where m is an integer from 3 to 12, and R ４ R represents a hydrogen atom or a methyl group. ５ R represents a methyl group, a methoxy group, or an ethoxy group. ６ and R ７ Each of these independently represents either a methoxy group or an ethoxy group, and * indicates the bond position.

6. The copolymer according to claim 5, having a repeating unit A represented by the following formula (1a), a repeating unit B represented by the following formula (2a), and a repeating unit C represented by the following formula (3a): (In the formula, n-Bu represents a butyl group, and * represents a bond position.) 7. The copolymer according to claim 5, having a repeating unit A represented by the following formula (1a), a repeating unit B represented by the following formula (2b), and a repeating unit C represented by the following formula (3a): (In the formula, n-Bu represents a butyl group, and * represents a bond position.) 8. A cell separation substrate having a base material and a layer formed on the base material from a copolymer according to any one of claims 1 to 7.

9. The cell separation substrate according to claim 8, wherein the material of the substrate is glass, silicone, synthetic resin, or synthetic rubber.

10. The cell separation substrate according to claim 8, wherein the thickness of the layer formed from the copolymer is 1 nm to 1000 nm.

11. A method for separating target cells from a group of cells containing target cells, comprising: step A, bringing a group of cells containing target cells into contact with a layer formed from a copolymer of the cell separation substrate described in claim 8 at a temperature higher than the lower critical solution temperature (LCST) of the copolymer to adhere the target cells to the layer; step B, washing the layer to which the target cells have adhered at a temperature higher than the lower critical solution temperature (LCST) of the copolymer; and step C, cooling the cell separation substrate having the washed layer to a temperature lower than the lower critical solution temperature (LCST) of the copolymer to detach and recover the target cells from the layer.

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