JP2017112910A - Cell separation material and use therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell separation material for selectively separating cells having CaSR on the surface from a cell containing sample such as body fluid.SOLUTION: The cell separating material comprises a substrate and an aminoglycoside antibiotic linked to the substrate, more preferably the substrate and the antibiotic are linked via a linker. A method for separating cells using the cell separating material is also provided.SELECTED DRAWING: None

Description

  The present invention relates to a cell separation material capable of selectively separating cells from a cell-containing sample such as a body fluid, a method for producing the cell separation material, and a cell separation method and a differentiation induction method using the cell separation material.

  In recent years, cell therapy for separating cells contained in body fluids such as blood and bone marrow and transplanting them for the purpose of tissue regeneration such as blood vessels and immunostimulation of cancer patients has been put into practical use, and cells useful for treatment have been selected selectively Various materials and devices for separation have been developed.

  As a method for fractionating cells, a specific gravity density gradient centrifugation method using a difference in specific gravity of cells is known.

  A method for separating cells using an antibody against a protein present on the surface of a target cell has also been developed. For example, a method is known in which a magnetic bead in which an antibody specific to the surface protein of a target cell is linked is brought into contact with a cell-containing sample, and the magnetic bead is recovered using magnetism.

  A method for separating cells using a cell separation filter has also been reported (Patent Documents 1 to 3). For example, Patent Document 3 discloses that in a method for separating and collecting cells using a nonwoven fabric filter, the capturing property of target cells (monocytes) is improved by defining the packing density and fiber diameter of the nonwoven fabric filter. .

  Furthermore, techniques for imparting chemical characteristics to physical cell separation filters have been developed (Patent Documents 4 to 8). For example, in Patent Document 4, cells existing in blood (particularly activated leukocytes such as granulocytes and monocytes), cancer cells, etc. are removed by an adsorbent carrier containing fibers having a specific structure with amino groups arranged on the surface. Is disclosed.

  On the other hand, it is known that calcium sensing receptor (CaSR) is expressed on the surface of human monocytes (Non-patent Document 1). In addition, it is conventionally known that CaSR is also expressed on the surface of human mesenchymal stem cells. Non-Patent Document 2 reports divalent metal ions, polyamines, aminoglycoside antibiotics and the like as ligands for CaSR, and specific examples of aminoglycoside antibiotics include neomycin, gentamicin, and tobramycin.

  Patent Document 9 discloses a method for producing a physiologically active substance-immobilized molded article in which a physiologically active substance having an amino group such as a polypeptide antibiotic or an aminoglycoside antibiotic is immobilized on a molded article carrier. Polymyxin B is disclosed as a polypeptide antibiotic. In Patent Document 9, a carrier having a functional group capable of chemically immobilizing a physiologically active substance having an amino group is used as a molded article carrier serving as a base material for fixing an antibiotic, and the physiological activity having the functional group and the amino group is used. It is disclosed that a physiologically active substance is immobilized on a molded article carrier by reaction with the substance. This physiologically active substance-immobilized molded article is described as being used for applications such as therapeutic blood purification agents, antibacterial agents, therapeutic imino modulators, and affinity chromatographic adsorbents.

  The applicant of Patent Document 9 also discloses, in Patent Document 10, as in Patent Document 9, a material that removes or detoxifies toxins derived from Gram-negative bacteria, and a material that removes or detoxifies toxins derived from Gram-positive bacteria. A liquid processing column comprising It is described that the material for removing or detoxifying toxins derived from Gram-negative bacteria contains polymyxin B or a polyamino compound, and that the material for removing or detoxifying toxins derived from Gram-positive bacteria contains urea bonds.

  Patent Document 11 describes an insoluble carrier on which a chemotherapeutic agent that inhibits protein synthesis such as aminoglycoside antibiotics is immobilized. In Patent Document 11, a carrier binding method or the like is used as a method for binding a chemotherapeutic agent (ligand) to a carrier, and the carrier and the ligand may be directly bonded or may be bonded via a spacer. Is described.

WO98 / 32840 JP-A-9-75076 JP 2004-129550 A JP 2008-80114 A JP 58-74611 A JP 2007-53950 A JP 2006-312804 A JP 2007-31737 A JP-A-5-305139 Japanese Patent Laid-Open No. 10-225515 JP 7-124252 A

Plos One, vol. 8, Issue 10, e74800, 2013 Molecular Pharmacology, vol. 76: 1131-1144, 2009

  The existing cell separation means listed in the background art are not always satisfactory for the following reasons.

  The specific gravity density gradient centrifugation method cannot separate a plurality of types of cells with similar specific gravity and needs to be used in combination with other cell separation means as necessary.

  Separation of cells using a complex of antibody and magnetic beads is an excellent method in that the target cells can be selectively obtained. However, since the binding force between the cells and the antibody is very strong, cell recovery is possible. There is a problem that is not easy. In addition, the treatment of cells takes a long time, is expensive, and when recovered cells are used for treatment, the risk of remaining magnetic beads needs to be eliminated. .

  The cell separation filters described in Patent Documents 1 to 3 are physical filtration filters, and cannot be said to be suitable for selectively collecting target cells.

  Patent Documents 4 to 8 disclose various cell separation materials to which chemical characteristics are added, but the number of cells to be separated is limited, and there is still a need for development according to the cells to be separated.

  Cells having differentiation ability such as monocytes and mesenchymal stem cells are expected to be applied to regenerative medicine. Therefore, a technique for selectively separating cells having differentiation potential such as monocytes and mesenchymal stem cells is required. Monocytes, mesenchymal stem cells and the like are known to express calcium-sensing receptor (CaSR) on the surface, but cells having CaSR on the surface are selectively separated using a CaSR-specific ligand. No attempt has been made in the past, and it is unclear whether or not selective separation of cells will succeed. Of course, it was also unclear what ligands would be suitable for the selective separation of these cells.

  Non-Patent Document 2 lists aminoglycoside antibiotics as agonists that activate CaSR, but aminoglycoside antibiotics can be used for specific capture of cells expressing CaSR on the cell surface. It has not been studied in the past. The fact that the aminoglycoside antibiotic acts as an agonist for CaSR is independent of whether the binding ability between the aminoglycoside antibiotic and CaSR is available for cell capture. In addition, aminoglycoside antibiotics are known to be incorporated into bacterial cells from cell membranes and inhibit protein synthesis systems, and as described in Non-Patent Document 2, aminoglycoside antibiotics are nephrotoxic. Since it is known to have (nephrotoxicity), it is not easily conceivable by those skilled in the art to use antibiotics for the purpose of separating cells for the purpose of regenerative medicine.

  On the other hand, Patent Documents 9 to 11 disclose carriers on which antibiotics are immobilized, but these carriers are not intended to adsorb cells as described below.

  Patent Document 9 describes that a physiologically active substance-immobilized molded article in which a physiologically active substance having an amino group such as an aminoglycoside antibiotic is immobilized on a molded article carrier as described above is used as a blood purification agent for treatment. . The therapeutic blood purification agent in Patent Document 9 is for neutralizing and detoxifying endotoxin that can be contained in therapeutic blood with an antibiotic contained in a physiologically active substance-immobilized molded article, and adsorbs cells in the blood. It doesn't let you. The use as an antibacterial agent in Patent Document 9 is also unrelated to cell adsorption. Patent Document 9 does not describe an experimental example in which blood is treated with a physiologically active substance-immobilized molded article having an aminoglycoside antibiotic.

  The applicants of Patent Documents 9 and 10 cited the liquid processing column of Patent Document 10 as background art in Patent Document 4 by themselves, and the liquid processing column of Patent Document 10 adsorbs toxins and cytokines. It is described that cell adsorption is not intended. From this, it is not easy for those skilled in the art to use the liquid processing column described in Patent Document 10 to adsorb cells.

  It is described that an insoluble carrier on which a chemotherapeutic agent that inhibits protein synthesis such as aminoglycoside antibiotics disclosed in Patent Document 11 is immobilized can adsorb negatively charged physiologically active substances such as DNA and RNA. However, there is no description about adsorbing cells. Patent Document 11 describes that a chemotherapeutic agent (ligand) and an insoluble carrier may be bound via a spacer, but the specific structure of the spacer is not disclosed.

  As described above, a cell separation material for selectively separating cells having CaSR on the surface thereof such as monocytes and mesenchymal stem cells has not been provided conventionally. In order to capture, separate, and collect cells with a cell separation material, appropriate binding (affinity) between the cells and the cell separation material is required. When the binding property between the cell and the cell separation material is too weak, the separation and adsorption of the cell is insufficient, and when the binding property is too strong, it becomes difficult to collect and detach the cells captured by the cell separation material. When the cells and the cell separation material have appropriate binding properties, the cell recovery rate can be increased. In addition, it is important not to damage the cells (for example, adverse effects on the cell viability, the therapeutic effect using the cells, etc.) in the process of capturing and collecting the cells by the cell separation material. No cell separation material that satisfies this requirement for cells having CaSR on the surface has been conventionally provided.

  Therefore, the present invention provides a cell separation material capable of selectively separating cells having CaSR on the surface from a cell-containing sample, and a cell separation method and differentiation induction method using the cell separation material. Providing is a problem to be solved.

  As a result of intensive studies, the inventors have obtained cells, particularly cells having a structure having an affinity for the antibiotic on the surface, by using a cell separation material containing the substrate and an aminoglycoside antibiotic linked to the substrate. The inventors have found that the cells can be selectively separated, and that when the separated cells are cells having differentiation ability, the differentiation ability is maintained and differentiation can be induced, and the present invention has been achieved. As mentioned above, there is no prior art suggesting that aminoglycoside antibiotics can be used for selective separation of cells with CaSR on the surface, but rather aminoglycoside antibiotics are used for cell separation because of their toxicity. In view of the fact that there is an inhibiting factor, the present invention should be a particularly remarkable invention exceeding the expectation of those skilled in the art.

Specifically, the present invention provides the following inventions.
(1) A cell separation material comprising a substrate and an aminoglycoside antibiotic linked to the substrate.
(2) The cell separation material according to (1), wherein the base material and the aminoglycoside antibiotic are linked via a linker.
(3) The cell separation material according to (2), wherein the base material and the linker are linked by a covalent bond, and the linker and the aminoglycoside antibiotic are linked by a covalent bond.
(4) The linker is a polymer,
The substrate is connected to a side chain of the polymer, and
The cell separation material according to (2) or (3), wherein the aminoglycoside antibiotic is linked to a side chain of the polymer.
(5) The cell separation material according to any one of (2) to (4), wherein the linker is a polymer including a main chain composed of carbon atoms linked by a carbon-carbon single bond.
(6) The linker is
A repeating unit 1 comprising a linking group linked to the substrate;
Any one of (2) to (5), which is a polymer comprising a repeating unit 2 containing a group containing the aminoglycoside antibiotic, wherein the repeating unit 1 and the repeating unit 2 are randomly copolymerized. The cell separation material described.

（式Ｉ中
Ｒ^１１は、水素、アルキル基、芳香族基又は芳香族アルキル基であり、
Ｒ^１２は、水素、アルキル基、芳香族基又は芳香族アルキル基であり、
Ｒ^１３は、水素、アルキル基、芳香族基又は芳香族アルキル基であり、
Ｌ^１４は、水素以外の原子の数が１以上で価数ｐの基であり、ここでｐは２以上４以下の整数であり、
Ｒ^１５は、基材に連結した連結基である）
で表される、（２）〜（６）のいずれかに記載の細胞分離材。 (7) The linker is
A repeating unit 1 comprising a linking group linked to the substrate;
A polymer comprising a repeating unit 2 containing a group containing the aminoglycoside antibiotic,
Said repeat unit 1 is represented by formula I:

(In Formula I, R ¹¹ is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group;
R ¹² is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group;
R ¹³ is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group,
L ¹⁴ is a group having 1 or more atoms other than hydrogen and a valence p, wherein p is an integer of 2 or more and 4 or less,
R ¹⁵ is a linking group linked to the substrate)
The cell separation material according to any one of (2) to (6), which is represented by:

（式ＩＩ中
Ｒ^２１は、水素、アルキル基、芳香族基又は芳香族アルキル基であり、
Ｒ^２２は、水素、アルキル基、芳香族基、芳香族アルキル基又はカルボキシ基であり、
Ｒ^２３は、水素、アルキル基、芳香族基又は芳香族アルキル基であり、
Ｌ^２４は、水素以外の原子の数が１以上で価数ｑの基であり、ここでｑは２以上４以下の整数であり、
Ｒ^２５は、アミノグリコシド系抗生物質を含む基である）
で表される、（２）〜（７）のいずれかに記載の細胞分離材。 (8) The linker is
A repeating unit 1 comprising a linking group linked to the substrate;
A polymer comprising a repeating unit 2 containing a group containing the aminoglycoside antibiotic,
The repeating unit 2 is represented by the formula II:

(In Formula II, R ²¹ represents hydrogen, an alkyl group, an aromatic group, or an aromatic alkyl group;
R ²² is hydrogen, an alkyl group, an aromatic group, an aromatic alkyl group or a carboxy group,
R ²³ is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group,
L ²⁴ is a group having 1 or more atoms other than hydrogen and a valence of q, where q is an integer of 2 or more and 4 or less,
R ²⁵ is a group containing an aminoglycoside antibiotic)
The cell separation material according to any one of (2) to (7), represented by:

(9) A method for producing a cell separation material according to any one of (2) to (8),
A substrate;
A linker comprising a linking group connectable to the substrate and a reactive group capable of forming a bond with the aminoglycoside antibiotic,
A linker linking step of linking via the linking group to form a substrate-linker conjugate;
The cell separation material according to any one of (2) to (8), wherein the reactive group in the substrate-linker conjugate is reacted with an aminoglycoside antibiotic to bind the aminoglycoside antibiotic to the linker. Forming an antibiotic binding step.
(10) In the linker linking step, the linking group and the substrate are linked by a covalent bond,
The method according to (9), wherein in the antibiotic binding step, the reactive group and the aminoglycoside antibiotic are linked by a covalent bond.
(11) The method according to (9) or (10), wherein the linker before linking to the base material is a polymer containing the linking group and the reactive group in a side chain.
(12) The linker according to any one of (9) to (11), wherein the linker before being connected to the base material is a polymer including a main chain composed of carbon atoms connected by a carbon-carbon single bond. the method of.
(13) The linker before being connected to the substrate is
Repeating unit 1 containing the linking group;
The method according to any one of (9) to (12), which is a polymer comprising a repeating unit 2 containing the reactive group, wherein the repeating unit 1 and the repeating unit 2 are randomly copolymerized.

（式Ｉ’中
Ｒ^１１は、水素、アルキル基、芳香族基又は芳香族アルキル基であり、
Ｒ^１２は、水素、アルキル基、芳香族基又は芳香族アルキル基であり、
Ｒ^１３は、水素、アルキル基、芳香族基又は芳香族アルキル基であり
Ｌ^１４は、水素以外の原子の数が１以上で価数ｐの基であり、ここでｐは２以上４以下の整数であり、
Ｒ^１６は、基材に連結可能な連結性基である）
で表される、（９）〜（１３）のいずれかに記載の方法。 (14) The linker before being connected to the substrate is
Repeating unit 1 containing the linking group;
A polymer comprising the repeating unit 2 containing the reactive group,
The repeating unit 1 is represented by the formula I ′:

(In Formula I ′, R ¹¹ represents hydrogen, an alkyl group, an aromatic group, or an aromatic alkyl group;
R ¹² is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group;
R ¹³ is hydrogen, an alkyl group, an aromatic group, or an aromatic alkyl group. L ¹⁴ is a group having 1 or more atoms other than hydrogen and a valence p, where p is 2 or more and 4 or less. An integer,
R ¹⁶ is a linking group that can be linked to a substrate)
The method in any one of (9)-(13) represented by these.

（式ＩＩ’中
Ｒ^２１は、水素、アルキル基、芳香族基又は芳香族アルキル基であり、
Ｒ^２３は、水素、アルキル基、芳香族基又は芳香族アルキル基であり、
Ｒ^２６は、水素、アルキル基、芳香族基又は芳香族アルキル基であり、
Ｒ^２７は、式ＩＩＩ

（式ＩＩＩ中、
Ｌ^２４は、水素以外の原子の数が１以上で価数ｑの基であり、ここでｑは２以上、４以下の整数であり、
Ｒ^２８は、前記アミノグリコシド系抗生物質により置換可能な脱離基又は前記アミノグリコシド系抗生物質が有する官能基と結合を形成可能な反応性基であり、
＊は結合を示す）
で表される基であり、
或いは、
Ｒ^２６とＲ^２７とは一体となって式ＩＶ

（式ＩＶ中、＊は、結合を示す）
で表される二価の基を形成している）
で表される、（９）〜（１４）のいずれかに記載の方法。 (15) The linker before being connected to the substrate is
Repeating unit 1 containing the linking group;
A polymer comprising the repeating unit 2 containing the reactive group,
Said repeating unit 2 is of formula II ′:

(In Formula II ′, R ²¹ represents hydrogen, an alkyl group, an aromatic group, or an aromatic alkyl group;
R ²³ is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group,
R ²⁶ is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group,
R ²⁷ is a compound of formula III

(In Formula III,
L ²⁴ is a group having 1 or more atoms other than hydrogen and a valence of q, where q is an integer of 2 or more and 4 or less,
R ²⁸ is a leaving group displaceable by the aminoglycoside antibiotic or a reactive group capable of forming a bond with a functional group of the aminoglycoside antibiotic,
* Indicates binding)
A group represented by
Or
R ²⁶ and R ²⁷ together form Formula IV

(In formula IV, * represents a bond)
A divalent group represented by
The method in any one of (9)-(14) represented by these.

(16) A method for separating cells in a sample,
(1) The method including the cell capture | acquisition process which makes the cell separation material in any one of (8) and a sample contact, and capture | acquires the cell in the said sample with the said cell separation material.
(17) A method for inducing differentiation of a cell having differentiation ability,
A cell separation step of separating cells having differentiation ability from the method according to (16);
A differentiation induction step of inducing differentiation of the cells separated in the cell separation step.

  Since the cell separation material of the present invention can selectively capture cells having CaSR on the surface and can collect the captured cells, selective separation of cells having CaSR on the surface is possible. Is possible.

  According to the method for producing a cell separation material of the present invention, it is possible to produce a cell separation material having a structure in which a base material and an aminoglycoside antibiotic are linked via a linker in a simple process.

  According to the cell separation method of the present invention, cells having CaSR on the surface can be easily and selectively separated from the cell-containing sample.

  In the method for inducing differentiation of a cell having differentiation ability according to the present invention, damage to the cell in the cell separation step can be reduced, so that differentiation of the cell can be efficiently induced.

In Experiment 2, XPS (Kratos AXIS-ULTRA DLD) was measured for the cell culture dish on which the polymer was immobilized, and the result of confirming the immobilization of neomycin is shown. FIG. 1 (A) shows the peak intensity of N (1s) in a cell culture dish in which the polymer is immobilized but the polymer is not modified with neomycin, and FIG. 1 (B) shows the polymer immobilized. The peak intensity of N (1 s) of a cell culture dish in which the polymer is modified with neomycin is shown.

Hereinafter, the present invention will be described in detail.
<Aminoglycoside antibiotics>
An aminoglycoside antibiotic is a general term for glycoside antibiotics containing amino sugar or aminocyclitol, and does not include antibiotic groups such as macrolides, nucleosides and anthracyclines. The present inventors have surprisingly been able to specifically capture cells by linking aminoglycoside antibiotics onto a substrate, and that the captured cells are highly efficient in inducing differentiation without being harmed by antibiotics. The inventors have found that this is possible and have completed the present invention.

  The aminoglycoside antibiotic is preferably at least one selected from the group consisting of neomycin, gentamicin, kanamycin, streptomycin and tobramycin, more preferably at least one selected from the group consisting of neomycin, gentamicin and tobramycin. One or more selected are particularly preferred. These antibiotics have moderate affinity with cells having CaSR on the surface, can capture the cells using a cell separation material containing the antibiotics, and easily collect the captured cells The following antibiotics are more preferable from this viewpoint.

  The neomycin may be any of neomycin A (= neamine), neomycin B (= fraradiomycin B, streptosricin B) and neomycin C (= fraradiomycin C), and a mixture of two or more of these. However, it is preferable to use at least one of neomycin B and neomycin C, and it is particularly preferable to use neomycin B.

Gentamicin C is particularly preferable as gentamicin. Gentamicin C includes at least one selected from the group consisting of gentamicin C ₁ , gentamicin C ₂ and gentamicin C _1a, and may be a mixture of two or more.

It is particularly preferable to use kanamycin A as kanamycin.
The aminoglycoside antibiotic may be linked to the base material in any form such as ion, salt, esterified product, amidated product and the like.

  The aminoglycoside antibiotic is preferably linked to the substrate in the form of a monovalent group in which one hydrogen atom is replaced by a bond in one of an amino group or a hydroxyl group. The aminoglycoside antibiotic may be directly connected to the base material, but more preferably is connected to the base material via a linker described later.

  In the present invention, the connection between the base material and the aminoglycoside antibiotic refers to the connection between the base material and the aminoglycoside antibiotic by chemical bonding. Examples of the chemical bond include one or more selected from the group consisting of a covalent bond, an ionic bond, a hydrogen bond, a brine bond and a coordination bond, and a plurality of types of bonds may be combined. In order to suppress the elimination of the antibiotic from the base material, a covalent bond with particularly high binding stability is preferred.

In the cell separation material of the present invention, the density of aminoglycoside antibiotics on the substrate is not particularly limited and can be appropriately adjusted according to the purpose. In a preferred embodiment of the cell separation material of the present invention, the aminoglycoside antibiotic is preferably 0.02 μmol / cm ² or more and 200 μmol / cm ^{2 in the} region of the surface of the substrate where the aminoglycoside antibiotic is linked. Or less, more preferably 0.05 μmol / cm ² or more, 0.10 μmol / cm ² or more, 0.11 μmol / cm ² or more, 0.12 μmol / cm ² or more, 0.13 μmol / cm ² or more, 0.14 μmol / cm ² or more, 0.15 μmol / cm ² or more, 0.16 μmol / cm ² or more, 0.17 μmol / cm ² or more, 0.18 μmol / cm ² or more, 0.19 μmol / cm ² or more, 0.20 μmol / cm ² or more , and the addition 190μmol / cm ² or less, 180μmol / cm ² or less, 170Myumol cm ² or less, 160 [mu] mol / cm ² or less, 150 [mu] mol / cm ² or less, 140μmol / cm ² or less, 130μmol / cm ² or less, 120 [mu] mol / cm ² or less, 110μmol / cm ² or less, 100 [mu] mol / cm ² or less, 90 [mu] mol / cm ² hereinafter, 80μmol / cm ² or less, 70μmol / cm ² or less, 60 [mu] mol / cm ² or less, 50 [mu] mol / cm ² or less, 40 [mu] mol / cm ² or less, 30 [mu] mol / cm ² or less, and is connected at a density of 20 [mu] mol / cm ² or less . The cell separation material of the present invention can capture cells particularly efficiently within the above numerical range.

<Base material>
The substrate used for the cell separation material of the present invention preferably has a surface property capable of reacting with a group possessed by an aminoglycoside antibiotic or a linking group possessed by a linker to form a chemical bond. Specifically, a hydroxyl group, a carboxy group, a carbonyl group, an aldehyde group, a thiol group, an epoxy group, a primary to quaternary amino group, a -SiH group, a vinyl group, Au, or other metal element, avidin It is preferable that a functional group such as a containing group or a streptavidin-containing group is present because it is easy to form a chemical bond with the linking group of the linker, and in particular, a silicon-containing group and a —Si—O— bond, which will be described later, etc. Therefore, it is preferable that a hydroxyl group, an epoxy group, an amino group, and a carboxy group exist on the surface of the substrate.

  In order to introduce a hydrophilic group such as a hydroxyl group, a carboxy group, a carbonyl group, an aldehyde group, a thiol group, an epoxy group, a primary to quaternary amino group, or a vinyl group on the surface of the substrate, for example, in an ozone atmosphere Ultraviolet irradiation treatment, plasma treatment (eg, treatment with oxygen plasma, nitrogen plasma, or a mixture thereof), high energy ray irradiation treatment (eg, gamma ray, electron beam irradiation treatment), chemical oxidation treatment using an oxidizing agent, etc. Can be used. SiH groups can be introduced onto the surface of a substrate by subjecting the surface of the substrate containing silicon, such as glass or various types of silicon-coated substrates, to hydrogen plasma treatment. Further, metal elements such as Au, avidin-containing groups, and streptavidin-containing groups can be introduced onto the substrate surface by methods known to those skilled in the art. These functional groups can also be introduced by grafting and bonding molecules containing the functional group to the surface of the base material into which the radical has been introduced. Examples of the method for introducing radicals on the surface of the substrate include a method of irradiating the surface of the substrate with a high energy beam such as an electron beam. For example, after irradiating a fiber material with an electron beam, after immersing it in a radical polymerizable compound solution in which a radical polymerizable compound is dissolved in a solvent to give the fiber material a radical polymerizable compound, a film is applied to both sides of the fiber material. The method of adhering and polymerizing is mentioned. Examples of the molecule containing a functional group include a molecule containing a radical reactive group such as a vinyl group. The molecule may contain the functional group before being bonded to the substrate surface, or may be further bonded to the functional group after being bonded to the substrate surface.

  When using a base material containing a synthetic resin such as polyolefin or polyester as the base material, examples of the functional group to be introduced onto the surface of the base material include a carbonyl group, a carboxy group, and a hydroxyl group. Can be introduced. For a substrate containing polyolefin, it is relatively easy to introduce a radical containing a functional group by grafting and bonding a molecule containing a functional group after introducing a radical by irradiation with high energy rays.

  When using a base material containing a polysaccharide such as cellulose on the surface as the base material, a hydroxyl group that is abundant in the polysaccharide can be used for the reaction with the antibiotic or the linker. In addition, as a method for introducing a functional group into a substrate containing a polysaccharide, the functional group is introduced through a covalent bond such as an ester bond or a silyl ether bond (Si—O—C) bond. Examples thereof include a method of binding a molecule further containing a functional group capable of binding to a hydroxyl group possessed by a polysaccharide to a hydroxyl group possessed by the polysaccharide.

  Examples of the form of the substrate of the cell separation material of the present invention include a substrate having at least a part of a surface, such as a dish, flask, plate, bag, cartridge, cassette, or a structure having a communication hole. The form is not particularly limited. For example, when differentiation is induced in a state where cells having differentiation ability are captured, a substrate containing at least a part of the surface portion can be preferably used, and the target cells are concentrated from the cell-containing sample. In this case, a structure having a communication hole can be preferably used, and the form of the base material of the cell separation material may be appropriately designed according to the purpose of those skilled in the art.

  The base material including at least a part of the surface part is a base material including at least a part (surface part) having a surface having a two-dimensional extension. The surface having a two-dimensional extension may be a flat surface, a curved surface, or a combination of a flat surface and a curved surface, but is preferably a flat surface. Here, the plane may be anything that can be regarded as a plane. The surface having a two-dimensional spread is preferably a surface spread so as to include a square having a side of 1 cm or more, for example, 2 cm or more when viewed from a direction perpendicular to the surface. For example, when the substrate is in the shape of a container (including a plate-like container), the inner surface of the container in contact with the cell-containing sample, specifically, the inner surface of the container for cell processing such as a dish or flask (bottom surface, side wall surface, etc.) At least part of the inner surface of the cell-containing sample mounting surface in the plate, the bag, the cartridge, or the cassette is an example of the surface having the two-dimensional extension. When using a base material containing at least a part of a surface part as the base material of the cell separation material of the present invention, an aminoglycoside antibiotic is linked to at least the surface of the surface part having the two-dimensional extension. It is preferable.

  When the base material of the cell separation material of the present invention is a base material including at least a part of the surface part, the form and size of the surface part are not particularly limited. Although it does not specifically limit as a material, Nonreactive polymer, a metal, glass etc. are used preferably. Alternatively, a material coated or plated with these non-reactive polymers, metal or glass may be used. Specifically, as the polymer, acrylonitrile polymers such as acrylonitrile butadiene styrene terpolymer, halogenated polymers such as polytetrafluoroethylene, polychlorotrifluoroethylene, copolymer tetrafluoroethylene or hexafluoropropylene, polyamide, polysulfone, polycarbonate, Examples thereof include polyethylene, polypropylene, polyvinyl chloride acrylic copolymer, polycarbonate acrylonitrile butadiene styrene, styrene butadiene copolymer, and polystyrene. Examples of the metal include stainless steel, titanium, platinum, tantalum, gold, or an alloy thereof, as well as gold-plated alloy iron, platinum-plated alloy iron, cobalt chromium alloy, or titanium nitride-coated stainless steel. The alloy is preferred. The glass may be silicon coated glass. Particularly preferred are materials having sterilization resistance, and specific examples include silicon-coated glass, polystyrene, polypropylene, polyvinyl chloride, polycarbonate, polysulfone, and polymethylpentene.

  In addition, when the base material of the cell separation material of the present invention is a base material including at least a part of a surface portion, a material having a gas permeable structure at least on the surface portion can be preferably used. Examples of the material having a gas permeable structure include a material having a fine hole structure and a slit structure capable of suppressing contamination and a material having a high gas permeability. Examples of the material having high gas permeability include polymethylpentene, cyclic polyolefin, olefin-based thermoplastic elastomer, styrene-based thermoplastic elastomer, polyamide-based thermoplastic elastomer, etc., but the present invention is not limited thereto. Absent.

  The “structure having communication holes” that can be used as a substrate may be a structure having communication holes that allow liquid such as water to pass through. For example, a fiber aggregate, a granular aggregate, and a porous structure. Examples of the aggregate of fibers include nonwoven fabrics, woven fabrics, felt-like structures formed by gathering fibers, and cotton-like structures. In the fiber assembly, communication holes are formed between the fibers. In the aggregate of granules, communication holes may be formed at least between the granules, and further, the inside of each particle may be porous and the communication holes may be formed inside. Examples of the porous body include those in which a large number of communication holes are formed in a tissue formed by a material exemplified later (for example, a sponge-like structure). In the structure having a communication hole, the hole diameter of the communication hole is not particularly limited, and as an embodiment having a hole diameter through which target cells to be captured and separated and other cells contained in the sample can pass, This is preferable because it enables selective capture and separation of cells by affinity with the target cells. The diameter of the communication hole can be, for example, 9 μm or more and 80 μm or less. Here, the “hole diameter” of the communication hole means that the cross-section of the structure can be observed by using a magnifying observation means such as a scanning electron microscope, along the direction substantially perpendicular to the length direction of the communication hole. The diameter of the cross section of the communication hole (the shortest distance among a pair of opposing points on a straight line passing through the center of gravity of the cross section on the periphery of the cross section, that is, the short diameter). An average value of the above pore diameters obtained by observing an appropriate number (eg, 10 or more, 20 or more, or 30 or more) of the cross-sections with the magnification observation means and comparing with an appropriate scale (for example, a scale on an observation image) ( That is, the average hole diameter) can be regarded as the hole diameter of the communication hole. The structure having the communication hole can be formed into various shapes, and the overall shape is not particularly limited. For example, the structure can be a sheet shape such as a disk shape, a cylindrical shape, a pipe shape, or the like.

  When the base material of the cell separation material of the present invention is a structure having a communicating hole, the material is not particularly limited. For example, polyurethane, polystyrene, polyolefin (for example, ethylene, propylene, 1-butene, Α-olefin homopolymers such as 1-pentene or two or more kinds of copolymers), polyacrylamide, polyester (for example, polybutylene terephthalate, polyethylene terephthalate), nylon, polyimide, aramid, polyamide, acrylic resin, etc. Synthetic polymers, chitin, chitosan, cellulose acetate, cellulose, rayon, agarose, alginic acid and other natural polymers, hydroxyapatite, glass, alumina and other inorganic materials, stainless steel, titanium and other metals Etc.

In an embodiment in which the structure having communication holes is an aggregate of fibers, the average fiber diameter is, for example, 50 μm or less, 40 μm or less, 30 μm or less, 20 μm or less, 10 μm or less, such as 1 μm or more, preferably 3 μm or more. it can. When the average fiber diameter is within this range, cells can be easily captured. The average fiber diameter is determined by, for example, observing the fiber or the aggregate of fibers with a magnification observation means such as a scanning electron microscope, and an appropriate number in the observation image, for example, 10 or more, 20 or more, or 30 or more locations (fiber end portions). In other words, the width of the fiber is obtained by comparison with an appropriate scale (for example, the scale on the observation image), and the average value thereof is obtained. The density of the fiber assembly is not particularly limited, but for efficient cell capture, for example, 1 × 10 ⁴ g / m ³ or more, 2 × 10 ⁴ g / m ³ or more, 3 × 10 ⁴ g / m ³ or more, 4 × 10 ⁴ g / m ³ or more, 5 × 10 ⁴ g / m ³ or more, 6 × 10 ⁴ g / m ³ or more, 7 × 10 ⁴ g / m ³ or more, 8 × 10 ⁴ g / m ³ or more, 9 × 10 ⁴ g / m ³ or more, for example, 1 × 10 ⁶ g / m ³ or less, 9 × 10 ⁵ g / m ³ or less, 8 × 10 ⁵ g / m ³ or less, 7 × 10 ⁵ g / M ³ or less, 6 × 10 ⁵ g / m ³ or less, 5 × 10 ⁵ g / m ³ or less, 4 × 10 ⁵ g / m ³ or less, 3 × 10 ⁵ g / m ³ or less, 2 × 10 ⁵ g / M ³ or less, and 1 × 10 ⁵ g / m ³ or less. As the material constituting the fiber, those exemplified above as the material of the structure having communication holes can be used, but polyolefin, polyester, polybutylene terephthalate, and polyamide are particularly used as existing cell separation filters. Therefore, it is preferable from the viewpoint of safety. In addition, as the aggregate of fibers used as the base material, non-woven fabric is particularly preferable from the viewpoint of safety because it has been used in many cases as an existing cell separation filter.

  In an embodiment in which the structure having communication holes is an aggregate of granules, the particle size of the granules is preferably 10 μm or more and 2000 μm or less, and more preferably 70 μm or more and 1000 μm or less in terms of processing efficiency. Here, the “particle size” of a particle means the diameter of an observation image observed when each individual particle is observed with a magnification observation means such as a scanning electron microscope (the observation image on the periphery of the observation image). The shortest distance among a pair of opposing points on a straight line passing through the center of gravity, that is, the short diameter). An average value of the above-mentioned particle diameters obtained by observing an appropriate number, for example, 10 or more, 20 or more, or 30 or more of the particles by the magnification observation means and comparing with an appropriate scale (for example, a scale on an observation image) (That is, the average particle diameter) can be regarded as the particle diameter of the granules. Examples of the material for the particles include inorganic materials such as glass beads and silica gel, crosslinked polyvinyl alcohol, crosslinked polyacrylate, crosslinked polyacrylamide, crosslinked polystyrene, and ethylene in addition to those exemplified as the material for the structure having communication holes. -Synthetic resin materials such as saponified vinyl acetate copolymer, crosslinked polyacrylic acid, crosslinked polymethacrylic acid, polymethyl methacrylate, polyacrylamide grafted polyethylene, crystalline cellulose, crosslinked cellulose, cellulose derivatives, crosslinked agarose, crosslinked dextrin , Polysaccharides such as chitin and chitosan, and organic-organic and organic-inorganic composite materials obtained by a combination thereof, but are not limited thereto.

<Linker>
The base and the aminoglycoside antibiotic are preferably linked via a linker. Through the linker, the distance from the surface of the base material of the antibiotic that becomes a capture point where the cells are captured becomes large, and the range in which the antibiotic that captured the cells can move is widened. It is considered that the steric hindrance at the time of binding to the protein is reduced, and the cells can be efficiently captured by the antibiotic.

  The structure of the linker is not particularly limited. Preferably, the linker has a structure in which at least one bond to the base material and at least one aminoglycoside antibiotic are bonded in one molecule of the linker. Has a structure in which one linker molecule has a plurality of bonds to the substrate and a plurality of the aminoglycoside antibiotics. Since the linker and the antibiotic are firmly bound to the base material by binding one molecule of the linker to the base material at a plurality of locations, the risk that they are separated from the base material and mixed into the cells to be separated can be reduced. . Moreover, it is possible to arrange | position antibiotics on a base-material surface with a moderate density by setting it as the structure which couple | bonds the said antibiotics with 1 linker molecule.

  The type of bond between the linker and the substrate is not particularly limited as long as it is one of the above-described chemical bonds, but is preferably a covalent bond because the bond stability is high. Similarly, the bond between the linker and the antibiotic is not particularly limited as long as it is one of the chemical bonds described above, but is preferably a covalent bond because of high stability of the bond.

  The linker may be a chemical structure formed by a reaction on the surface of the base material, or may be prepared by binding a separately prepared compound to the base material and an antibiotic, more preferably the latter. In some cases, the separately prepared compound is referred to as “linker compound” or “linker precursor” for convenience.

  The linker compound usually has at least a linking group that can be linked to the substrate and a reactive group that can form a bond with the antibiotic in a state before being bonded to the substrate and the antibiotic. A compound containing one. The linker compound contains a linking group, and when the linker compound forms a substrate-linker conjugate with the substrate, the linking group is linked to a functional group on the substrate surface to form a bond. In the present invention, a structure in which a connecting group is connected to a base material to change the structure is referred to as a “linking group”. Similarly, the linker compound before binding to the antibiotic contains a reactive group, and the reactive group changes its structure when it forms a bond with the group of the antibiotic.

The linking group possessed by the linker compound is not particularly limited as long as it can be linked to the substrate, but in order to make the bond between the linker and the substrate sufficiently stable, R described in detail below. Particularly preferred is a group selected from the range of ¹⁶ . For the same reason, the linking group possessed by the linker after bonding to the substrate is particularly preferably a group selected from the range of R ¹⁵ described in detail below.

  The reactive group possessed by the linker compound before binding to the substrate is not particularly limited as long as it is a group capable of forming a bond with the aminoglycoside antibiotic, and the functional group (primary primary) possessed by the aminoglycoside antibiotic. Any of an amino group, a secondary amino group, a hydroxyl group, an aldehyde group, etc.) may be formed, but preferably a bond is formed with a primary amino group. If the antibiotic can be bound to the linker via its primary amino group, it is presumed that appropriate binding of the bound antibiotic to the cells can be realized. The reactive group is a group selected from the range of groups represented by Formula III or Formula IV described in detail below in order to sufficiently bond the linker and the antibiotic. It is more preferable.

  The size of the linker is not particularly limited, and may be a low molecular weight structure in which the number of constituent atoms excluding hydrogen is less than 20, or less than 10, or a high molecular weight structure in which the number of constituent atoms exceeds the above range. Also good.

  The linker can have any shape such as a straight chain, a branched chain, a cyclic chain, a combination of two or more thereof, and may have a side chain.

  The linker is particularly preferably a polymer containing one or more repeating units. The polymer may have a linear structure or a branched structure having three or more terminals. The linker which is a polymer may be one in which main chains are cross-linked.

  The polymer constituting the linker is not particularly limited, and may be an addition polymerization polymer or a condensation polymerization polymer. Specifically, polymers obtained by addition polymerization of monomers having radical polymerizable double bonds, epoxy polymers, polyester polymers, polyurethane polymers, polyamide polymers, polycarbonate polymers, polyurea polymers, polyether polymers, polyimides A polymer having a radical polymerizable double bond added thereto is particularly preferable. In addition, naturally occurring polysaccharides such as chitin, chitosan, cellulose and the like or derivatives thereof can also be used as the polymer constituting the linker. Examples of the polymer obtained by addition polymerization of a monomer having a radical polymerizable double bond include polyacrylate, polymethacrylate and the like.

  The polymer constituting the linker is particularly preferably a polymer containing a main chain composed of carbon atoms linked by carbon-carbon single bonds. The main chain having such a structure has high stability, is hardly cleaved even under the influence of heat or chemical treatment, and can stably hold the antibiotic on the base material. Examples of the polymer having such a main chain structure include a polymer obtained by addition polymerization of a monomer having a radical polymerizable double bond. In this polymer, each repeating unit has two carbons constituting the main chain. Note that “a polymer containing a main chain composed of carbon atoms linked by a carbon-carbon single bond” means that at least a part of the main chain has such a structure, but the chemical stability of the polymer From the viewpoint of safety, except for both terminal portions, the bond connecting adjacent atoms constituting the main chain is preferably 70 mol% or more, more preferably 80 mol% or more, more preferably 90 mol% or more, more preferably 95. More than mol%, more preferably 98 mol% or more, more preferably 100 mol% is a carbon-carbon single bond.

  The polymer constituting the linker preferably has an aminoglycoside antibiotic linked to the side chain, or has the reactive group in the side chain in a state before linking. In this case, a large amount of aminoglycoside antibiotic can be efficiently arranged on the substrate.

  The polymer constituting the linker preferably has the above-mentioned connecting group in the side chain when the side chain is connected to the base material or in a state before connection. In this case, the polymer chain can be firmly bonded to the substrate surface as compared with the case where the polymer chain is bonded only at one or both ends of the main chain.

  In a more preferred embodiment of the polymer constituting the linker, after the base and the antibiotic are linked, the base is linked to the side chain of the polymer, and the aminoglycoside antibiotic is It is a polymer linked to the side chain of the polymer, or a polymer containing the linking group and the reactive group in the side chain before linking with the base material and the antibiotic. In this embodiment, the linker is immobilized along the surface of the substrate and the antibiotic is disposed along the polymer chain, so that a large amount of aminoglycoside antibiotic can be placed at different positions on the substrate surface. It is preferable because it can be efficiently disposed on the substrate and the binding stability of the linker to the substrate is enhanced.

  The linker is preferably a polymer containing at least repeating unit 1 and repeating unit 2, and at least repeating unit 2 includes a group containing the aminoglycoside antibiotic. In this case, the embodiment in which the repeating unit 1 includes a linking group linked to the substrate is preferable because the synthesis of the polymer is easy and the bonding of the polymer to the substrate surface is sufficiently strong. In particular, in an embodiment in which the repeating unit 1 includes a linking group linked to the substrate and the repeating unit 1 and the repeating unit 2 are randomly copolymerized, the group is located at positions dispersed along the length of the polymer chain. This is preferred because there can be a site of connection to the material.

  In another preferred embodiment, it has a structure in which the antibiotic is not bonded to the repeating unit 1 and the antibiotic is bonded to the repeating unit 2. By including the repeating unit 1 that does not contain the antibiotic, the antibiotic that serves as a trapping point for cells can be sparsely arranged, and the cells can be efficiently removed by reducing interference between the antibiotic and the bound cells. It becomes possible to capture. In order to enhance this effect, it is particularly preferable that the repeating unit 1 and the repeating unit 2 are polymerized by random copolymerization.

  The repeating unit 1 does not need to include only one type of structure, and may include a plurality of types of units having different structures. Similarly, the repeating unit 2 does not need to include only one type of structure, and may include a plurality of types of units having different structures.

  The amount ratio of the repeating unit 1 and the repeating unit 2 in the polymer is not particularly limited, but preferably, the repeating unit 2 is 50 mol or more and 990 mol or less with respect to 100 mol of the repeating unit 1. More preferably, it is 100 mol or more, 150 mol or more, 200 mol or more, More preferably, it is 900 mol or less, 500 mol or less. In this numerical range, the cell adsorption rate to the cell separation material is particularly high.

  The polymer is preferably bonded to the substrate surface via one or both side chains of the repeating unit 1 and the repeating unit 2, and is bonded to at least the side chain of the repeating unit 1. More preferably. In this case, the polymer is suitable because it can be firmly bonded to the surface of the substrate and a large amount of aminoglycoside antibiotic can be efficiently disposed on the surface of the substrate.

<Preferred Embodiment of Polymer>
A preferred embodiment of the polymer constituting the linker is a polymer containing at least the repeating unit 1 and the repeating unit 2. Therefore, a more preferred embodiment of this polymer will be described below.

  In the following description, the monomer compound that forms repeating unit 1 is referred to as monomer 1, and the monomer compound that forms repeating unit 2 is referred to as monomer 2.

  Each of the monomer 1 and the monomer 2 preferably includes at least one radical polymerizable double bond in each molecule because a main chain composed of a carbon-carbon single bond can be formed by polymerization. Any monomer having a structure capable of forming various polymers as described above may be used.

  The repeating unit 1 and the monomer 1 may have the linking group in a state before being bonded to the base material.

  The repeating unit 2 and the monomer 2 are not particularly limited as long as the repeating unit 2 and the monomer 2 have the reactive group in a state before binding to the aminoglycoside antibiotic.

  The repeating unit 2 and the monomer 2 may have the linking group in a state before being bonded to the base material.

A part of the repeating unit 1 contained in the polymer may remain unreacted with the base group even after the polymer and the base material are connected. Similarly, a part of the repeating unit 2 contained in the polymer has the reactive group remaining unreacted with the aminoglycoside antibiotic even after the polymer and the aminoglycoside antibiotic are linked. Also good. When the repeating unit 2 contains the reactive group that has not reacted with the aminoglycoside antibiotic, it is reacted with another compound having a functional group that can form a bond with the reactive group, and the other compound may be introduced in place of the groups containing aminoglycoside antibiotics group containing, R ²⁵ in formula II repeating unit 2 is described later in this case is based on including the other compounds. As the other compound, a compound having the same functional group (for example, primary amino group, hydroxyl group) as that of aminoglycoside antibiotics and having no specific adsorptivity to the cell to be separated can be used. Amines can be used. The preferred range for the quantitative ratio between the repeating unit 1 and the repeating unit 2 in the polymer is the unreacted linkage in addition to the repeating unit 1 having the linking group linked to a base material. In addition to all repeating units derived from monomer 1, such as repeating unit 1 having a reactive group, and repeating unit 2 linked to an aminoglycoside antibiotic, unreacted reactive group or reactive group and other compound Is a preferable range of the quantitative ratio with all the repeating units derived from the monomer 2, such as the repeating unit 2 having a group containing the other compound formed by reaction.

Hereinafter, particularly preferred embodiments of each unit and monomer will be described.
<Especially Preferred Embodiment of Repeating Unit 1 and Monomer 1>
The repeating unit 1 preferably has a structure represented by the formula I in the polymer linked to the base material, and is represented by the formula I ′ in the polymer before bonding to the base material. It has a structure.

式Ｉ’’及び式Ｉ’において、並びに、該当する基が含まれる場合は式Ｉにおいても、
Ｒ^１１は、水素、アルキル基、芳香族基又は芳香族アルキル基であり、
Ｒ^１２は、水素、アルキル基、芳香族基又は芳香族アルキル基であり、
Ｒ^１３は、水素、アルキル基、芳香族基又は芳香族アルキル基であり
Ｌ^１４は、水素以外の原子の数が１以上で価数ｐの基であり、ここでｐは２以上４以下の整数であり、
Ｒ^１６は、基材に連結可能な連結性基である。 The repeating unit 1 having the structure represented by the formula I ′ is composed of a monomer 1 having a radical polymerizable double bond represented by the following formula I ″ and other radical polymerizable monomers such as the monomer 2 described later. It can be formed by radical polymerization together with the monomer component.
Formula I ″:

In formula I '' and formula I ', and also in formula I if the relevant group is included,
R ¹¹ is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group,
R ¹² is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group;
R ¹³ is hydrogen, an alkyl group, an aromatic group, or an aromatic alkyl group. L ¹⁴ is a group having 1 or more atoms other than hydrogen and a valence p, where p is 2 or more and 4 or less. An integer,
R ¹⁶ is a linking group that can be linked to a substrate.

In R ¹¹ , R ¹² and R ¹³ , the number of carbon atoms of the “alkyl group” is not particularly limited. However, the steric hindrance at the time of polymerization and cell capture can be reduced, so that the carbon number is preferably small, and the carbon number is preferably 1 or more. 6 or less, more preferably 4 or less, more preferably 3 or less, still more preferably 1 or 2, and most preferably 1. The alkyl group may be linear or branched, but is preferably a linear alkyl group from the viewpoint of reducing steric hindrance during polymerization and cell capture.

In R ¹¹ , R ¹² and R ¹³ , the number of atoms of the “aromatic group” is not particularly limited, but in order to reduce steric hindrance during polymerization and cell capture, the number of atoms other than hydrogen is preferably 5 or more, 15 Or less, more preferably 10 or less, and more preferably 5 or 6 aromatic groups. The type of atoms other than hydrogen is not particularly limited, but typically includes at least carbon, and may further include nitrogen, oxygen, and sulfur. As long as the total number of atoms other than hydrogen is within the above range, the aromatic group may have one or more substituents, and examples of the substituent include an alkyl group, and examples of the alkyl group include R ¹¹ , Similar to those described above for R ¹² and R ¹³ can be used. As the aromatic group, a phenyl group is particularly preferred because of its small steric hindrance.

In R ¹¹ , R ¹² and R ¹³ , the “aromatic alkyl group” is a group in which one or more of the alkyl groups, preferably one hydrogen atom is substituted with an aromatic group, and the alkyl group part of the aromatic alkyl group Is preferably selected from the range of alkyl groups defined above, and the aromatic group portion of the aromatic alkyl group is preferably selected from the range of aromatic groups defined above. The aromatic alkyl group is most preferably a benzyl group. When the aromatic alkyl group is selected from the range exemplified here, steric hindrance during polymerization of the linker compound and during cell capture can be reduced.

R ¹¹ is more preferably hydrogen, an alkyl group, or a phenyl group, and particularly preferably hydrogen or an alkyl group. As the alkyl group for R ¹¹ , a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, and a tert-butyl group are preferable, and a methyl group or an ethyl group is particularly preferable. It is. When R ¹¹ is selected from the range exemplified here, the steric hindrance during polymerization of the linker compound and during cell capture can be reduced.

R ¹² is more preferably hydrogen, an alkyl group, or a phenyl group, and particularly preferably hydrogen or an alkyl group. As the alkyl group for R ¹² , a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, and a tert-butyl group are preferable, and a methyl group or an ethyl group is particularly preferable. It is. When R ¹² is selected from the range exemplified here, steric hindrance during polymerization of the linker compound and during cell capture can be reduced.

From the viewpoint of reactivity during polymerization of the linker compound, at least one of R ¹¹ and R ¹² is preferably hydrogen, and particularly preferably both.

R ¹³ is more preferably hydrogen, an alkyl group, or a phenyl group, and particularly preferably hydrogen or an alkyl group. As the alkyl group for R ¹³ , a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, and a tert-butyl group are preferable, and a methyl group or an ethyl group is particularly preferable. It is. When R ¹³ is selected from the range exemplified here, steric hindrance during polymerization of the linker compound and during cell capture can be reduced.

L ¹⁴ is a group having 1 or more atoms other than hydrogen and a valence of p, where p is an integer of 2 or more and 4 or less. L ¹⁴ has the role as a spacer to reduce the possibility of conflict with the linker backbone and the substrate a distance significantly to cell substrate surface, this by the number of atoms other than hydrogen is 2 or more The role is played more prominently. The number of atoms other than hydrogen of L ¹⁴ is 1 or more. For example, it is preferably 2 or more or 3 or more as a spacer, and the upper limit is not particularly limited, but is preferably 12 or less, 10 or less or from the viewpoint of ease of handling. 8 or less. In particular, among the atoms other than hydrogen constituting L ¹⁴ , the bond from the atom to which the carbon to which R ¹³ is bonded to the atom to which R ¹⁶ (or R ^{15 in} formula I) is bonded becomes the minimum number of atoms. It is preferable from the same viewpoint that the number of atoms (namely, the chain length) is 1 or more or 2 or more and 12 or less, 10 or less, 8 or less or 6 or less. The valence valence p of L ¹⁴ may be 2 or more and 4 or less, but p is preferably 2 or 3 and more preferably 2 for ease of production. Examples of L ¹⁴ include those represented by the following formula.

（式中、Ｘ^１は酸素又は硫黄を表し、好ましくは酸素であり、Ｘ^２は酸素、硫黄又はＮＨを表し、ｍ_１は０以上、８以下、好ましくは１以上、６以下の整数を表し、＊_１はＲ^１３が結合しているのと同一の炭素への結合を示し、＊_２はＲ^１６への結合を示す。炭素鎖の部分は１つ以上の水素原子が水酸基、ハロゲン原子、アルキル基等により置換されていてもよい。或いは、Ｌ^１４全体で上記の原子数となる範囲内及び／又は鎖長が上記の原子数となる範囲内で＊_１とＲ^１３が結合しているのと同一の炭素との間に更に２価以上４価以下の基が介在していてもよく、＊_２とＲ^１６との間に更に２価以上４価以下の基が介在していてもよい。）

(In the formula, X ¹ represents oxygen or sulfur, preferably oxygen, X ² represents oxygen, sulfur or NH, and m ₁ represents an integer of 0 or more and 8 or less, preferably 1 or more and 6 or less. , * ₁ represents a bond to the same carbon to which R ¹³ is bonded, and * ₂ represents a bond to R ^{16. In the} carbon chain portion, one or more hydrogen atoms are a hydroxyl group, a halogen atom, It may be substituted by an alkyl group, etc. Alternatively, * ₁ and R ¹³ are bonded within the range where the total number of atoms of L ¹⁴ is the above number and / or the chain length is the number of atoms described above. Noto same may be separated by further bivalent or tetravalent following groups between the carbons, * ₂ and it is interrupted more divalent or tetravalent following groups between R ¹⁶ Good.)

R ¹⁶ may be a linking group that can be linked to the substrate, can be appropriately selected according to the group present on the surface of the substrate, and is not limited to a specific structure.

  Examples of the group present on the surface of the substrate include metal elements such as a hydroxyl group, a carboxy group, a carbonyl group, an aldehyde group, a thiol group, an epoxy group, a primary to quaternary amino group, a —SiH group, a vinyl group, and Au. , Avidin-containing groups, streptavidin-containing groups, and the like. These groups may be groups that the substrate has in advance, or may be groups introduced on the surface of the substrate by the method described above.

  The linking group may be any group that can be linked to a group on the substrate surface, and includes a covalent bond, an ionic bond, a hydrogen bond, a brine bond, and a coordinate bond with the group on the substrate surface. It is preferable that one or more selected from the group can be formed, and it is particularly preferable that a covalent bond can be formed from the viewpoint of bond stability. For example, when a hydroxyl group is present on the surface of the substrate, the linking group is preferably a group capable of forming a covalent bond such as a —Si—O— bond, an ester bond, or an ether bond by reaction with the hydroxyl group. When a carboxy group is present on the surface of the substrate, the linking group is preferably a group capable of forming a covalent bond such as an ester bond or an amide bond by reaction with the carboxy group. When a carbonyl group is present on the surface of the substrate, the linking group is preferably a group capable of forming a covalent bond by reaction with the carbonyl group. Examples of groups capable of forming a covalent bond by reaction with a carbonyl group include groups capable of forming an imine by reaction with a carbonyl group. When an aldehyde group is present on the substrate surface, the linking group is preferably a group capable of forming a covalent bond such as an amide bond or an ester bond by reaction with the aldehyde group. Other examples of groups that can form a covalent bond by reaction with an aldehyde group include groups that can form a Schiff base by reaction with an aldehyde group. When a thiol group is present on the substrate surface, the linking group can form a covalent bond such as a thioester bond, -SS-bond, sulfur-metal bond, or S-C bond by reaction with the thiol group. Are preferred. The S—C bond with a thiol group is a reaction between a thiol group and a maleimidyl group by a thiol-ene reaction when the linking group is an alkenyl group such as a vinyl group, and when the linking group is a maleimidyl group. Can be formed respectively. When an epoxy group is present on the surface of the substrate, the linking group is preferably a group capable of forming a covalent bond by reaction with the epoxy group, and specific examples include an amino group and a thiol group. When a primary to quaternary amino group is present on the substrate surface, the linking group is preferably a group capable of forming a covalent bond such as an amide bond by reaction with the primary to quaternary amino group. When a -SiH group is present on the substrate surface, the linking group is preferably a group capable of forming a covalent bond such as a Si-C bond by reaction with the -SiH group. Examples of the linking group capable of forming a Si—C bond by reaction with —SiH group include a vinyl group capable of forming a Si—C bond by radical reaction with —SiH group. When a vinyl group is present on the surface of the substrate, the linking group is preferably a group capable of forming a covalent bond by reaction with the vinyl group. Examples of the linking group capable of forming a covalent bond by reaction with a vinyl group include a thiol group that can form an S—C bond by a thiol-ene reaction with a vinyl group. When a metal element such as Au is present on the substrate surface, the linking group is preferably a group capable of forming a covalent bond such as a sulfur-metal bond by reaction with the metal element. When an avidin-containing group or a streptavidin-containing group is present on the substrate surface, the linking group is preferably a group containing biotin or a derivative thereof.

When using a base material having a group such as a hydroxyl group, an epoxy group, an amino group, or a carboxy group on the surface, R ¹⁶ and the linking group contained in the linker compound in a state before being connected to the base material are particularly preferably , A group having a total of 1 to 3 —OR ¹⁷ and / or silicon atoms bonded to a halogen atom, wherein R ¹⁷ is an alkyl group. The halogen atom is preferably chlorine, fluorine, iodine or bromine, more preferably chlorine. Such groups can be hydrolyzed to form silanols with the silicon atoms, and can react with the groups on the substrate to form covalent bonds (such as -Si-O- bonds). In addition, a covalent bond (-Si-O- bond or the like) can be formed by directly reacting with the above on the substrate without passing through silanol under heating conditions.

（式中
Ｘ^３は−ＯＲ^１７及び／又はハロゲン原子であり、
Ｒ^１７はアルキル基であり、
Ｙは水素又はアルキル基であり、
ｎは１以上、３以下の整数であり、
＊^３は結合を表す） As the linking group having a silicon atom bonded to a total of 1 to 3 —OR ¹⁷ and / or a halogen atom, those represented by the following formula V are preferable.
Formula V:

(Wherein X ³ is —OR ¹⁷ and / or a halogen atom,
R ¹⁷ is an alkyl group;
Y is hydrogen or an alkyl group,
n is an integer of 1 to 3,
* ³ represents a bond)

As the alkyl group for R ¹⁷ and Y, each independently preferably has a carbon number of preferably 1 or more and 6 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and most preferably 1. A chain or branched chain, preferably a linear alkyl group, preferably a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, or a tert-butyl group, A methyl group or an ethyl group is preferable. When the alkyl group in R ¹⁷ and Y is within this range, the reactivity at the time of reaction between a group such as a hydroxyl group on the surface of the substrate and the linking group is preferable.

Other suitable examples of the linking group contained in the linker compound in a state prior to linking to R ¹⁶ and the substrate include a vinyl group, a thiol group, and biotin. When the linking group in the repeating unit 1 is a vinyl group, it can react to form a covalent bond when an —SH group or —SiH group is present on the substrate. When the linking group is a thiol group, a -SS-bond can be formed if a thiol group is present on the substrate, and -Au-S if gold (Au) is present on the substrate. A bond can be formed; When the linking group is biotin, specific binding can be formed if avidin or streptavidin is present on the substrate.

The linker compound before linking to the base material contains the linking group (R ¹⁶ or the like), and when the linker compound forms a base material-linker conjugate together with the base material, the linking group becomes a base material. And form a connection.

The linking group is typically a group represented by R ¹⁵ in the embodiment of the repeating unit 1 represented by the formula I. The linking group is a group formed as a result of the linking group reacting with a group on the substrate surface, and its structure is determined according to the combination of the linking group and the group on the substrate surface. For example, a group having a silicon atom bonded to a total of 1 to 3 —OR ¹⁷ and / or a halogen atom, for example, a group represented by Formula V, and a hydroxyl group that reacts with the group on the substrate surface , Epoxy groups, amino groups, carboxy groups and the like, the linking group is preferably a group containing a silicon atom having at least one bond to the surface of the substrate, typically In the group represented by V, at least one of X ³ is a group substituted with a bond to the surface of the substrate (for example, a single bond of —O— to oxygen).

<Especially Preferred Embodiment of Repeating Unit 2 and Monomer 2>
The repeating unit 2 preferably has a structure represented by the formula II in a polymer to which an aminoglycoside antibiotic is bound, and preferably in the polymer before being bound to an aminoglycoside antibiotic. It has a structure represented by II ′.

式ＩＩ’’及び式ＩＩ’において、並びに、該当する基が含まれる場合は式ＩＩにおいても、
Ｒ^２１は、水素、アルキル基、芳香族基又は芳香族アルキル基であり、
Ｒ^２３は、水素、アルキル基、芳香族基又は芳香族アルキル基であり、
Ｒ^２６は、水素、アルキル基、芳香族基又は芳香族アルキル基であり、
Ｒ^２７は、式ＩＩＩ

（式ＩＩＩ中、
Ｌ^２４は、水素以外の原子の数が１以上で価数ｑの基であり、ここでｑは２以上、４以下の整数であり、
Ｒ^２８は、アミノグリコシド系抗生物質により置換可能な脱離基又はアミノグリコシド系抗生物質が有する官能基（例えば第一級アミノ基、水酸基）と結合を形成可能な反応性基であり、
＊は結合を示す）
で表される基であり、
或いは、
Ｒ^２６とＲ^２７とは一体となって式ＩＶ

（式ＩＶ中、＊は、結合を示す）
で表される二価の基を形成している。 The repeating unit 2 having the structure represented by the formula II ′ is a monomer having a radically polymerizable double bond represented by the following formula II ″ and other radically polymerizable monomers such as the monomer 1. It can be formed by radical polymerization together with the monomer component.
Formula II '':

In formula II '' and formula II ', and also in formula II if the relevant group is included,
R ²¹ is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group,
R ²³ is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group,
R ²⁶ is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group,
R ²⁷ is a compound of formula III

(In Formula III,
L ²⁴ is a group having 1 or more atoms other than hydrogen and a valence of q, where q is an integer of 2 or more and 4 or less,
R ²⁸ is a reactive group capable of forming a bond with a functional group (for example, primary amino group, hydroxyl group) that a leaving group that can be substituted by an aminoglycoside antibiotic or an aminoglycoside antibiotic has,
* Indicates binding)
A group represented by
Or
R ²⁶ and R ²⁷ together form Formula IV

(In formula IV, * represents a bond)
The bivalent group represented by these is formed.

The carbon number of the “alkyl group” in R ²¹ , R ²⁶ and R ²³ is not particularly limited, but it is preferable that the steric hindrance at the time of polymerization and cell capture can be reduced, and the carbon number is preferably small, and the carbon number is preferably 1 or more. 6 or less, more preferably 4 or less, more preferably 3 or less, still more preferably 1 or 2, and most preferably 1. The alkyl group may be linear or branched, but is preferably a linear alkyl group from the viewpoint of reducing steric hindrance during polymerization and cell capture.

In R ²¹ , R ²⁶ and R ²³ , the number of atoms of the “aromatic group” is not particularly limited, but the number of atoms other than hydrogen is preferably 5 or more, in order to reduce steric hindrance during polymerization and cell capture. Or less, more preferably 10 or less, and more preferably 5 or 6 aromatic groups. The type of atoms other than hydrogen is not particularly limited, but typically includes at least carbon, and may further include nitrogen, oxygen, and sulfur. As long as the total number of atoms other than hydrogen is within the above range, the aromatic group may have one or more substituents, and examples of the substituent include an alkyl group, and examples of the alkyl group include R ²¹ , Similar to those described above for R ²⁶ and R ²³ can be used. As the aromatic group, a phenyl group is particularly preferred because of its small steric hindrance.

The “aromatic alkyl group” in R ²¹ , R ²⁶ and R ²³ is a group in which one or more of the alkyl groups, preferably one hydrogen atom is substituted with an aromatic group, and the alkyl group part of the aromatic alkyl group Is preferably selected from the range of alkyl groups defined above, and the aromatic group portion of the aromatic alkyl group is preferably selected from the range of aromatic groups defined above. The aromatic alkyl group is most preferably a benzyl group. When the aromatic alkyl group is selected from the range exemplified here, steric hindrance during polymerization of the linker compound and during cell capture can be reduced.

R ²¹ is more preferably hydrogen, an alkyl group, or a phenyl group, and particularly preferably hydrogen or an alkyl group. As the alkyl group for R ²¹ , a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, and a tert-butyl group are preferable, and a methyl group or an ethyl group is particularly preferable. It is. When R ²¹ is selected from the range exemplified here, steric hindrance during polymerization of the linker compound and during cell capture can be reduced.

R ²⁶ is more preferably hydrogen, an alkyl group, or a phenyl group, and particularly preferably hydrogen or an alkyl group. As the alkyl group for R ²⁶ , a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, and a tert-butyl group are preferable, and a methyl group or an ethyl group is particularly preferable. It is. When R ²⁶ is selected from the range exemplified here, steric hindrance during polymerization of the linker compound and during cell capture can be reduced.

From the viewpoint of reactivity during polymerization of the linker compound, at least one of R ²¹ and R ²⁶ is preferably hydrogen, and particularly preferably both.

R ²³ is more preferably hydrogen, an alkyl group, or a phenyl group, and particularly preferably hydrogen or an alkyl group. Alkyl The methyl group at R ^23, ethyl, n- propyl group, an isopropyl group, n- butyl group, sec- butyl group, an isobutyl group, preferably a tert- butyl group, particularly preferably a methyl group or an ethyl group It is. When R ²³ is selected from the ranges illustrated herein, it is possible to reduce the polymerization time and sterically hindered at the time of cell capture linker compound.

Here, an embodiment in which R ²⁷ is a group represented by Formula III will be described.
L ²⁴ is a group having 1 or more atoms other than hydrogen and a valence of q, where q is an integer of 2 or more and 4 or less. L ²⁴ has a role as a spacer that reduces the possibility of cells coming into contact with the substrate surface by increasing the distance between the linker main chain and the antibiotic and the distance between the substrate surface and the antibiotic. When the number of atoms is 1 or more and 2 or more, it is more preferable as a spacer. The upper limit of the number of atoms other than hydrogen of L ²⁴ is not particularly limited, but is preferably 12 or less, 10 or less, 8 or less, or 5 or less from the viewpoint of easy handling. In particular, among atoms other than hydrogen constituting L ²⁴ , the bond from the atom to which the carbon to which R ²³ is bonded to the atom to which R ²⁸ (or R ^{25 in} formula II) is bonded is the minimum number of atoms. From the viewpoint similar to the above, the number of atoms (namely, the chain length) is preferably 1 or more and 12 or less, 10 or less, 8 or less, or 5 or less. The valency q of L ²⁴ may be 2 or more and 4 or less, but q is preferably 2 or 3 and more preferably 2 for ease of production. Examples of L ²⁴ include those represented by the following formula.

（式中、Ｘ^４は酸素又は硫黄を表し、好ましくは酸素であり、＊_４はＲ^２３が結合しているのと同一の炭素への結合を示し、＊_５はＲ^２８への結合を示す。或いは、Ｌ^２４全体で上記の原子数となる範囲内で＊_４とＲ^２３が結合しているのと同一の炭素との間に更に２価以上４価以下の基が介在していてもよく、＊_５とＲ^２８との間に更に２価以上４価以下の基が介在していてもよい。）

(Wherein X ⁴ represents oxygen or sulfur, preferably oxygen, * ₄ represents a bond to the same carbon to which R ²³ is bonded, and * ₅ represents a bond to R ²⁸ . . Alternatively, be interposed more divalent or tetravalent following groups between the same carbon and the above number of atoms and in the range of * ₄ and R ²³ are bonded across L ²⁴ well, * ₅ and tetravalent following groups further divalent or between R ²⁸ may be interposed.)

（式中、＊_４はＲ^２３が結合しているのと同一の炭素への結合を示し、＊_５はＲ^２８への結合を示す。或いは、Ｌ^２４全体で上記の原子数となる範囲内で＊_４とＲ^２３が結合しているのと同一の炭素との間に更に２価以上４価以下の基が介在していてもよく、＊_５とＲ^２８との間に更に２価以上４価以下の基が介在していてもよい。）

(In the formula, * ₄ represents a bond to the same carbon to which R ²³ is bonded, and * ₅ represents a bond to R ^28. Alternatively, within the range where the total number of L ²⁴ is the above number of atoms. in * ₄ and may be separated by further bivalent or tetravalent following groups between the same carbon and the R ²³ is attached, * ₅ and further divalent or between R ²⁸ A tetravalent or lower group may be present.)

（式中、＊_４はＲ^２３が結合しているのと同一の炭素への結合を示し、＊_５はＲ^２８への結合を示す。或いは、Ｌ^２４全体で上記の原子数となる範囲内で＊_４とＲ^２３が結合しているのと同一の炭素との間に更に２価以上４価以下の基が介在していてもよく、＊_５とＲ^２８との間に更に２価以上４価以下の基が介在していてもよい。）

(In the formula, * ₄ represents a bond to the same carbon to which R ²³ is bonded, and * ₅ represents a bond to R ^28. Alternatively, within the range where the total number of L ²⁴ is the above number of atoms. in * ₄ and may be separated by further bivalent or tetravalent following groups between the same carbon and the R ²³ is attached, * ₅ and further divalent or between R ²⁸ A tetravalent or lower group may be present.)

In formula III, R ²⁸ is a reactive group capable of forming a bond with a leaving group displaceable by an aminoglycoside antibiotic or a functional group (eg, primary amino group, hydroxyl group) possessed by the aminoglycoside antibiotic. The “reactive group” in R ²⁸ is a group other than a leaving group and a group capable of forming a bond with a functional group possessed by an aminoglycoside antibiotic.

The leaving group that can be substituted with aminoglycoside antibiotics as R ²⁸ is not limited to aminoglycoside antibiotics, but may be any leaving group that can be substituted with a primary amine compound. Examples of groups that are easy to handle include groups represented by the following formulas and halogen atoms. At this time, when the terminal atom of L ²⁴ to which R ²⁸ is bonded is an atom that undergoes a nucleophilic reaction by the amino group or hydroxyl group of the antibiotic and the leaving group can be replaced by the antibiotic, the antibiotic This is preferable because the bonding of substances becomes easier. Examples of the terminal atom of L ²⁴ include a carbon atom of —C (═O) — group, a carbon atom of —C (═S) — group, a sulfur atom of —S (═O) — group, and —S. (= O) ₂ -group sulfur atom and the like. The halogen atom can be fluorine, chlorine, bromine or iodine, preferably bromine or chlorine.

（式中、Ａ^１〜Ａ^５はそれぞれ独立に同一又は異なるハロゲン原子であることができ、好ましくは全てフッ素であり、＊はＬ^２４への結合を示す）

(In the formula, A ^{1 to} A ⁵ can be independently the same or different halogen atoms, preferably all are fluorine, and * represents a bond to L ²⁴ ).

A reactive group capable of forming a bond with a functional group (for example, primary amino group, hydroxyl group) possessed by an aminoglycoside antibiotic that can be used as R ²⁸ is highly reactive and easily available. The reactive group containing group can be illustrated. Examples of the reactive group containing an epoxy group include a group having a structure represented by the following formula.

（式中、Ｌ^２９は水素以外の原子数が１〜１０個、好ましくは１〜４個の二価の基又は結合を表し、＊はＬ^２４への結合を表す）

(Wherein L ²⁹ represents a divalent group or bond having 1 to 10, preferably 1 to 4 atoms other than hydrogen, and * represents a bond to L ²⁴ )

In the above formula, although the structure of the L ²⁹ is not particularly limited, for example, L ²⁹ may have oxygen, sulfur or NH in a position to bind to L ^24, the remainder is a hydrocarbon chain of bonds or 1-9 carbon atoms.

In the formula II ″, when R ²⁶ and R ²⁷ are combined to form the group represented by the formula IV, the monomer 2 represented by the formula II ″ is a maleic anhydride derivative. In this embodiment, in order to improve the reactivity of the polymerization reaction between the monomers, preferably at least one of R ²¹ and R ²³ is hydrogen, and more preferably both are hydrogen.

  In the polymer formed by radical copolymerization of the monomer 2 represented by the formula II ″ together with other monomers having a radical polymerizable double bond such as the monomer 1 of the formula I ″, The repeating unit derived from the represented monomer 2 is the repeating unit 2 having a structure represented by the formula II ′. Each group in the repeating unit 2 represented by the formula II ′ is as described above for the monomer 2 represented by the formula II ″.

  When an aminoglycoside antibiotic is reacted with a polymer having the repeating unit 2 represented by the formula II ', the repeating unit 2 is changed into a structure represented by the formula II.

In Formula II above, R ²⁵ is a monovalent group comprising an aminoglycoside antibiotic in embodiments where R ²⁸ in Formula II ′ and Formula II ″ is a leaving group that can be substituted with an aminoglycoside antibiotic. For example, when R ²⁸ is a leaving group displaceable by a primary amino group or a hydroxyl group, R ²⁵ in the above formula II is formed by removal of one hydrogen atom of the amino group or hydroxyl group of an aminoglycoside antibiotic. Is a monovalent group. ^{L 24} in this case formula II is the same as ^{L 24} in the formula II 'and Formula II''.

Further, R ²⁵ in formula II above, in embodiments R ²⁸ in formula II 'and Formula II''are the reactive groups, and one functional group of the reactive group and the aminoglycoside antibiotic reacts Is a monovalent group. This monovalent group usually includes a monovalent group containing an aminoglycoside antibiotic as its partial structure. Again ^{L 24} in the formula II is the same as ^{L 24} in the formula II 'and Formula II''.

In the above formula II, R ²⁵ is an aminoglycoside antibiotic in the embodiment in which R ²⁶ and R ²⁷ in the formula II ′ and formula II ″ together form a group represented by formula IV (Typically, a monovalent group formed by removing one hydrogen atom from an amino group or a hydroxyl group of an aminoglycoside antibiotic). In this case, L ²⁴ in Formula II is a carbonyl group (—C (═O) —). In this case, R ²² in Formula II is a carboxy group.

In formula II ′ and formula II ″, when R ²¹ is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group, in formula II, R ²¹ is the same as in formula II ′ and formula II ″. It is.

In formula II ′ and formula II ″, when R ²⁶ is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group, R ²² in formula II is the same as R ²⁶ in formula II ′ and formula II ″. It is.

In formula II ′ and formula II ″, when R ²³ is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group, in formula II, R ²³ is the same as in formula II ′ and formula II ″. It is.

<Polymerization>
Monomer 1 and monomer 2 as exemplified above can be copolymerized to form a polymer.

  By reacting monomer 1 and monomer 2 in a suitable solvent in the presence of a radical polymerization initiator, a polymer before linking the substrate and the antibiotic as a random copolymer can be obtained. Various conditions such as a solvent, a radical polymerization initiator, a reaction temperature, and a reaction time can be appropriately set by those skilled in the art. Preferably, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO) are used as the solvent. , Tetrahydrofuran (THF), dioxane, water / dimethyl sulfoxide (DMSO) mixed solvent and the like can be used, and DMF or DMSO is particularly preferable. However, when the linking group in the monomer 1 is the silicon-containing group, a solvent containing no water is preferable. Examples of radical polymerization initiators include azo compounds such as azobisisobutyronitrile (AIBN) and 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPD), peroxides such as benzoyl peroxide, and the like. AIBN is particularly preferable. The reaction temperature can be, for example, in the range of 40 ° C. or higher and 120 ° C. or lower, more preferably 50 ° C. or higher, more preferably 100 ° C. or lower, still more preferably 60 ° C. or higher, and still more preferably 80 ° C. or lower. The reaction time can be, for example, 0.5 hours or more and 50 hours or less, more preferably 1.0 hour or more and 25 hours or less, further preferably 6 hours or more, 12 hours or less, and further preferably 10 hours or more. can do. The polymerization reaction is preferably performed in a nitrogen atmosphere. By setting the reaction conditions within this range, a polymer having a molecular weight that can be used as a linker compound can be efficiently produced.

  In the copolymerization, in addition to the monomer 1 and the monomer 2, one or more other monomers may be added. Other monomers are not particularly limited as long as they have at least one radical polymerizable double bond, and vinyl monomers such as acrylate monomers, methacrylate monomers, acrylamide monomers, methacrylamide monomers, styrene monomers. Etc. can be used. For example, for the purpose of imparting hydrophilicity to the polymer, a hydrophilic monomer such as 2-methacryloyloxyethyl phosphorylcholine (MPC) can be added as another monomer. More preferably, the repeating unit 1 (repeating unit derived from monomer 1) and the repeating unit 2 (repeating unit derived from monomer 2) in all repeating units contained in one molecule of the polymer before linking the base material and the antibiotic. The total is preferably 50 mol% or more, more preferably 60 mol% or more, and more preferably 70 mol%, from the viewpoint of sufficiently securing the amount of the antibiotics linked to the linker and the linker site to the substrate. More preferably, it is 80 mol% or more, more preferably 90 mol% or more, more preferably 95 mol% or more, and most preferably 100 mol%.

  The ratio of the monomer 1, the monomer 2, and the other monomer used as raw materials in the polymerization can be set to a molar ratio according to the molar ratio of the desired repeating unit as described above.

  The molecular weight of the polymer before linking the base material and the antibiotic is not particularly limited, but is preferably 1,000 or more and 1,000,000 or less, more preferably 2,000 or more as the weight average molecular weight (Mw). More preferably, it is 500,000 or less, 200,000 or less, or 100,000 or less. Here, as the weight average molecular weight, the weight average molecular weight converted as standard polyethylene glycol can be adopted, and the method for measuring the molecular weight is not particularly limited, but is based on the dynamic light scattering method, the static light scattering method, and the viscosity measurement. A method based on viscosity measurement is particularly suitable for measuring the molecular weight of a polymer comprising a main chain composed of carbon atoms linked by carbon-carbon single bonds. When Mw is within this range, it is easy to handle the polymer when connecting to the base material and / or when connecting the antibiotic, and the polymer and / or antibiotic is efficiently arranged on the surface of the base material. can do. Further, the value (Mw / Mn) obtained by dividing the weight average molecular weight (Mw) by the number average molecular weight (Mn) is a molecular weight distribution, and the range of the molecular weight distribution is not particularly limited, but is preferably 2 or less.

  The terminal structure of the polymer is not particularly limited, and a group derived from the polymerization initiator as described above may be bonded, or the terminal may be sealed with another appropriate group.

<Method for producing cell separation material>
The method for producing the cell separation material of the present invention is not particularly limited, but as a preferred embodiment,
A substrate;
A linker (“linker compound” or “linker precursor”) comprising a linking group that can be linked to the substrate, and a reactive group that can form a bond with the aminoglycoside antibiotic,
A linker linking step of linking via the linking group to form a substrate-linker conjugate;
Examples include performing an antibiotic binding step of reacting the reactive group in the substrate-linker conjugate with an aminoglycoside antibiotic to bind the aminoglycoside antibiotic to the linker.

  When the linker compound is a polymer as mentioned above, it can be synthesized by polymerization of monomer molecules as exemplified in the section <Polymerization> above.

In the linker linking step, conditions can be appropriately set according to a combination of at least one linking group possessed by the linker and a group on the substrate surface. Specific examples of the bond formed with the combination of the connectivity group and the group on the substrate surface are as described above. Moreover, when reacting the linking group and the group on the substrate surface, one or both may be derivatized to a highly reactive functional group and then reacted. For example, when the linker linking group is a group having a total of 1 to 3 —OR ¹⁷ and / or silicon atoms bonded to a halogen atom, and the group on the substrate surface is a hydroxyl group, the polymer is treated with DMSO. A solution dissolved in an aprotic polar solvent such as the above can be contacted with a substrate and incubated to form a Si—O bond, and a temperature of 60 ° C. or higher, for example, about 100 ° C. during or after incubation. The formation of Si—O bond can be promoted by desorbing —OR ¹⁷ or halogen atom bonded to the silicon atom as R ¹⁷ OH or halogen ion by heating with and / or microwave irradiation.

  In an embodiment in which the final substrate is an aggregate of fibers or an aggregate of granules, the linker and the antibiotic are used after performing the linker linking step and the antibiotic binding step using the fibers or granules as a base. A method for producing a cell separation material of the present invention by forming a fiber aggregate or a granular aggregate using fibers or granules to which substances are connected, and the fiber or granule as a base material as described above. And then forming a fiber aggregate or a granular aggregate using the fibers or granules to which the linker is linked, and then performing the antibiotic binding step of the present invention. A method for producing a cell separation material (Aspect 2), a method for producing the cell separation material of the present invention by performing the linker linking step and the antibiotic binding step using a fiber aggregate or a granular aggregate as a base material ( It can be set as various aspects, such as aspect 3)The method for producing a cell separation material of the present invention includes any of these aspects.

  In the antibiotic binding step, the reaction conditions for reacting the substrate-linker conjugate with the aminoglycoside antibiotic can also be appropriately determined according to the combination of the reactive group of the linker and the aminoglycoside antibiotic. Aminoglycoside antibiotics have a plurality of primary amino groups, each having a different pKa value. Therefore, when the reactive group of the linker is reacted with the primary amino group of the aminoglycoside antibiotic, in the antibiotic binding step, the lowest pKa among the primary amino groups contained in the aminoglycoside antibiotic to be used is used. It is preferable to react the substrate-linker conjugate with the aminoglycoside antibiotic under a pH value condition higher than the value.

A washing step may be appropriately performed after the linker linking step and the antibiotic binding step.
In the antibiotic binding step, when a part of the reactive group in the substrate-linker conjugate remains without reacting with the aminoglycoside antibiotic, it further binds to the remaining reactive group after the antibiotic binding step. A remaining reactive group treatment step of introducing a group containing the other compound by reacting the substrate-linker conjugate with another compound having a functional group capable of forming a group may be performed. Specific examples of the other compounds are as described above.

In the cell separation material of the present invention, the binding amount of the linker on the substrate is not particularly limited and can be appropriately adjusted according to the purpose. In an embodiment in which the cell separation material of the present invention comprises the polymer as a linker, the polymer is preferably 0.2 μmol in terms of a constituent monomer in the region of the surface of the substrate where aminoglycoside antibiotics are linked. / cm ² are connected in the above density, said seal degree is more preferably 2 [mu] mol / cm ² or more, 5 [mu] mol / cm ² or more and 10 .mu.mol / cm ² or more, the upper limit is not particularly limited example 2,000Myumol / cm ² or less , preferably 200 [mu] mol / cm ² or less, 100 [mu] mol / cm ² or less, 50 [mu] mol / cm ² or less. The cell separation material of the present invention can capture cells particularly efficiently when the binding density of the polymer on the substrate surface is in the above range.

<Form of cell separation material>
The cell separation material of the present invention can be used for cell separation as it is. In particular, when the form of the substrate of the cell separation material is a substrate containing at least a part of a surface such as a dish, flask, plate, bag, cartridge, cassette, etc., the cell separation material is used as it is for cell separation. Suitable for

  The cell separation material of the present invention may also be used by filling a container having a liquid inlet and / or a liquid outlet. A combination of the container and the cell separation material of the present invention filled in the container may be particularly referred to as a cell separation device.

  When the base material of the cell separation material is a structure having communication holes, such as an aggregate of fibers, an aggregate of granules, or a porous body, it is preferable to fill the container with the cell separation material.

  The form, material, and size of the container are not particularly limited, and may be any form such as a container, bag, tube, column, cassette, or flask. As an example of a cell separation device, a spherical carrier or the like communicates with a transparent or translucent cylindrical container having a capacity of about 0.1 ml or more and about 500 ml or less and a diameter of about 0.1 cm or more and about 10 cm or less. A structure in which a structure having pores is packed into a column shape can be used, and the column is useful as a cell separation device. The device is preferably mounted and filled in a container having a body fluid inlet and a body fluid outlet so as to partition the liquid inlet and the liquid outlet to form a filter or a cross flow module, and the filter and the cross flow module Is also a form of cell separation device. In addition, in the case of mounting and filling a cell separation material composed of a structure having a communication hole in the base material, the cell separation material can be laminated into a single layer or multiple layers in the container, It can also be mounted and filled in combination with a material that has no aminoglycoside antibiotics linked to its surface.

  The material of the container constituting the cell separation device is not particularly limited, but non-reactive polymer, biocompatible metal or alloy thereof, glass or the like is used. Alternatively, a material coated or plated with these non-reactive polymers or biocompatible metals may be used. Specifically, as the polymer, acrylonitrile polymers such as acrylonitrile butadiene styrene terpolymer, halogenated polymers such as polytetrafluoroethylene, polychlorotrifluoroethylene, copolymer tetrafluoroethylene or hexafluoropropylene, polyamide, polysulfone, polycarbonate, Examples include polyethylene, polypropylene, polyvinyl chloride acrylic copolymer, polycarbonate acrylonitrile butadiene styrene, and polystyrene. Examples of the metal material include stainless steel, titanium, platinum, tantalum, gold, and alloys thereof, as well as gold-plated alloy iron, platinum-plated alloy iron, cobalt chromium alloy, and titanium nitride-coated stainless steel. The glass may be silicon-coated glass. Particularly preferred are materials having sterilization resistance, and specific examples include silicon-coated glass, polystyrene, polypropylene, polyvinyl chloride, polycarbonate, polysulfone, and polymethylpentene.

<Capture target cells>
The cells captured and separated by the cell separation material of the present invention are not particularly limited as long as the cells have a structure capable of binding to aminoglycoside antibiotics on the surface, but preferably, calcium sensing receptor (CaSR) is present on the surface. It is a cell that exists. Since the affinity between CaSR and aminoglycoside antibiotics is high enough to capture cells but low enough to be easily detached, the cells containing CaSR on the surface captured by the cell separation material of the present invention are It can be collected without damaging the cells, and is suitable for using the collected cells.

  Examples of cells having CaSR on the surface include monocytes, mesenchymal stem cells, parathyroid cells, and ovarian epithelial cells. In many cases, cells isolated from the pancreas or kidney are also cells having CaSR on the surface. Monocytes are monocytic cells derived from hematopoietic stem cells. The origin of the cell is preferably a mammal such as a human, but is not particularly limited.

<Sample containing cells>
The sample to be treated with the cell separation material of the present invention is not particularly limited as long as it is a cell-containing sample. Body samples such as blood, lymph and bone marrow fluid, biological samples containing cells, and cell suspensions prepared therefrom Liquid. The sample containing cells is preferably substantially free of calcium ions, for example, even if calcium ions are present, the concentration is less than 1 mM, in particular less than 0.5 mM, and preferably free of calcium ions.

  The sample containing monocytes refers to a cell suspension containing at least monocytes, and usually refers to a cell suspension containing lymphocytes in addition to monocytes, ie, containing monocytes. As a sample containing monocytes, typically, monocyte-containing body fluids such as peripheral blood, bone marrow fluid, and umbilical cord blood, or these body fluids are subjected to centrifugation treatment, or if desired, Ficoll, Percoll, Bactiner tube, Examples thereof include cell suspensions that have been subjected to specific gravity centrifugation using prep, HES (hydroxyethyl starch), or the like. When collecting monocyte-containing samples from monocyte-containing body fluids, commonly used anticoagulants include heparin, low molecular weight heparin, nafamostat mesylate, gabexate mesylate, argatroban, and acid citrate dextrose solution. Citric acid-containing anticoagulants such as (ACD solution) and citrate phosphate dextrose solution (CPD solution) may be used. Among them, citric acid-containing anticoagulant or heparin can be mentioned as an anticoagulant generally most preferably used.

  An example of a sample containing mesenchymal stem cells is a cell suspension containing at least mesenchymal stem cells. Specific examples of the cell suspension include cell suspensions containing erythrocytes in addition to leukocytes, that is, blood cells. As a sample containing mesenchymal stem cells, typically, mesenchymal stem cell-containing biological samples such as peripheral blood, bone marrow fluid, umbilical cord blood, placenta, and amniotic membrane, or cells prepared by processing these biological samples Suspensions are mentioned. Examples of the cell suspension include centrifugation, or cell suspension subjected to specific gravity centrifugation using Ficoll, Percoll, Bactiner tube, Refoprep, HES (hydroxyethyl starch) or the like, if desired. It is done. When collecting samples containing mesenchymal stem cells from biological samples containing mesenchymal stem cells, commonly used anticoagulants include heparin, low molecular weight heparin, nafamostat mesylate, gabexate mesylate, argatroban, Citric acid-containing anticoagulants such as acid citrate dextrose solution (ACD solution) and citrate phosphate dextrose solution (CPD solution) may also be used. Among them, citric acid-containing anticoagulant or heparin can be mentioned as an anticoagulant generally most preferably used. When collecting a sample containing mesenchymal stem cells from a mesenchymal stem cell-containing biological sample, collagenase, metalloprotein thermolysin and dispase may be used as commonly used enzymes.

<Cell separation method>
The present invention also provides
A method for separating cells in a sample, comprising:
A cell capturing step of bringing the cell separation material of the present invention into contact with a sample and capturing the cells in the sample with the cell separation material;
Relates to a method comprising:

The method may further include a washing step of washing and removing non-captured cells from the cell separation material with a washing solution after the cell trapping step.
The method can further include a recovery step of recovering the captured cells from the cell separation material.

  In the cell capturing step, the conditions for bringing the cell separation material into contact with the sample containing the cells to be captured are not particularly limited. Examples of the contact time include 5 minutes or more and 120 minutes or less, and preferably 30 minutes or more and 90 minutes or less from the viewpoint of stable capture of cells and less damage to the cells. The pH of the sample to be contacted and the contact temperature are not particularly limited, but the pH (for example, 7 or more and 8 or less) and the temperature (for example, 32 ° C. or more and 40 ° C. or less, more preferably 36 or more and 38 ° C. or less) equivalent to the internal environment. Most preferably, 37.0 ± 0.5 ° C. is desirable. The cell trapping step is preferably performed under conditions in which calcium ions are substantially absent (for example, conditions in which calcium ions are present at less than 1 mM or less than 0.5 mM). In an embodiment in which the cell separation material includes a base material including at least a part of the surface part and an aminoglycoside antibiotic linked to the surface part, the cell capturing step using the same may include a surface of the surface part. It is preferable to leave it in contact with the cell-containing sample placed on the bottom. In an embodiment in which the cell separation material is a cell separation material based on a structure having communication holes, the sample is allowed to contact the cell separation material by allowing the sample to permeate or impregnate the sample. Can do.

  When the washing step is performed after the cell capturing step, the sample brought into contact with the cell capturing step is first removed from the cell separation material. After removing the sample, non-captured cells remaining on the cell separation material can be washed away using an appropriate washing solution. Examples of the washing solution include, but are not limited to, a physiological saline solution, a Ringer solution, a buffer solution such as a phosphate buffer, and a cell culture liquid medium such as RPMI 1640. The pH and contact temperature of the cleaning solution to be contacted are not particularly limited, but the pH (for example, 7 or more and 8 or less) and temperature (for example, 32 ° C or more and 40 ° C or less, more preferably 36 ° C or more and 38 ° C or less) equivalent to the internal environment. And most preferably 37.0 ± 0.5 ° C.).

  The recovery step of recovering the cells captured in the cell capturing step from the cell separation material is preferably performed after the washing step. The recovery step is performed by bringing an appropriate cell recovery solution into contact with the cell separation material that has captured the cells. Examples of the cell recovery solution include physiological saline, liquid medium, cell preservation solution, and a solution containing a divalent cation chelating agent such as acid citrate dextrose solution (ACD solution) or citrate phosphate dextrose solution (CPD solution). It is done. The cell recovery operation is performed by bringing the cell recovery solution into contact with the cell separation material that has captured the cells and allowing them to stand, shake, or circulate, and then pipetting, adding vibration, and damaging the captured cells. The cells can be recovered by a method of extruding the cell recovery solution vigorously to such an extent that it is not given (for example, flushing or the like). Examples of the means for extruding include air, inert gas, physiological saline, and the like, but the present invention is not limited to these. At this time, in order to increase the recovery efficiency, cooling to 4 ° C. or heating to 37 ° C. may be used in combination. In addition, when the collection of monocytes is insufficient, the above collection operation may be repeated.

<Method for inducing differentiation of cells having differentiation ability>
The present invention also provides
A method for inducing differentiation of a cell having differentiation ability,
A cell separation step of separating cells having differentiation ability from the cell-containing sample by the method using the cell separation material of the present invention;
And a differentiation induction step of inducing differentiation of the cells separated in the cell separation step.

  Here, the “cell having differentiation ability” includes a cell having pluripotency and a cell having unipotency, and includes not only undifferentiated cells but also cells in the process of differentiation. For example, mesenchymal stem cells that can differentiate into mesenchymal cells; monocytes that can differentiate into dendritic cells, osteoclasts, macrophages, etc .; A typical example is a cell that has not reached a cell.

  “Differentiation induction” refers to causing differentiation of cells having differentiation ability, and includes differentiation to a state in the middle of differentiation and differentiation to mature cells. When inducing differentiation of a cell having differentiation ability to a mature cell, the differentiation of the cell can be advanced to a state where a differentiation marker specific to the mature cell is detected.

  The cell separation step is a step of capturing and separating the cells from the sample containing cells having differentiation potential by the cell separation material of the present invention by the above method using the cell separation material of the present invention. The cell separation step includes at least the cell capture step, and may further include one or both of the washing step and the recovery step.

  The “cells separated in the cell separation step” in the differentiation induction step may be any cell having a differentiation ability separated from a sample in the cell separation step, and preferably after the cell capture step (preferably Furthermore, it may be cells that have been captured by the cell separation material (after the washing step), or from the cell separation material (preferably further after the washing step) after the cell capture step. It may be a cell recovered by a recovery step.

  When the differentiation inducing step is a step of inducing differentiation of cells captured by the cell separation material, a cell collection step of collecting cells from the cell separation material after differentiation induction can be further performed.

  For example, when monocytes are separated using the cell separation material of the present invention, it is preferable to induce dendritic cells by further inducing differentiation of the separated monocytes. Here, the dendritic cells are immature dendritic cells whose monocytes are induced to differentiate by cytokines such as interleukin-4 (IL-4) and granulocyte macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor α. Mature dendritic cells, cancer antigens induced by adding cytokines such as (TNFα), interleukin 1-β (IL1-β), interleukin-6 (IL-6), prostaglandin E2 (PGE2) It means a dendritic cell or the like on which a cancer antigen is presented through a step of acquiring an antigen presenting ability represented by the addition of.

  When the monocytes captured by the cell separation material are induced to differentiate into dendritic cells, the cell separation material with the monocytes still captured is a liquid medium containing cytokines such as IL-4 and GM-CSF. Can be induced to differentiate into immature dendritic cells by culturing for about 3 days to 7 days. Thereafter, the liquid medium is replaced with a liquid medium containing cytokines such as TNFα, IL1-β, IL-6, and PGE2, and further matured by culturing for about 1 day to 3 days in the medium. Differentiation can be induced in dendritic cells. In addition, if necessary, it can be induced to differentiate into dendritic cells on which the cancer antigen is presented through a step of acquiring the antigen presenting ability represented by the addition of the cancer antigen. As a method for recovering dendritic cells after differentiation induction from the cell separation material, the same method as described above can be used as a method for recovering monocytes from the cell separation material.

  When monocytes recovered from the cell separation material through the recovery step are induced to differentiate into dendritic cells, the recovered monocytes are moved to an appropriate container, for example, a dish or flask for cell culture, and the container A liquid medium containing cytokines such as IL-4 and GM-CSF can be added to the cells and cultured for about 3 days to 7 days to induce differentiation into immature dendritic cells. Thereafter, the liquid medium is replaced with a liquid medium containing cytokines such as TNFα, IL1-β, IL-6, and PGE2, and further matured by culturing for about 1 day to 3 days in the medium. Differentiation can be induced in dendritic cells. In addition, if necessary, it can be induced to differentiate into dendritic cells on which the cancer antigen is presented through a step of acquiring the antigen presenting ability represented by the addition of the cancer antigen. As a method for recovering the dendritic cells after differentiation induction from the container, a method similar to that described above as a method of recovering by pipetting or the like, or a method of recovering monocytes from the cell separation material can be used.

（式中、ｍはＴＭＳＭＡの重合度を表し、ｎはＮＡＳの重合度を表す） Experiment 1: Polymer synthesis

(Where m represents the degree of polymerization of TMSMA and n represents the degree of polymerization of NAS)

  By the following procedure, trimethoxysilylpropyl methacrylate (SIGMA-ALDRICH) (hereinafter sometimes referred to as “TMSMA”) and N-acryloxysuccinimide (Tokyo Chemical Industry Co., Ltd. (TCI)) (hereinafter referred to as “NAS”). In some cases, a polymer (hereinafter sometimes referred to as “poly (TMSMA-NAS)”) was synthesized.

  Three types of poly (TMSMA-NAS) having a molar ratio of TMSMA to NAS of 5: 5, 3: 7, and 1: 9 were prepared.

  First, a Schlenk tube that had been dried by applying heat while sucking with a diagram pump was replaced with nitrogen.

  As TMSMA, TMSMA in which a polymerization inhibitor was removed in advance through an alumina column was used.

  When the molar ratio of TMSMA to NAS is 5: 5, 2380 μL (10 mmol) of TMSMA and 1690 mg (10 mmol) of NAS are dissolved in 20 mL of N, N-dimethylformamide (DMF), and the Schlenk tube Put in. The monomer concentration in this solution was 198 g / L.

  When the molar ratio of TMSMA to NAS was 3: 7, 1430 μL (6 mmol) of TMSMA and 2370 mg (14 mmol) of NAS were dissolved in 20 mL of DMF and placed in the Schlenk tube. The monomer concentration in this solution was 185 g / L.

  When the molar ratio of TMSMA to NAS was 1: 9, 475 μL (2 mmol) of TMSMA and 3040 mg (18 mmol) of NAS were dissolved in 20 mL of DMF and placed in the Schlenk tube. The monomer concentration in this solution was 172 g / L.

  In each Schlenk tube, each solution was bubbled with nitrogen for 30 minutes under a nitrogen atmosphere, and then 0.2 mmol of azobisisobutyronitrile (AIBN) dissolved in 0.5 mL of DMF was added, and polymerization was performed. Started.

  Thereafter, the diagram pump was again used to suck in the air in the Schlenk tube, and then nitrogen was charged, and the reaction mixture was reacted in an oil bath at 70 ° C. The reaction was terminated by opening after 20 hours.

  When the molecular weight of the synthesized three types of poly (TMSMA-NAS) was estimated using a dynamic light scattering measurement device (Malvern, Zetasizer), compared with a polyethylene glycol (PEG) standard sample, All had a weight average molecular weight of about 10,000.

Experiment 2: The plastic container used as the substrate for the production of the substrate-polymer conjugate and the cell separation material was an easy grip cell culture dish made of Falcon having a diameter of 35 mm and a height of 10 mm (material: polystyrene). ).

  The plastic container was irradiated with UV for 30 minutes in an ozone atmosphere to make the surface hydrophilic. At this time, it is considered that at least one group out of a hydroxyl group, a carbonyl group and a carboxylic acid group was introduced to the surface of the container, and in particular, at least a hydroxyl group was assumed to be introduced.

  The reaction mixture containing each poly (TMSMA-NAS) obtained in Experiment 1 was diluted with dimethyl sulfoxide (DMSO) to prepare a solution having a concentration of 20 g / L in terms of the weight of the raw material monomer. About 2 mL of this solution was added to the vessel after UV / ozone treatment.

It left still at normal temperature (about 25 degreeC) overnight, and bound poly (TMSMA-NAS) to the surface in the said container.
Thereafter, the solution in the container was discarded, and the container was rinsed in the order of DMSO and distilled water.

  Water remaining in the container was removed by an air flow and heated for 1 minute in a range of 500 W or more to immobilize poly (TMSMA-NAS).

Thus, a plastic container (base material-polymer conjugate) in which three types of poly (TMSMA-NAS) were respectively immobilized was produced. Assuming that the total amount of poly (TMSMA-NAS) is bound to the inner bottom surface of the culture dish, when the TMSMA: NAS molar ratio of poly (TMSMA-NAS) is 1: 9, NAS When the unit is about 21 μmol / cm ² and the ratio is 3: 7, the inner bottom surface contains NAS unit about 15 μmol / cm ^2, and when the ratio is 5: 5, the inner bottom surface is NAS. It can be calculated when the unit is contained at about 10 μmol / cm ² .

Neomycin was then conjugated to poly (TMSMA-NAS).
As neomycin was used neomycin sulfate commercially available from Nacalai Tesque _{(C 23 H 46 N 6 O} 13 · 3H 2 SO 4 = 908.88). This neomycin sulfate is considered to consist essentially of the neomycin B sulfate.

Neomycin was adjusted with phosphate buffered saline (PBS) at pH 7.2 to a concentration of 10 mM to prepare a neomycin solution. About 2 mL of this neomycin solution was placed in a plastic container in which poly (TMSMA-NAS) was immobilized, and left overnight in an incubator adjusted to 37 ° C.
Thereafter, the solution in the container was rinsed, the container was washed with distilled water, and air was applied to remove moisture.

Next, in order to remove the unreacted succinimide group, about 2 mL of a 1% by volume ethanolamine aqueous solution was put in a container and allowed to stand for 2 hours.
After standing for 2 hours, the liquid in the container was discarded, washed with distilled water, and water was removed with an air stream.

In this way, a plastic container (cell separation material) in which neomycin was bonded via a polymer was prepared. In the above treatment, if the total amount of neomycin is bound to the inner bottom surface of the plastic container, neomycin will be present on the surface at a density of about 2 μmol / cm ² .

  The XPS (Kratos AXIS-ULTRA DLD) was measured on the cell culture dish on which the polymer was immobilized to confirm neomycin immobilization. In FIG. 1, (A) shows the peak intensity of N (1s) in a cell culture dish in which the polymer is immobilized but the polymer is not modified with neomycin, and (B) is the polymer immobilized and The peak intensity of N (1 s) in a cell culture dish in which the polymer is modified with neomycin is shown. Since the peak intensity of N (1s) increased significantly before and after modification with neomycin, it was confirmed that neomycin was immobilized (FIG. 1).

Experiment 3: Separation of monocytes using cell separator and differentiation into dendritic cells (Example 1)
Mononuclear cells were collected by the Ficoll method from human peripheral blood (human peripheral blood derived from a specific human used herein is referred to as “human peripheral blood A”). After washing the mononuclear cells with PBS, they were resuspended in RPMI 1640 medium, and the number and composition of mononuclear cells were measured with a SYSMEX blood cell counter.

Next, the prepared mononuclear cell suspension was added to a polystyrene cell culture dish on which neomycin-modified poly (TMSMA-NAS) (TMSMA: NAS = 3: 7 (molar ratio)) manufactured in Experiment 2 was fixed. After adding 6 × 10 ⁵ PBMCs (peripheral blood mononuclear cells) / cm ² , the plate was allowed to stand for 60 minutes in a 37 ° C., 5% CO ₂ incubator. After standing for 60 minutes, the supernatant was collected, and the cell culture dish was washed twice with 2 mL of physiological saline. The collected liquid was concentrated, and the cell number and composition were measured with a blood cell counter.

The adsorption rate of monocytes was determined by the following formula (1).
Monocyte adsorption rate (%)
= (1- (number of monocytes recovered from the container / number of monocytes before seeding)) × 100 (1)
As a result, it was found that the monocyte adsorption rate was 73.95%.

(Example 2)
A neomycin-modified poly (TMSMA-NAS) (TMSMA: NAS = 3: 7 (molar ratio) was obtained by the same procedure as in Experiment 2 except that a slide glass (MATSANAMI) was used instead of the polystyrene cell culture dish as a substrate. The slide glass which fixed)) was manufactured.

  Mononuclear cell suspension from human peripheral blood derived from a human different from human peripheral blood A used in Example 1 (referred to as “human peripheral blood B”) in the same procedure as described in Example 1 Was prepared.

  Monocytes were separated from the mononuclear cell suspension using the slide glass, and the adsorption rate of monocytes was determined. As a result, it was found that the monocyte adsorption rate was 73.10%.

(Example 3)
The molar ratio of TMSMA: NAS was 1: 9, 3: 7, 5: 5 by the same procedure as in Experiment 2 except that a slide glass (MATSANAMI) was used instead of the polystyrene cell culture dish as the substrate. A slide glass on which a kind of neomycin-modified poly (TMSMA-NAS) was fixed was produced.

  Human peripheral blood-derived mononuclear cells prepared from human peripheral blood (referred to as “human peripheral blood C”) derived from a human different from the above-mentioned human peripheral blood using the slide glass in the same procedure as in Example 1 above. Monocytes were separated from the suspension, and the adsorption rate of monocytes was determined. As a result, when the ratio is 1: 9, the monocyte adsorption rate is 80.47%, 3: 7, the monocyte adsorption rate is 85.51%, and when the ratio is 5: 5, the monocyte adsorption rate is 73. It was found to be 39%.

(Comparative Example 1)
From the suspension of mononuclear cells derived from human peripheral blood A in the same procedure as in Example 1 except that the polystyrene cell culture dish used as the base material in Experiment 2 was used without fixing poly (TMSMA-NAS). Monocytes were separated and the adsorption rate of monocytes was determined. As a result, the adsorption rate of monocytes was 55.27%.

(Comparative Example 2)
A monocyte from a human peripheral blood B-derived mononuclear cell suspension was prepared in the same manner as in Example 2 except that the slide glass used as a base material in Example 2 was used without fixing poly (TMSMA-NAS). And the adsorption rate of monocytes was determined. As a result, the adsorption rate of monocytes was 24.39%.

Example 4
(1) Preparation of Cell Separation Material and Monocytes Neomycin-modified poly (TMSMA-NAS) was prepared in the same manner as in Experiment 2 except that a glass dish (TOP) was used instead of the polystyrene cell culture dish as the base material. ) (TMSMA: NAS = 3: 7 (molar ratio)) fixed glass dish was produced.

In accordance with the method described in Example 1, a mononuclear cell suspension prepared from human peripheral blood derived from a human different from the above human peripheral blood (referred to as “human peripheral blood D”) is used as the neomycin-modified poly. (TMSMA-NAS) (TMSMA: NAS = 3: 7 (molar ratio)) After adding 6 × 10 ⁵ pieces of PBMC / cm ^{2 to} a fixed glass dish (TOP), a 37 ° C., 5% CO ₂ incubator It was left still for 60 minutes. After standing for 60 minutes, the supernatant was removed, 2 mL of phosphate buffered saline (PBS) was added, and the cell culture dish was gently stirred to perform a washing operation to remove the supernatant. By repeating this washing operation twice, the non-captured cells were washed away. The adsorption rate of monocytes was 73.88%.

Thereafter, RPMI 1640 (GIBCO, 10% of fetal bovine serum (FBS) prepared so that interleukin 4 (IL-4): 500 IU / mL, granulocyte macrophage colony stimulating factor (GM-CSF): 500 IU / mL was added. (Completed) 2 mL of the medium was added and cultured in a 37 ° C., 5% CO ₂ incubator for 5 days. This culture promoted the differentiation of monocytes into dendritic cells.

  Thereafter, in order to mature dendritic cells, tumor necrosis factor α (TNFα): 10 ng / mL and prostaglandin E2 (PGE2): 1 μg / mL were added to a medium supplemented with IL-4 and GM-CSF. Reagents were added, the medium was changed, and culture was further performed for 2 days (7 days in total).

(2) Collection of dendritic cells After 7 days in total, the cells in the cell culture dish were collected by pipetting, and the number of cells was determined with a hemocytometer.

(3) Identification of dendritic cells 100 μL of the cell suspension obtained by the dendritic cell collection operation is taken into a polypropylene test tube, and an anti-CD83 antibody (FITC labeled) solution and an anti-CD86 antibody (PE labeled) 5 μL of each solution (manufactured by Biolegend) was added and allowed to stand for 30 minutes in a cool dark place. After standing for 30 minutes, centrifugation was performed at 1500 rpm for 5 minutes, and after removing the supernatant, 0.5 mL of PBS was added and measurement was performed with a flow cytometer. As a result of the measurement, the surface antigens CD83 + and CD86 + were used as mature dendritic cells, and the positive rate was determined. Positive rate means the percentage of the total number of cells stained by fluorescence.

The number of mature dendritic cells was determined from the following formula (2).
(Number of mature dendritic cells recovered from petri dishes)
= (Total number of cells collected from petri dish) × (dendritic cell positive rate) (2)
As a result, the number of collected dendritic cells was 4.3 × 10 ⁵ .
Dendritic cell positive rate was 76.5%.

(Comparative Example 3)
A mature cell was prepared from a human peripheral blood D-derived mononuclear cell suspension by the same procedure as in Example 4 except that the glass dish was used without fixing poly (TMSMA-NAS) in Example 4. The number of mature dendritic cells was determined. As a result, the adsorption rate of monocytes was 60.75%. The number of collected dendritic cells was 3.0 × 10 ⁵ .
Dendritic cell positive rate was 76.2%.

Experiment 4: Preparation of polymer from NAS and compound having radical polymerizable double bond N-acryloxysuccinimide (NAS) has not only trimethoxysilylpropyl methacrylate (TMSMA) but also radical polymerizable double bond It can also be polymerized with other compounds to form a polymer. Therefore, as an example of a compound having a radical polymerizable double bond, 2-methacryloyloxyethyl phosphorylcholine (MPC) is used to randomly copolymerize NAS and MPC to form a polymer (hereinafter referred to as “poly (NAS-MPC)”). Experimental results are shown in which the NAS unit is modified with neomycin. From this experimental result and the experimental result obtained by synthesizing poly (TMSMA-NAS) in Experiments 1 and 2 above and modifying the NAS unit with neomycin, a poly which is a polymer obtained by random copolymerization of NAS, TMSMA and MPC. (TMSMA-NAS-MPC) is synthesized and shows that the NAS unit can be modified with neomycin.
The NAS and neomycin used in the following tests are the same as those used in Experiments 1 and 2.

（式中、ｎはＮＡＳの重合度を表し、ｐはＭＰＣの重合度を表す）

(Where n represents the degree of polymerization of NAS and p represents the degree of polymerization of MPC)

  NAS 5 mmol (846 mg), MPC (Sigma Aldrich) 5 mmol (1480 mg) and 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPD) 27 mg were dissolved in 10 mL of water / DMSO, and under nitrogen atmosphere, When polymerized at 70 degrees for 1.5 hours, poly (NAS-MPC) was obtained. The resulting polymer was dialyzed and purified.

  A neomycin 10 mM aqueous solution (PBS pH 7.2) was added to 23 mg of poly (NAS-MPC), and the mixture was stirred for 12 hours. Thereafter, dialysis was performed with purified water for 1 day to remove unreacted neomycin and salts contained in the buffer. Analysis by NMR confirmed that all succinimide groups contained in the NAS unit were replaced with neomycin.

Experiment 5: Binding of neomycin under various pH conditions It is known that neomachine B has 6 amino groups and has different pKa values. Ozen et al. , J .; Am. Chem. Soc. According to 2006, 128, 15248-15254, the pKa value of each amino group in the neomycin B of the free form is 1st position: 7.9, 3rd position: 5.7, 2 'position: 7.4, 6' position: 8.1, 2 ″ ″ position: 7.7, 6 ″ ″ position: 8.7. Therefore, in order to confirm the pH dependency of the modification reaction of the NAS unit in the polymer by neomycin, the reactivity with neomycin was confirmed using poly (NAS-MPC) produced in Experiment 4 above under different pH values. did. The results of this experiment are equally valid for the pH dependence in the modification reaction of poly (TMSMA-NAS) immobilized on a substrate with neomycin. In this experiment, poly (TMSMA-NAS) was not used because the poly (TMSMA-NAS) was fixed to the substrate via a trimethylsilyl group and whether or not neomycin was bonded to the NAS unit. This is because it cannot be confirmed.

The neomycin used in the following tests is the same as that used in experiments 1 and 2.
(Reaction at pH 3)
Poly (NAS-MPC) 23 mg was dissolved in purified water and 10 mL of neomycin 10 mM solution was added (purified water). The pH of the resulting solution was examined without adjusting the pH, and it became 3-4 acidic. . After reacting for 12 hours, dialyzed with purified water and freeze-dried, the introduction of neomycin was confirmed by NMR, but neomycin was not introduced.

(Reaction at pH 5)
23 mg of poly (NAS-MPC) was dissolved in an acetate buffer (around pH 5.0), and 10 mL of a neomycin 10 mM solution was added (acetate buffer). After reacting for 12 hours, dialyzed with purified water and freeze-dried, the introduction of neomycin was confirmed by NMR, but neomycin was not introduced.

(Reaction at pH 7.2)
As described in Experiment 4, all succinimide groups contained in the NAS unit were replaced with neomycin.

(Reaction at pH 8.0)
Poly (NAS-MPC) 23 mg was dissolved in sodium carbonate buffer (pH around 8.0), and 10 mL of neomycin 10 mM solution was added (sodium carbonate buffer). After reacting for 12 hours, dialysis was performed with purified water, and after freeze-drying, the introduction of neomycin was confirmed by NMR. Neomycin was introduced quantitatively by the amount of NAS.

Claims (17)

  A cell separation material comprising a substrate and an aminoglycoside antibiotic linked to the substrate.   The cell separation material according to claim 1, wherein the base material and the aminoglycoside antibiotic are linked via a linker. The cell separation material according to claim 2, wherein the substrate and the linker are linked by a covalent bond, and the linker and the aminoglycoside antibiotic are linked by a covalent bond. The linker is a polymer;
The substrate is connected to a side chain of the polymer, and
The cell separation material according to claim 2 or 3, wherein the aminoglycoside antibiotic is linked to a side chain of the polymer.   The cell separation material according to any one of claims 2 to 4, wherein the linker is a polymer containing a main chain composed of carbon atoms linked by a carbon-carbon single bond. The linker is
A repeating unit 1 comprising a linking group linked to the substrate;
The repeating unit 2 containing a group containing the aminoglycoside antibiotic, and the repeating unit 1 and the repeating unit 2 are polymers obtained by random copolymerization according to any one of claims 2 to 5. The cell separation material described. The linker is
A repeating unit 1 comprising a linking group linked to the substrate;
A polymer comprising a repeating unit 2 containing a group containing the aminoglycoside antibiotic,
Said repeat unit 1 is represented by formula I:

(In Formula I, R ¹¹ is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group;
R ¹² is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group;
R ¹³ is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group,
L ¹⁴ is a group having 1 or more atoms other than hydrogen and a valence p, wherein p is an integer of 2 or more and 4 or less,
R ¹⁵ is a linking group linked to the substrate)
The cell separation material of any one of Claims 2-6 represented by these. The linker is
A repeating unit 1 comprising a linking group linked to the substrate;
A polymer comprising a repeating unit 2 containing a group containing the aminoglycoside antibiotic,
The repeating unit 2 is represented by the formula II:

(In Formula II, R ²¹ represents hydrogen, an alkyl group, an aromatic group, or an aromatic alkyl group;
R ²² is hydrogen, an alkyl group, an aromatic group, an aromatic alkyl group or a carboxy group,
R ²³ is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group,
L ²⁴ is a group having 1 or more atoms other than hydrogen and a valence of q, where q is an integer of 2 or more and 4 or less,
R ²⁵ is a group containing an aminoglycoside antibiotic)
The cell separation material of any one of Claims 2-7 represented by these. A method for producing a cell separation material according to any one of claims 2 to 8,
A substrate;
A linker comprising a linking group connectable to the substrate and a reactive group capable of forming a bond with the aminoglycoside antibiotic,
A linker linking step of linking via the linking group to form a substrate-linker conjugate;
The cell separation material according to any one of claims 2 to 8, wherein the reactive group in the substrate-linker conjugate is reacted with an aminoglycoside antibiotic, and the aminoglycoside antibiotic is bound to the linker. Forming an antibiotic binding step. In the linker linking step, the linking group and the substrate are linked by a covalent bond,
The method according to claim 9, wherein in the antibiotic binding step, the reactive group and the aminoglycoside antibiotic are linked by a covalent bond.   The method according to claim 9 or 10, wherein the linker before linking to the substrate is a polymer containing the linking group and the reactive group in a side chain.   The method according to any one of claims 9 to 11, wherein the linker before linking to the substrate is a polymer including a main chain composed of carbon atoms linked by a carbon-carbon single bond. The linker before connecting to the substrate is
Repeating unit 1 containing the linking group;
The method according to any one of claims 9 to 12, which is a polymer comprising a repeating unit 2 containing the reactive group, wherein the repeating unit 1 and the repeating unit 2 are randomly copolymerized. The linker before connecting to the substrate is
Repeating unit 1 containing the linking group;
A polymer comprising the repeating unit 2 containing the reactive group,
The repeating unit 1 is represented by the formula I ′:

(In Formula I ′, R ¹¹ represents hydrogen, an alkyl group, an aromatic group, or an aromatic alkyl group;
R ¹² is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group;
R ¹³ is hydrogen, an alkyl group, an aromatic group, or an aromatic alkyl group. L ¹⁴ is a group having 1 or more atoms other than hydrogen and a valence p, where p is 2 or more and 4 or less. An integer,
R ¹⁶ is a linking group that can be linked to a substrate)
The method of any one of Claims 9-13 represented by these. The linker before connecting to the substrate is
Repeating unit 1 containing the linking group;
A polymer comprising the repeating unit 2 containing the reactive group,
Said repeating unit 2 is of formula II ′:

(In Formula II ′, R ²¹ represents hydrogen, an alkyl group, an aromatic group, or an aromatic alkyl group;
R ²³ is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group,
R ²⁶ is hydrogen, an alkyl group, an aromatic group or an aromatic alkyl group,
R ²⁷ is a compound of formula III

(In Formula III,
L ²⁴ is a group having 1 or more atoms other than hydrogen and a valence of q, where q is an integer of 2 or more and 4 or less,
R ²⁸ is a leaving group displaceable by the aminoglycoside antibiotic or a reactive group capable of forming a bond with a functional group of the aminoglycoside antibiotic,
* Indicates binding)
A group represented by
Or
R ²⁶ and R ²⁷ together form Formula IV

(In formula IV, * represents a bond)
A divalent group represented by
The method of any one of Claims 9-14 represented by these. A method for separating cells in a sample, comprising:
A method comprising a cell capturing step of contacting the cell separation material according to any one of claims 1 to 8 with a sample and capturing cells in the sample with the cell separation material. A method for inducing differentiation of a cell having differentiation ability,
A cell separation step of separating cells having differentiation ability from the method according to claim 16;
A differentiation induction step of inducing differentiation of the cells separated in the cell separation step.

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