WO2019103103A1 - Method for separating cells, and particles and kit for cell separation or concentration - Google Patents

Abstract

One embodiment of the present invention relates to a method for separating cells, and to particles and a kit for cell separation or concentration. Said method is for separating target cells or non-target cells and comprises the following steps 1 and 2. The following organic polymer-containing magnetic particles comprise an organic polymer and magnetic particles. The content of the magnetic particles in the organic polymer-containing magnetic particles is ≥40 mass%. The volume-average particle diameter of the organic polymer-containing magnetic particles is 10-1000 nm. Step 1 is for bringing a material containing the target cells into contact with organic polymer-containing magnetic particles.　Step 2 is for magnetically separating complexes of organic polymer-containing magnetic particles and target cells or non-target cells that are produced in step 1.

Description

Method of separating cells, particle and kit for cell separation or enrichment

One embodiment of the present invention relates to a method of separating cells, particles for cell separation or enrichment, and a kit.

Identification and separation of target cells (hereinafter also referred to as "target cells") from a mixture containing cells and undesirable impurities and the like are performed. Examples of methods for identifying and separating such cells include the following methods 1) to 3).

1) Identification and separation of cells by cell-permeant dyes: Each cell uses cell-permeant dyes according to the form of whole cells, the ratio of nuclei occupied in whole cells, and the type and amount of granules present in cytoplasm. Can be identified. When nuclei or intracellular granules are targeted for identification, they are often detected by staining with a dye. For example, when it is desired to observe nuclei, methods such as staining with orcein acetate or carmine acetate, papanicolaou stain, DAPI stain and the like can be mentioned. In addition to staining with a dye and detection with visible light, it can also be observed as a fluorescent image by fluorescent staining. For detection, a method of visual identification by visual observation under a microscope and a method of identification as an image by a CCD camera or the like have been put to practical use. For example, tests such as bladder cancer and urethral cancer by atypical cell test appearing in urine, identification of atypical cells in blood, and cancer tests by cytology in tissues can be mentioned.

2) Identification and separation of cells by cell surface antigen (marker) staining by fluorescent antibody method: Generally, cell separation or flow by cell sorter is a method of staining cell surface antigens called CD markers with a specific fluorescently labeled antibody. It is used for cancer examinations by cytometer and tissue staining. Of course, these are often used not only in the medical field but also in cell physiology research and industrial use of cells.

The methods 1) and 2) are extremely useful methods if they only identify cells and examine their morphological differences. In particular, the method 2) can be finely classified, and is an indispensable method for histologic research and examination, and cell separation by a cell sorter. However, there are problems in separating cells and then culturing and using the separated cells. That is, since modification of a cell surface antigen with a fluorescently labeled antibody is usually irreversible, the fluorescently labeled antibody on the cell surface remaining after cell separation may impair cellular function. In particular, depending on the fluorescent substance used, this often prevents contact with other antibodies or ligands, as it is necessary to attach a sufficient amount of fluorescently labeled antibody to the cell surface to identify the cell. And cell function is impaired.

Separation and concentration of target cells from cells and mixtures containing undesirable impurities is a difficult task. When the target cells are present in the culture solution, in the biological sample, or in a mixture thereof, to separate and concentrate the target cells, the target cells are captured and substantially all the cells are intact, That is, this is particularly difficult because it is necessary to capture without killing cells that may lead to the release of cell fragments that further contaminate the mixture etc., or to capture without lysis of the target cells. For this reason, reagents used in the cell separation and concentration steps can ideally capture cells efficiently and in a sufficient cell density range, so that cell wall lysis does not occur, and further unintended cell activity And differentiation induction does not occur. In addition, reagents used are cells, and analysis of cells themselves and / or cell secretions and extracts, etc., downstream of flow cytometry analysis, ELISpot assay, PCR or other analysis methods, etc. It is also required not to affect the process.

As a method suitable for such separation and concentration of target cells, the following method 3) can be mentioned.
3) Identification and separation of cells by affinity to a carrier on which another substance that specifically binds to a target substance is immobilized: separation and separation of another substance to which the target substance specifically binds, such as antibody, sugar chain and lectin Affinity separation of a target substance can be performed by using a ligand bound to a carrier such as microparticles or beads. Generally, a material that facilitates separation by magnetic force or gravity can be used for this purpose, but in recent years, separation means by magnetic force (magnetic substance-containing particles) is selected because of high specificity and easy handling. ing.

The separation using the magnetic substance-containing particles can be classified based on the size (volume average particle diameter) of the magnetic substance-containing particles. That is, it can be classified into large (more than 1.5 μm to about 50 μm), medium (0.1 μm to 1.5 μm), and small (<100 nm) also called nanoparticles.

Typical examples of large magnetic substance-containing particles (in particular, volume average particle diameter of more than 1.5 μm and about 50 μm or less) are described in, for example, Patent Document 1 and manufactured by Thermo fisher scientific. Such a magnetic substance-containing particle can be easily separated by a simple laboratory magnet since the content of the magnetic substance contained in one particle is large. In addition, they are considered to be suitable for cell separation as they can be easily dispersed when the magnetic field is removed.

However, on the other hand, there are also many problems with the large magnetic substance-containing particles. For example, when cells are incubated with large magnetic substance-containing particles such as Dynabeads (manufactured by Thermo fisher scientific), the collisions caused by mixing the systems because the magnetic substance-containing particles are too large to effectively diffuse. It is necessary to label the cells by Thus, when the cells are only slightly present in the population, the probability of labeling the target cells is considered to be related to the number of magnetic substance-containing particles added to the system and the length of mixing time. Since mixing cells with the magnetic substance-containing particles for a considerable period of time is harmful to the cells, it is necessary to increase the concentration of magnetic substance-containing particles as much as possible, but depending on the amount of magnetic substance-containing particles that can be used. There is a limit. In addition, when the amount of the magnetic substance-containing particles used increases, the cost increases. Furthermore, even though large magnetic-containing particles are very simple in design and can be magnetically separated by a relatively low magnetic field, large magnetic-containing particles tend to form cage-like clusters around the cells. Tend to be difficult to observe and analyze. Therefore, it is necessary to release the magnetic substance-containing particles from the target cells before analysis, and when the magnetic substance-containing particles are released, other problems tend to occur.

Here, Non-Patent Documents 1 and 2 show that a large continuous surface contact area is extremely important for effective CTL activation. Optimal stimulation is provided using class I alloantigens immobilized on latex microspheres of particle size 4 to 5 μm, the response decreases rapidly as particle size decreases, and even large numbers of small particles are effective In terms of CTL activation, it is described that it does not extend to particles having a large surface. That is, according to the descriptions in these documents, it is found that when large magnetic substance-containing particles (micro size) are used, the influence on the cells is large, and unintended cell activation or differentiation induction may occur. .

As a method of solving the above-mentioned problems, it is conceivable to use small magnetic substance-containing particles (<100 nm). A typical example of such a particle is described in US Pat. No. 5,677, 544, manufactured by Miltenyi Biotec (Germany). It is thought that the problem can be solved by using such magnetic material-containing particles. However, since such conventional small magnetic substance-containing particles have relatively weak magnetic response, it is necessary to use a method or the like that generates a large magnetic field gradient such as high gradient magnetic separation (HGMS).

The HGMS method uses a column packed with fine steel wool, steel gauze or steel microbeads, etc., and the column is placed adjacent to a magnet to form a very steep gradient system to enhance the magnetism How to However, with the HGMS column, there is a problem that components in the sample can be trapped in the column. Therefore, methods using HGMS columns etc. are undesirable, especially when the cells being captured at low frequency are the purpose of separation. Furthermore, there is also the problem that using an HGMS column increases the cost.

U.S. Pat. No. 4,654,267 U.S. Pat. No. 5,693,539

Molecular Therapy, Vol. 16, No. 4, 765-772, April 2008 J Immunol 149: 2402-2405, 1992

One embodiment of the present invention is capable of separating and concentrating cells while suppressing cell death and cell activation, such as an HGMS column causing errors in separation performance and causing cost increase. Provided are particles and methods capable of sufficiently separating and concentrating cells without using consumables.

As a result of earnest studies to solve the above problems, the inventor of the present invention has found that the problems can be solved according to the following configuration example and the like, and has completed the present invention.
In the present specification, the descriptions “A to B” representing the numerical value range and the like mean A or more and B or less, and include A and B in the numerical value range.

[1] including the following steps 1 and 2,
The following organic polymer-containing magnetic particles contain organic polymer and magnetic particles, and the content of magnetic particles in the organic polymer-containing magnetic particles is 40% by mass or more, and the volume average particle size of the organic polymer-containing magnetic particles 10 to 1000 nm in diameter,
A method of separating target cells or non-target cells.
Step 1 of contacting the sample containing the target cells with the organic polymer-containing magnetic particles
Step 2 of magnetically separating the complex of the organic polymer-containing magnetic particle and the target cell or the non-target cell, generated in the step 1

[2] The method according to [1], wherein the organic polymer-containing magnetic particles have a polymer layer on the surface of at least a part of particles containing the organic polymer and the magnetic particles.
[3] The method according to [2], wherein the polymer is a hydrophilic polymer.
[4] The hydrophilic polymer is (meth) acrylic acid, methyl (meth) acrylic acid, diacetone (meth) acrylamide, (meth) acrylamide, dimethyl (meth) acrylamide, dimethylaminopropyl (meth) acrylamide, dimethylaminopropyl (Meth) acrylamide hydrochloride, N, N-diethyl (meth) acrylamide, N-isopropyl (meth) acrylamide, glycerol (meth) acrylamide, (meth) acrylamide-N-glycolic acid, hydroxyethyl (meth) acrylate, hydroxybutyl (Meth) acrylate, ethylene glycol mono (meth) acrylate, 2-sulfoethyl (meth) acrylate, phosphorus-containing (meth) acrylic ester, dimethylaminoethyl (meth) acrylate, diethylamide Noethyl (meth) acrylate, t-butylaminoethyl (meth) acrylate, glycidyl (meth) acrylate, glycerol (meth) acrylate, vinyl sulfone, styrene sulfonic acid, 2- (meth) acrylamido-2-methylpropane sulfonic acid, Meta) acryloyl morpholine, N-vinyl pyrrolidone, N-vinyl caprolactam, N-vinyl oxazolidone, N-vinyl succinimide, vinyl pyridine, vinyl acetate, itaconic acid, crotonic acid, N-vinyl imidazole, vinyl benzyl ammonium salt, vinyl benzyl The method according to [3], which is a polymer comprising a structural unit derived from at least one monomer selected from the group consisting of alcohol and hydroxystyrene.

[5] The method according to any one of [1] to [4], wherein the coefficient of variation of the volume average particle diameter of the organic polymer-containing magnetic particles is 25% or less.

[6] The method according to any one of [1] to [5], wherein the organic polymer is crosslinked.

[7] The method according to any one of [1] to [6], wherein the volume average particle diameter of the magnetic particles is 5 to 25 nm.

[8] The method according to any one of [1] to [7], wherein at least one ligand is physically adsorbed or chemically bonded to the organic polymer-containing magnetic particle.
[9] At least one member selected from the group consisting of antibodies, antigens, nucleic acids, nucleotides, nucleotides, nucleosides, proteins, peptides, amino acids, polysaccharides, sugars, lipids, vitamins, drugs, substrates, hormones and neurotransmitters The method described in [8].

[10] An organic polymer-containing particle comprising an organic polymer and an inorganic particle,
The content of inorganic particles in the organic polymer-containing particles is 40% by mass or more,
The volume average particle size of the organic polymer-containing particles is 10 to 1000 nm,
Particles for cell separation or concentration.
[11] The particle according to [10], wherein the inorganic particle is a magnetic particle.

[12] The particle according to [10] or [11], wherein the organic polymer-containing particle has a polymer layer on the surface of at least a part of a particle containing an organic polymer and an inorganic particle.
[13] The particle according to [12], wherein the polymer is a hydrophilic polymer.

[14] The particle according to any one of [10] to [13], and
Including a member for separating the particles,
kit.

According to one embodiment of the present invention, cells can be separated and concentrated with little influence on cells (cell death and cell activation).
Furthermore, according to an embodiment of the present invention, since cells can be sufficiently separated and concentrated without using consumables such as HGMS columns, errors in cell separation performance can be minimized. Also, cost reduction effects can be expected.

FIG. 1 is a plot by a flow cytometer showing the results of cell separation and concentration in Example 1 (vertical axis: FSC, horizontal axis: FL-2, left view: plot using adjusted PBMC solution, right Figure: Plot after cell separation and concentration).

«Particle for cell separation or concentration»
The particle for cell separation or concentration according to one embodiment of the present invention (hereinafter also referred to as "the present separated particle") is an organic polymer-containing particle containing an organic polymer and an inorganic particle, wherein the organic polymer-containing particle The content of the inorganic particles therein is 40% by mass or more, and the volume average particle diameter of the organic polymer-containing particles is 10 to 1000 nm. The separated particles are also particles used to separate or concentrate cells.
Such present separated particles are particles having less influence on cells. Further, since the present separated particles contain a high content of inorganic particles, cells can be separated at a high recovery rate by using the particles.

In addition, the influence on the cells is to cause an unintended cell death or stimulation response of the cells to the target cells before and after use. The allowable range of the rate at which the target cells are affected is 10% or less, preferably 5% or less, more preferably 1% or less. If the ratio exceeds 10%, it tends to be difficult to secure the required amount of cells. The rate at which the target cells are affected is, for example, the relationship between the cell viability before cell separation and the cell viability after cell separation based on the method described in Example 3 below ((Before cell separation Cell viability-Cell viability after cell separation / cell viability before cell separation x 100) or the degree of cell activation before and after cell separation based on the method described in Example 4 below (| It can be judged from the number of stained cells before cell separation−the number of stained cells after cell separation | / the number of stained cells before cell separation × 100).

The present separated particles can be used to separate target cells from a system in which target cells are present together with impurities, in order to separate target cells and non-target cells (hereinafter also referred to as “non-target cells”). More specifically, the target cell is separated, and then it is used for stimulation of proliferation to the separated cells, cell processing such as differentiation induction or gene transfer, cell analysis such as cell classification based on cell surface antigen analysis, etc. be able to.
Since the separated particles have little influence on the target cells (the possibility of changes in the target cells) before and after the separation and concentration of the cells, not only cell analysis in basic research, but also in the clinical and diagnostic fields using cells Are also suitably used. In particular, it can be expected to be used in the production of specific cell processed products, regenerative medical products and the like used in cell therapy.

The volume-average particle size (hereinafter, also simply referred to as “particle size”) of the separated particles has less influence on the target cells, can sufficiently separate and concentrate the cells, is excellent in handleability, and has inorganic particles 10 nm or more, preferably 50 nm or more, more preferably 100 nm or more, and 1000 nm or less, preferably less than 1000 nm, from the viewpoint of easily obtaining particles in which aggregation of the particles is difficult to occur even though the content is high. More preferably, it is 500 nm or less, still more preferably 200 nm or less.
If the particle size exceeds 1000 nm, the reactivity of the present separated particles with target cells and non-target cells is poor, so that the separation performance is achieved when the proportion of target cells contained in a sample such as a biological sample is small. Is worse. On the other hand, when the proportion of target cells contained in a sample such as a biological sample is high, the amount of main separation particles used is sufficient to secure the number of main separation particles necessary to separate the target cells. It increases, and the cost for separation and concentration increases. In addition, when the particle size is smaller than 10 nm, the Browning motion of the separated particles tends to deteriorate the magnetic response, and in order to sufficiently separate and concentrate the cells, external magnetic enhancement such as HGMS method May be required. When the particle size is 200 nm or less, the present separated particles can pass through a 0.22 μm sterile filter, which is advantageous in clinical use.
The particle size can be measured by a measuring device based on a dynamic light scattering method, for example, Nanotrac UPA-EX 150 (manufactured by Nikkiso Co., Ltd.).

The present separated particles having a particle size in the above range can increase the binding amount of the ligand that can be bound per unit mass of the particles. In particular, when the inorganic particle is a magnetic particle, even if it is a particle of the above-mentioned particle size, it becomes a particle excellent in the magnetic separation performance, so it is difficult to increase the ligand binding amount, which was conventionally difficult. It is compatible with magnetic separation.

The separated particles preferably have a coefficient of variation (CV value) of 25% or less, and more preferably 20% or less.
When the CV value is in the above range, particles having little variation and easily exhibiting desired characteristics can be easily obtained, and in particular, when the inorganic particles are magnetic particles, separation occurs during magnetic separation. It is preferable because time does not easily vary.
The CV value can be measured, for example, using a dynamic light scattering type particle size distribution measuring apparatus (Nanotrac UPA-EX 150, manufactured by Nikkiso Co., Ltd.), and specifically calculated by the following formula Can.
CV (%) = [particle size standard deviation (σ) / volume average particle size (D _n )] × 100

The present separated particles are not particularly limited as long as they include an organic polymer and inorganic particles, and other components other than these, for example, in the case of using a magnetic fluid when producing the particles, the conventional particles contained in the magnetic fluid It may contain known ingredients.

<Inorganic particles>
The inorganic particles are not particularly limited, but magnetic particles are preferable. The use of magnetic particles as the inorganic particles is a simple method for separating and concentrating cells, and is preferable because the cells can be separated and concentrated by magnetic separation with little influence on the target cells.
Although the present separated particles are also organic polymer-containing particles, preferably, the inorganic particles in the particles are organic polymer-containing magnetic particles (hereinafter also referred to as "present particles"), which are magnetic particles, and the following book The separation method is characterized by using the present particles (organic polymer-containing magnetic particles).

Specifically, the material of the inorganic particles is selected from the group consisting of iron, titanium, cobalt, zinc, copper, manganese, nickel, single substances such as nickel or gadolinium, oxides thereof, or alloys thereof; and ferrites Preferred are one or two or more inorganic materials and the like. Among them, metal oxides such as hematite which is an iron oxide; ferrites such as magnetite, manganese ferrite, nickel ferrite or manganese zinc ferrite from the viewpoint of obtaining the separated particles which are excellent in magnetic separation performance; cobalt alloy; And one or more selected from nickel alloys.

From the viewpoint of obtaining the separated particles excellent in magnetic separation performance, a material having superparamagnetism without residual magnetism is preferable. The material having superparamagnetic properties is not particularly limited, and examples thereof include various ferrites such as iron trioxide (Fe ₃ O ₄ ) and γ-iron trioxide (γ-Fe ₂ O ₃ ). In particular, metal oxides are preferred, and iron trioxide (Fe ₃ O ₄ ) is particularly preferred.

As the magnetic particles, a mixed solution containing Fe ²⁺ and Fe ^{3+ in} a ratio of 1: 2 is dropped to a basic solution, and Fe ₃ O ₄ or the like obtained by coprecipitation reaction is used. it can. In addition, magnetic particles contained in magnetic fluid such as EMG 2001 (manufactured by Farotech Co., Ltd.), and commercially available products such as ferricolloid HC-50 (manufactured by Taiho Kozai Co., Ltd.) can also be used.

The particle diameter of the inorganic particles in the present separated particles is preferably 5 nm or more, from the viewpoint of obtaining particles capable of sufficiently exhibiting the properties of the inorganic particles, and in particular obtaining the present particles excellent in magnetic separation performance. More preferably, it is 8 nm or more, preferably 25 nm or less, more preferably 20 nm or less, and further preferably 15 nm or less.

The dispersion diameter of the inorganic particles in the organic polymer is preferably 1 nm or more, and preferably 30 nm or less. If the dispersion diameter is less than 1 nm, in addition to the difficulty in producing the inorganic particles itself, the magnetic response characteristics in the case where the inorganic particles are magnetic particles tend to deteriorate and the magnetic separation performance tends to deteriorate. In addition, when the dispersion diameter exceeds 30 nm, when the inorganic particles are magnetic particles, residual magnetism tends to occur and self-aggregation tends to occur, and the magnetic particles are easily exposed on the surface of the separated particles. .
The dispersion diameter is more preferably 5 nm or more, and more preferably 20 nm or less.
The dispersion diameter can be measured using a transmission electron microscope (TEM).

[Content of inorganic particles]
The present separated particles have a content of inorganic particles of 40% by mass or more, preferably 50% by mass or more, and more preferably, from the viewpoint of little influence on the target cells and sufficient separation and concentration of the cells. It is 55% by mass or more, preferably 95% by mass or less, and more preferably 92% by mass or less.
The content of the inorganic particles is a value calculated from the weight change before and after heating when the main separated particles are heated at 500 ° C. for 20 minutes to volatilize the polymer component and use only the inorganic particles. In this measurement, for example, a differential type differential thermal balance (manufactured by Rigaku Corporation, TG-8120) can be used. When the content of the inorganic particles is less than 40% by mass, the cell separation / concentration performance is poor, and in particular, when magnetic particles are used as the inorganic particles, the magnetic response is poor, so the cells are sufficiently separated / enriched In order to achieve this, it is necessary to carry out means for improving separation, such as the HGMS method.

<Organic polymer>
The organic polymer has a role as a matrix of the present separated particles. The organic polymer does not include the following polymer layer, ligand, blocking agent and the like that can be contained in the present separated particles.
A known polymer can be used as the organic polymer, and is not particularly limited. For example, a polymer obtained by polymerizing a monomer having an ethylenically unsaturated bond is preferable. Examples of such monomers include styrenic monomers, vinyl chloride, vinyl esters, unsaturated nitriles, (meth) acrylic acid esters and derivatives thereof.

More specifically, styrene-based monomers such as styrene, α-methylstyrene, p-methylstyrene, p-chlorostyrene, chloromethylstyrene and the like; vinyl chloride; vinyl esters such as vinyl acetate and vinyl propionate; Unsaturated nitriles such as acrylonitrile; methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, stearyl (meth) acrylate, ethylene glycol (meth) (Meth) acrylic acid esters such as acrylate, trifluoroethyl (meth) acrylate, pentafluoropropyl (meth) acrylate, cyclohexyl (meth) acrylate, tetrahydrofurfuryl (meth) acrylate and derivatives thereof, etc. It is not limited to these examples.
These monomers may be used alone or in combination of two or more.

The organic polymer is preferably a polymer containing a structural unit derived from a styrenic monomer, from the viewpoint of obtaining the separated particles excellent in dispersibility in an aqueous medium and the like.
The content of the structural unit derived from the styrenic monomer is preferably 100% by mass of the total amount of the organic polymer contained in the present separated particles, from the viewpoint that particles excellent in dispersibility in the aqueous medium can be obtained. 60 mass% or more, more preferably 70 mass% or more, further preferably 80 mass% or more, preferably 100 mass% or less, more preferably 97 mass% or less, still more preferably 93 mass% or less.

The organic polymer is preferably crosslinked, and the degree of crosslinking is preferably 3% or more, more preferably 5% or more, and still more preferably 7% or more. When the degree of crosslinking is equal to or more than the above-mentioned value, it is possible to reduce that the hydrophilic polymer which may be contained in the polymer layer described later behaves as a movable matrix, and the separated particles are adsorbed or adsorbed and aggregated. It can be reduced.
The degree of crosslinking can be calculated, for example, from the preparation amount when using the following crosslinking monomer (when using the following crosslinking monomer, use of the crosslinking monomer with respect to the total amount of monomers used when synthesizing the organic polymer) Percentage of mass (mass%)).

The method for crosslinking the organic polymer is not particularly limited, and examples thereof include a method using a crosslinking monomer and a method using a crosslinking agent.
Examples of the crosslinkable monomer include divinylbenzene, ethylene glycol di (meth) acrylate, 1,6-hexanediol di (meth) acrylate, neopentyl glycol di (meth) acrylate, trimethylolpropane tri (meth) acrylate, Examples thereof include, but are not limited to, tetramethylolmethane tri (meth) acrylate, tetramethylolpropane tetra (meth) acrylate, diallyl phthalate and its isomer, triallyl isocyanurate and its derivative.
Examples of the crosslinking agent include organic peroxides, phenol resins, sulfur, sulfur compounds, p-quinones, derivatives of p-quinone dioximes, bismaleimide compounds, epoxy compounds, silane compounds, amino resins, polyols, and polyamines. Although a triazine compound and a metal soap are mentioned, it is not limited to these examples.
These crosslinking monomers and crosslinking agents may be used alone or in combination of two or more.

The content of the organic polymer in the present separated particles is such that the inorganic particles are magnetic particles, in that the present invention can be easily obtained by keeping the inorganic particles firmly and having excellent physical strength. In the case where the content of the inorganic particles is in the above range and the amount is capable of retaining the inorganic particles, in particular, from the viewpoint that the present particles excellent in magnetic separation performance can be obtained, etc. Although not preferred, it is preferably less than 20% by mass, more preferably 18% by mass or less, particularly preferably 15% by mass or less, preferably 5% by mass or more, more preferably 8% by mass or more, particularly preferably 10% by mass or more It is.

<Other ingredients>
When the magnetic particle is used in producing the particle, the present separated particle contains a conventionally known component contained in the magnetic fluid, and a conventionally known component such as a surfactant used in producing the particle. It may be. Examples of such conventionally known components include surfactants, and stabilizers other than the surfactants, such as acid group-containing compounds, amino group-containing compounds, silane group-containing compounds and titanium atom-containing compounds. It is not limited to these examples.
These conventionally known components may be used alone or in combination of two or more.

The surfactant is not particularly limited, and conventionally used compounds can be appropriately used. For example, oleate, carboxylate, sulfonate, sulfate, phosphate ester and the like can be used. Anionic surfactant; Cationic surfactant such as amino acid salt, quaternary ammonium salt; Ester type such as glycerin fatty acid ester, Ether type such as polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, fatty acid polyethylene glycol And nonionic surfactants such as ester and ether type; and amphoteric surfactants such as alkyl betaine.

Examples of the acid group-containing compound include compounds having a carboxy group or a sulfo group described in JP-A-2008-258564 and inorganic acids, but the invention is not limited thereto.
Examples of the amino group-containing compound include, but are not limited to, fluorine-containing amines described in JP-A-7-94315.
Examples of the silane group-containing compound include a silane group-containing surface treatment agent. Examples of the surface treatment agent include alkoxysilanes described in JP-A-10-4006, and JP-A-2004-205481. Although the silane compound of description is mentioned, it is not limited to these examples.
Examples of the titanium atom-containing compound include titanium coupling agents, and examples of the coupling agent include titanium triisostearoyl isopropoxide, (2-n-butoxycarbonylbenzoyloxy) tributoxytitanium, titanium acetylacetoate Nitrate, iso-butoxytitanium ethyl acetoacetate, tetraisopropyl titanate, tetra n-butyl titanate, but is not limited to these.

The present separated particles can be used as they are in various applications, but in order to provide a particle surface according to the desired application, particles containing an organic polymer and inorganic particles (the particles are also referred to as “base particles”). It is preferable to have a polymer layer on at least a part of the surface of the (a particle having a polymer layer on at least a part of the base particle hereinafter also referred to as "coated particle").
By the present separated particles having the polymer layer, unnecessary activation of the target cells can be further suppressed, and particles having lower toxicity to cells can be obtained.

[Polymer layer]
The components of the polymer layer are not particularly limited, but vinyl polymers are preferable, and as vinyl monomers used for the synthesis, aromatic vinyl monomers such as styrene, α-methylstyrene, halogenated styrene, divinylbenzene and the like Vinyl esters such as vinyl acetate and vinyl propionate; unsaturated nitriles such as acrylonitrile; methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, lauryl Ethylenically unsaturated carboxylic acid esters such as acrylate, ethylene glycol di (meth) acrylate, cyclohexyl (meth) acrylate and the like; acrolein; and the like, but not limited thereto.
The vinyl polymer may be a homopolymer or a copolymer of two or more monomers.

Further, the polymer layer may be the vinyl monomer and a conjugated diolefin such as butadiene and isoprene; (meth) acrylic acid, itaconic acid, a maleic anhydride, a mono- or dicarboxylic acid compound such as crotonic acid or an acid anhydride thereof; (Meth) acrylamide, glycidyl (meth) acrylate, N-methylol (meth) acrylamide, N-isopropyl (meth) acrylamide, 2-hydroxyethyl (meth) acrylate, glycerol mono (meth) acrylate, polyethylene having 2 to 40 chains Glycol or polypropylene glycol side chain (meth) acrylate, diallyl phthalate, allyl (meth) acrylate, trimethylolpropane tri (meth) acrylate, styrene sulfonic acid and its sodium salt, 2- Acrylamide-2-methylpropanesulfonic acid and its sodium salt, such as isoprene sulfonic acid and its sodium salt, may be a layer comprising a copolymer of the vinyl monomer copolymerizable with monomers.

The polymer layer is desirably a layer in which 30% by mass or more, preferably 50% by mass or more, more preferably 70% by mass or more of the polymer layer is composed of a hydrophilic polymer. When the amount of hydrophilic polymer in the polymer layer is less than 30% by mass, adsorption of cells other than target cells (target cells or non-target cells) is likely to occur and nonspecific cell separation may occur. There is sex.

In the present specification, hydrophilic means that the affinity to water is strong. Specifically, a polymer which dissolves by 1 g or more in 100 g of pure water at normal temperature (25 ° C.) is referred to as a hydrophilic polymer.

The polymer layer containing the hydrophilic polymer may be a homopolymer or a copolymer layer formed by polymerizing a hydrophilic monomer, or a layer obtained by hydrophilizing the formed polymer layer by chemical conversion, a hydrophilic polymer May be a directly coated layer.
Examples of the hydrophilic monomers include (meth) acrylic acid, methyl (meth) acrylic acid; diacetone (meth) acrylamide, (meth) acrylamide, dimethyl (meth) acrylamide, dimethylaminopropyl (meth) acrylamide, dimethylaminopropyl (meth) ) (Meth) acrylamide compounds such as acrylamide hydrochloride, N, N-diethyl (meth) acrylamide, N-isopropyl (meth) acrylamide, glycerol (meth) acrylamide, (meth) acrylamide-N-glycolic acid, etc .; Meta) acrylate, hydroxybutyl (meth) acrylate, ethylene glycol mono (meth) acrylate, 2-sulfoethyl (meth) acrylate, phosphorus-containing (meth) acrylate, dimethylaminoeth (Meth) acrylate compounds such as (meth) acrylate, diethylaminoethyl (meth) acrylate, t-butylaminoethyl (meth) acrylate, glycidyl (meth) acrylate, glycerol (meth) acrylate, etc .; vinyl sulfone, styrene sulfonic acid, 2 Sulfone compounds such as (meth) acrylamido-2-methylpropane sulfonic acid; (meth) acryloyl morpholine, N-vinyl pyrrolidone, N-vinyl caprolactam, N-vinyl oxazolidone, N-vinyl succinimide, vinyl pyridine, vinyl acetate Itaconic acid, crotonic acid, N-vinylimidazole, vinylbenzyl ammonium salt, vinyl benzyl alcohol, hydroxystyrene and the like, but not limited to these examples, and It may be used or two or more of them may be using a seed.

As the hydrophilic monomer, (meth) acrylic acid, methyl (meth) acrylic acid, hydroxyethyl (meth) acrylate, ethylene glycol mono (meth) acrylate, phosphorus-containing (meth) acrylic acid from the viewpoint of easy polymerization on the surface of the base particles, etc. More preferred is at least one selected from meta) acrylic ester, glycidyl (meth) acrylate, glycerol (meth) acrylate, (meth) acryloyl morpholine and itaconic acid.

The polymer layer is preferably crosslinked, and examples of the method of crosslinking include the same methods as the method of crosslinking the organic polymer described above.

The polymer layer preferably does not contain a movable matrix. According to JP-A-2015-533493, a magnetic substance-containing particle having a mobile polymer matrix (non-solid surface) such as Microbeads (manufactured by Miltenyi Biotec) adheres to the cell surface by its mobility. By deforming and acting like a large particle, it may induce stimulation to the cells, and the magnetic substance-containing particles having a movable matrix may cause unintended activation of cells and induction of differentiation. It is because there is.
As the movable matrix, a layer composed of polysaccharides such as dextran and polysaccharide is represented, and the characteristics are shown in Langmuir 2006, 22 (12), pages 5485-5490 (Bertholon et al.).

Whether or not to have a movable matrix is determined based on whether the values of the sizes obtained by the respective methods coincide or not when the particle size (particle diameter) is measured by two or more different methods. be able to. The particles having the movable matrix do not have the same size value obtained by each method.
Particles with mobile matrices are measured by Transmission Electron Microscopy (TEM) to have a diameter of a certain size, but Dynamic Light Scattering (DLS) is measured to have a much larger diameter than that. AMI-25 (manufactured by Advanced Magnetics), a clinical contrast reagent consisting of dextran matrix and embedded iron oxide nanoparticles, has a diameter of 5-10 nm according to TEM, According to DLS, it is 80-150 nm, which means that more than 99% of the total volume of particles in the aqueous solution is occupied by the mobile matrix (Wang et al. Eur. Radiol. 2001, page 2323)). That is, if the difference between the particle sizes measured by TEM and DLS or the like is small, it can be said that the matrix is not a movable matrix.
In the present separated particles, the ratio of the particle size measured by TEM to the particle size measured by DLS is preferably in the range of 0.5 or more and 1.5 or less.

The separated particles preferably have a surface capable of chemically bonding at least one ligand, and specifically, preferably have a surface containing a reactive functional group.
The reactive functional group is preferably a group to which an antigen, an antibody, or the like can be bound by covalent bonding, and may be appropriately selected depending on the desired application, for example, a carboxy group, NHS (N-hydroxysuccinimide Group, amino group, tosyl group, thiol group, maleimide group, dimethylamino group and sulfonic acid group.

The reactive functional group may be introduced using a monomer having the functional group, or may be introduced by chemical conversion.
Examples of the monomer having a functional group include the same compounds as the hydrophilic monomer, but the present invention is not limited thereto, and one or two or more of these may be used.

The content (thickness) of the polymer layer is not particularly limited, and it is preferable that the content of the inorganic particles in the obtained coated particles be in the above range.

The separated particles are particles in which at least one ligand is physically adsorbed, preferably a hydrophobic interaction, in that they can easily provide an excellent reaction field, particularly a specific reaction field, in reaction with cells. It is preferred that the particles be adsorbed or particles in which at least one ligand is chemically bonded, preferably, particles in which the ligand is covalently bonded to reactive functional groups on the particle surface.

[Ligand]
The type of the ligand is not particularly limited as long as it has an appropriate affinity for the cell to be separated (target cell or non-target cell). Specific examples of the ligand include proteins such as protein A, protein G, protein L, Fc binding protein, avidin, streptavidin, lectin, functional variants thereof, etc .; amino acids; peptides such as insulin; antibodies such as monoclonal antibodies; Antigens; Enzymes; Hormones; Nucleic acids such as DNA and RNA; Nucleotides; Nucleosides; Saccharides or polysaccharides such as Heparin, Lewis X, Gangliosides; Lipids; Vitamins such as Biotin; Drugs; Substrates; Neurotransmitters; And low molecular weight compounds such as 2-aminophenylboronic acid, 4-aminobenzamidine, glutathione and their derivatives. Although these ligands may be used as the compound as they are, fragments thereof obtained by enzyme treatment of these compounds may be used. Also, the ligand may be an artificially synthesized peptide or peptide derivative, or a recombinant.

<Method for producing main separated particles>
The method for producing the separated particles is not particularly limited, and examples thereof include methods using suspension polymerization, microsuspension polymerization, miniemulsion polymerization, dispersion polymerization and the like. Among them, since a particle having a small particle size can be easily produced, a method to which a miniemulsion polymerization method is applied is preferable.

Furthermore, as a method for producing the present particles, it is excellent in magnetic separation performance, and in terms of being able to easily produce particles of desired shape (the occurrence of aggregates is suppressed and which is substantially spherical) with high production efficiency. A method including the following steps (A) to (C) (hereinafter also referred to as “the present production method”) is more preferable.
Step (A): A step of mixing a magnetic fluid, a monomer and a polymerization initiator to prepare a fluid mixture (hereinafter referred to as "monomer mixture") Step (B): dispersing the monomer mixture to form an emulsion Step (C): Step of polymerizing the monomers in the emulsion

<Step (A)>
The said process (A) is a process of mixing a magnetic fluid, a monomer, and a polymerization initiator, and preparing a monomer liquid mixture.
In the step (A), such a monomer mixture liquid is prepared, and this particle mixture is used to produce the present particles through the subsequent steps, so a high content of magnetic particles (inorganic particles) is contained, particularly in the above range The present particles can be easily produced with high production efficiency. In particular, since the magnetic fluid is used in the step (A), the present particles can be produced in a state where the magnetic particles contained in the fluid are uniformly dispersed, and the magnetic particles (inorganic particles) in the organic polymer The present particles having a dispersion diameter in the above range can be easily produced, and desired particles in which the aggregation of magnetic particles is suppressed can be easily produced. Further, in the above step (A), in order to prepare a mixed solution containing a monomer and a polymerization initiator, the magnetic particles are uniformly dispersed in a matrix made of an organic polymer, and aggregation of the magnetic particles is less likely to occur. And particles having a magnetic particle content of 40% by mass or more can be easily obtained. In addition, since aggregation of magnetic particles is difficult to occur, control of the particle diameter and shape of the particles is easy, and the particle size distribution can be narrowed.

On the other hand, in the conventional method for producing organic polymer-containing magnetic particles, the magnetic particles are first dispersed in an organic solvent in order to finely disperse the magnetic particles in the organic polymer with a predetermined dispersion diameter. A particle dispersion is prepared, and specifically, magnetic particles are separated by removing water or an organic solvent which is a liquid medium from a magnetic fluid, and magnetic particles are dispersed by adding another solvent to the separated magnetic particles. A liquid was prepared, and a monomer mixture was prepared by adding monomers, a polymerization initiator and a cosurfactant thereto.
However, as a result of intensive investigations conducted by the present inventor, in such a conventional manufacturing method, the magnetic particles are aggregated with each other when removing the liquid medium from the magnetic fluid, and the dispersion state is deteriorated. It turned out that the particles contained in the content can not be easily obtained with high production efficiency.

In another conventional method for producing organic polymer-containing magnetic particles, a monomer and a polymerization initiator have been added to an emulsion containing magnetic particles.
However, as a result of intensive investigations conducted by the present inventor, in such a conventional production method, when polymerizing the monomer, it becomes a shape other than substantially spherical or aggregates are generated, and the magnetic particle content is high. It has been found that the organic polymer-containing magnetic particles of the desired shape contained in can not be easily produced with high production efficiency.

Therefore, in the present manufacturing method, the magnetic fluid is processed, for example, without using the step of removing the liquid medium from the magnetic fluid, the magnetic fluid is used as it is, and the fluid is mixed with the monomer and the polymerization initiator. It is preferred to prepare a mixture.

(Magnetic fluid)
The magnetic fluid used in step (A) contains magnetic particles.
Normally, the magnetic fluid is stabilized by using (a) magnetic particles having a diameter of several nm to several tens of nm, (b) a liquid (dispersion medium) such as water, an organic solvent or oil, and (c) magnetic particles as a dispersion medium. And a stabilizer for dispersing.
In the magnetic fluid, a stabilizer layer such as a surfactant is usually present on the surface of the magnetic particles, so that repulsion works between the magnetic particles, causing no aggregation or sedimentation, and the magnetic particles are stable in the fluid. Maintain a balanced state. The magnetic fluid behaves as a normal liquid when no magnetic field is generated, but when the magnetic field is applied, the viscosity of the liquid changes, and the whole liquid behaves as if it has ferromagnetism. have. In addition, even when an external force such as a magnetic field, gravity, or centrifugal force is applied from the outside, the dispersed state of the magnetic particles in the fluid is maintained, and therefore, the magnet is attracted to the magnet despite the liquid.

(A) As magnetic particles having a diameter of several nm to several tens of nm, the magnetic particles having a diameter of several nm to several tens of nm, preferably 5 to 25 nm, can be used. Etc.) are also the same as mentioned in the column of the inorganic particles.
The magnetic particles may be used alone or in combination of two or more.

(B) It is preferable that the liquid (dispersion medium) such as water, an organic solvent or oil is excellent in the dispersibility of the magnetic particles, does not dissolve the magnetic particles, and can be mixed with the monomer. As such a dispersion medium, an organic solvent is preferable, and as the organic solvent, it is suitable to contain an aliphatic hydrocarbon solvent. As the aliphatic hydrocarbon solvent, a linear or branched compound having 5 to 20 carbon atoms is preferable because the dispersibility of the magnetic particles is particularly excellent, and a linear or branched compound having 5 to 7 carbon atoms is preferable. Is more preferred. Specific examples thereof include pentane, hexane, heptane, isobutane, isopentane and the like, but are not limited to these examples.
The dispersion medium may be used alone or in combination of two or more.

The content of the aliphatic hydrocarbon solvent in the organic solvent is preferably 80% by mass or more. It is excellent in the dispersibility of a magnetic particle as it is 80 mass% or more, aggregation of the magnetic particle in this particle can be suppressed, and the dispersion | variation in the magnetic particle content in this particle can also be suppressed.

The content of the organic solvent in the magnetic fluid is preferably 20 parts by mass or more, and preferably 500 parts by mass or less, with respect to 100 parts by mass of the magnetic particles. If the content of the organic solvent is less than 20 parts by mass, the magnetic particles may not be sufficiently dispersed. If it exceeds 500 parts by mass, removal of the residual solvent is required after the following step (C), and the production of the present particles The operation of may be complicated. The content of the organic solvent is more preferably 30 parts by mass or more, and more preferably 300 parts by mass or less.

(C) As a stabilizer for stably dispersing the magnetic particles in the dispersion medium, those conventionally used in the magnetic fluid can be appropriately used, and the explanation will be made in the column of other components of the main separated particles. Stabilizers and the like similar to the above-mentioned stabilizers.
The stabilizing agent may be used alone or in combination of two or more. For example, the magnetic fluid may be a surfactant, an acid group-containing compound, an amino group-containing compound, a silane group-containing compound and titanium. It is preferable to include at least one selected from the group consisting of atom-containing compounds.

(monomer)
As a monomer used at a process (A), the monomer similar to the monomer used when synthesize | combining said organic polymer is mentioned, A preferable monomer is also the same.
The monomers may be used alone or in combination of two or more.

The amount of the monomer used is not particularly limited, but it is preferable that the content of the magnetic particles in the obtained particles is in the above range, specifically, with respect to 100 parts by mass of the magnetic particles in the magnetic fluid, The amount is preferably 1 part by mass or more, more preferably 3 parts by mass or more, still more preferably 5 parts by mass or more, particularly preferably 10 parts by mass or more, preferably 100 parts by mass or less, more preferably 50 parts by mass or less, more preferably 20 It is less than parts by mass.
When the monomer is used in such an amount, the content of the magnetic particles is within the above range, and the present particles excellent in the magnetic separation performance and the physical strength can be easily obtained.

(Polymerization initiator)
As the polymerization initiator used in the step (A), a thermally polymerizable radical polymerization initiator is preferable, and, for example, 2,2'-azobisisobutyronitrile, 2,2'-azobis- (2-methylpropane nitrile) ), 2,2′-azobis- (2,4-dimethylpentanenitrile), 2,2′-azobis- (2-methylbutanenitrile), 1,1′-azobis- (cyclohexanecarbonitrile), 2,2 '-Azobis- (2,4-dimethyl-4-methoxyvaleronitrile), 2,2'-azobis- (2,4-dimethylvaleronitrile), 2,2'-azobis- (2-amidinopropane) hydrochloride Azo initiators such as benzoyl peroxide, cumene hydroperoxide, hydrogen peroxide, acetyl peroxide, lauroyl peroxide, persulfate (eg ammonium persulfate), peroxy ester (eg t- butyl peroxide octoate, alpha-cumyl peroxypivalate) radical polymerization initiator of peroxide type, such as, can be mentioned, but are not limited to these examples.
The polymerization initiator may be used alone or in combination of two or more.

The use amount of the polymerization initiator is not particularly limited, but is preferably 0.1 parts by mass or more, more preferably 0.5 parts by mass or more, and still more preferably 1 part by mass or more with respect to 100 parts by mass of the monomer. Preferably it is 30 mass parts or less, More preferably, it is 20 mass parts or less, More preferably, it is 10 mass parts or less.

The order of mixing in mixing the magnetic fluid, the monomer and the polymerization initiator is not particularly limited, and a surfactant may be used in this mixing, if necessary.

<Step (B)>
The step (B) is a step of dispersing the monomer mixture to prepare an emulsion. In this step (B), preferably, the monomer mixture is dispersed in an aqueous medium in which a surfactant is dissolved.

The aqueous medium is not particularly limited, and usually water such as distilled water or ion exchanged water is used.
The aqueous medium refers to a medium in which water occupies at least 50% by mass or more.

The surfactant is not particularly limited, and any of anionic surfactants, cationic surfactants, and nonionic surfactants can be used. Among them, anionic surfactants are preferred.
The surfactant may be used alone or in combination of two or more.

The anionic surfactant is not particularly restricted but includes sodium, potassium or ammonium salts such as dodecyl sulfuric acid, dodecyl benzene sulfuric acid, decyl benzene sulfuric acid, undecyl benzene sulfuric acid, tridecyl benzene sulfuric acid, nonyl benzene sulfuric acid and the like.

The cationic surfactant is not particularly restricted but includes cetyltrimethylammonium bromide, hexadecylpyridinium chloride and hexadecyltrimethylammonium chloride.

The nonionic surfactant is not particularly limited, and examples thereof include polyvinyl alcohol. Moreover, as nonionic surfactant, for example, Triton X-100, X-114, X-305, N-101 (above, union carbide company make), Tween 20, 40, 60, 80, 85 (above, ICi Inc.), Brij 35, 58, 76, 98 (above, iCi Inc.), Nonidet P-40 (Shell), Igepol CO 530, CO 630, CO 720, CO 730 (Rhone · Rhone Commercially available products such as those manufactured by Poulain can be used.

Moreover, as said surfactant, the reactive surfactant which has a reactive group which can be superposed | polymerized with the said monomer can also be used. As the reactive group, for example, an ethylenically unsaturated group such as a vinyl group, an allyl group or a (meth) acryloyl group is preferable.

The amount of the surfactant used is not particularly limited, but is preferably 0.01 parts by mass or more, more preferably 100 parts by mass of the monomer mixture, from the viewpoint of easily preparing an emulsion. It is 0.1 parts by mass or more, preferably 100 parts by mass or less, and more preferably 5 parts by mass or less.

As a method of dispersing the monomer mixture, for example, a method of adding the monomer mixture into an aqueous medium containing a surfactant and emulsifying it by a shear mixing device which generates high shear force can be mentioned.

The shear mixing device is not particularly limited, and, for example, a homogenizer (manufactured by IKA), Histocolon (manufactured by Microtech Nichion), Polytron (manufactured by Kinematica), and a TK autohomomixer (manufactured by Tokushu Kika Kogyo Co., Ltd.) Etc .; Ebara Milder (made by Taihei Kiko Co., Ltd.), TK film mix, TK pipeline homomixer (made by Tokushu Kika Kogyo Co., Ltd.), colloid mill (Shinko Environmental Solutions Co., Ltd.) ), Clairemix (M-Technology Co., Ltd.), Thrasher, Trigonal Wet Pulverizer (Nippon Coke Industry Co., Ltd.), Cavitron (Eurotech Co., Ltd.), Fine Flow Mill (Pacific Kiko Co., Ltd.) etc. Continuous emulsification machine; Microfluidizer (made by Mizuho Kogyo Co., Ltd.), Nanomizer (Nanomizer Inc.) And high pressure emulsifiers such as APV Gaulin (manufactured by Gaulin); membrane emulsifiers such as a film emulsifier (manufactured by Chilling Industry Co., Ltd.); and a vibrating emulsifier such as a vibro mixer (manufactured by Chilling Industry Co., Ltd.) An ultrasonic emulsifier such as an ultrasonic homogenizer (manufactured by Branson); and among them, a probe type ultrasonic disperser is suitably used.

In the step (B), in view of the same effects as described above, the monomer mixture is dispersed so that the particle size of the droplets in the obtained emulsion is about the same as the desired particle size of the present particles. Specifically, it is preferable to disperse the monomer mixture liquid so as to obtain an emulsion containing droplets having a particle diameter similar to (in the same preferable range as) the particle diameter of the present particles.

As a dispersion condition of such a monomer mixture, for example, when using an ultrasonic disperser when dispersing the monomer mixture, the ultrasonic output is preferably 5 W or more, and preferably 200 W or less. . When the ultrasonic output is less than 5 W, large droplets may be generated due to insufficient dispersing power, and the polymerization reaction in step (C) may be difficult. When it exceeds 200 W, desired particles may not be obtained.
Further, as the irradiation time of the ultrasonic wave, although it depends on the ultrasonic wave output, the time of one ultrasonic wave irradiation is preferably 10 seconds or more, more preferably 30 seconds or more, and still more preferably 1 minute or more. Preferably it is 10 minutes or less, More preferably, it is 5 minutes or less, More preferably, it is 3 minutes or less.
The ultrasonic wave may be applied once or a plurality of times.

<Step (C)>
The step (C) is a step of polymerizing the monomers in the emulsion. By this process, the present particles can be obtained.

The polymerization conditions may be appropriately selected depending on the monomers to be used and the like, but the heating is usually performed by heating at 50 ° C. or more and 95 ° C. or less for 5 hours or more and 24 hours or less.

In the conventional method for producing organic polymer-containing magnetic particles, particles having different magnetic particle contents are produced. Therefore, after polymerizing a monomer, organic polymer-containing magnetic particles having a desired magnetic particle content are fractionated. However, according to the present production method, since the present particles having a substantially constant magnetic particle content can be obtained according to the present production method, the magnetic particles can be made high without requiring such a fractionation step. The particles contained in the content can be easily prepared. For this reason, it can be said that this manufacturing method is a method excellent in productivity.

<Other process>
The present manufacturing method may optionally include other steps other than the steps (A) to (C).
As the other step, for example, a step of forming a polymer layer on the surface of at least a part of the present particles obtained in the step (C), the coated particles obtained in the step or the step obtained in the step (C) The step of adsorbing or binding the ligand to the present particles, the step of blocking the particles to which the ligand is bound, and the washing step of washing the particle dispersion obtained in each of the above steps with water or the like by the magnetic separation method.

Step of Forming a Polymer Layer The polymer layer may be, for example, a monomer mentioned as a monomer for forming the polymer layer described above in the presence of the base particle, preferably the present particle dispersion obtained in the step (C). If necessary, it can be formed by (co) polymerization in a liquid in the presence of a polymerization initiator, an emulsifier, a dispersant, a surfactant, an electrolyte, a crosslinking agent, a molecular weight regulator and the like. By forming the polymer layer in this manner, it is preferable because coated particles having desired surface characteristics can be easily obtained, such as a desired functional group can be introduced to the surface of the polymer layer. Further, after forming the polymer layer, it is also possible to modify functional groups which may be present on the surface of the coated particles by a method such as alkaline hydrolysis of ethylenic unsaturated carboxylic acid alkyl ester or alkaline saponification of vinyl ester.
Furthermore, the formation of the polymer layer may be performed twice or more. That is, the coated particles may have two or more polymer layers.

The method of bringing the base particle and the monomer into contact in the case of forming the polymer layer in this way is not particularly limited, and for example, the base particle may be base particles by any of batch method, division method or continuous addition method. Or the method of adding to base particle dispersion liquid is mentioned.

The polymerization conditions may be appropriately selected depending on the monomers, polymerization initiator and the like used, but the polymerization temperature is usually 10 ° C. or more, preferably 30 ° C. or more, and usually 90 ° C. or less, preferably 85 ° C. or less The polymerization time is usually about 1 hour or more and about 30 hours or less.

As the polymerization initiator, oil-soluble polymerization initiators are preferable when classified from the viewpoint of solubility in water. The use of a water-soluble polymerization initiator tends to produce a large amount of new particles consisting only of a polymer layer not containing base particles, not polymerization on the surface of the base particles.

Examples of the oil-soluble polymerization initiator include peroxides such as benzoyl peroxide, lauroyl peroxide, t-butylperoxy 2-ethylhexanate, di (3,5,5-trimethylhexanoyl) peroxide, azobisisobutyronitrile and the like. Although a thing * azo compound etc. can be mentioned, it is not limited to these examples.
The polymerization initiators may be used alone or in combination of two or more.
The amount of the polymerization initiator used is preferably 0.01 parts by mass or more and 8 parts by mass or less with respect to 100 parts by mass of the monomer.

As the emulsifying agent, a commonly used anionic surfactant or nonionic surfactant can be used.
The emulsifying agents may be used alone or in combination of two or more.

Examples of the anionic surfactant include alkali metal salts of higher alcohol sulfuric acid esters, alkali metal salts of alkylbenzene sulfonic acids, alkali metal salts of succinic acid dialkyl ester sulfonic acids, alkali metal salts of alkyl diphenyl ether disulfonic acid, and polyoxyethylene alkyls. In addition to sulfuric acid ester salts of (or alkylphenyl) ethers, phosphoric acid ester salts of polyoxyethylene alkyl (or alkylphenyl) ethers, formalin condensates of sodium naphthalene sulfonate, etc., Latemul S-180A (manufactured by Kao Corporation) And Eleminol JS-2 (manufactured by Sanyo Chemical Industries, Ltd.), Aqualon HS-10 (manufactured by Daiichi Kogyo Seiyaku Co., Ltd.), Adekaria Soap SE-10N (manufactured by ADEKA), etc. Limited to There.

Further, as nonionic surfactants, in addition to polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, etc., Aqualon RS-20 (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd.), Adekaria Soap NE-20 ( And the like) and the like.

The step of adsorbing or binding the ligand The step of adsorbing or binding the ligand to the present particles obtained in the step (C) or the coated particles obtained in the step of forming the polymer layer is not particularly limited, and It may be carried out by a known method.
The particle to which the ligand is physically adsorbed can be obtained, for example, by contacting the ligand with a particle obtained using a monomer such that the organic polymer becomes a hydrophobic polymer.
The method of chemically bonding the ligand may be performed according to a conventional method, but is preferably performed by the covalent bonding method. For example, when the particle surface has a carboxy group and the ligand has an amino group, they may be bound using a dehydrating condensing agent.

-Step of blocking the particle to which the ligand is bound The production method may include the step of binding a blocking agent to the present particle to which the ligand is adsorbed or bound.
By using the present particles obtained through such a process, it is possible to reduce nonspecific reaction, to improve the dispersibility of the present particles, to suppress denaturation of the ligand, and to further reduce the influence on the target cells.
Examples of blocking agents include bovine serum albumin (BSA), skimmed milk, gelatin, casein, synthetic polymers and the like.

As a blocking agent using a synthetic polymer, a copolymer of a vinyl monomer having a hydrophilic polymer such as polyoxyethylene in the side chain, a block copolymer of a hydrophilic monomer such as polyoxyethylene with another monomer, a functional group at an end And hydrophilic polymers such as polyoxyethylene having a group. In particular, a synthetic polymer having a structure having a polyamine at one end of polyoxyethylene as disclosed in JP-A-2008-170417 not only suppresses nonspecific adsorption to the surface of the present particles but also aligns the orientation of the ligand, thereby causing reactivity. It can be preferably used because the effect of improving the

The blocking agent may be physically adsorbed or chemically bound to the surface of the present particle to which the ligand is adsorbed or bound. The method of adsorbing or binding the blocking agent may be appropriately selected depending on the particles and the blocking agent to be used, and may be performed by a conventionally known method.

<< Method for separating target cells or non-target cells >>
A method of separating a target cell (target cell) or a non-target cell (non-target cell) according to an embodiment of the present invention (hereinafter also referred to as “the present separation method”) includes the following steps 1 and 2 ,
A method using the present particles, which comprises an organic polymer and magnetic particles, the content of the magnetic particles is 40% by mass or more, and the volume average particle diameter is 10 to 1000 nm.
Step 1: Contacting the sample containing the target cell with the present particle Step 2: Magnetic separation of the complex of the present particle and the target cell or non-target cell generated in the step 1

According to this separation method, cells can be separated with little influence on cells (cell death or activation of cells) without using HGMS method etc., and cells are separated with high recovery rate. be able to.

The present separation method may include, prior to the step 1, a step of obtaining a sample (biological specimen) containing a target cell from a subject.
The biological sample includes urine, plasma, serum, saliva, semen, stool, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid, peripheral blood sample, bone marrow aspirate, fine needle aspirate, lymph node biopsy, Biologically-derived samples such as specimens collected from epithelial tissue, connective tissue including bone and cartilage, muscle tissue and nerve tissue, and further, cell suspensions such as primary cells and cultured cells, and the like.

The target cells include all cells. Examples include T cells, regulatory T cells, B cells, NK cells, dendritic cells, monocytes, granulocytes, and hematopoietic stem cells.
The non-target cells may be cells other than the target cells contained in the sample, and the target cells in the sample can be separated and concentrated by selectively separating and removing the non-target cells.

The step of contacting the sample with the present particles in step 1 is preferably a step of contacting the present particles with target cells or non-target cells to be separated contained in the sample.
As a method of the contact, the sample and the present particles may be mixed, and in that case, if necessary, it may be subjected to end-over mixing, stirring or the like.

The contact conditions in Step 1 are not particularly limited as long as a complex of the present particle and a target cell or non-target cell is obtained, but the contact temperature is preferably 1 ° C. or more, more preferably 2 ° C. or more The temperature is 30 ° C. or less, more preferably 25 ° C. or less, and the contact time is preferably 1 minute or more, more preferably 5 minutes or more, preferably 60 minutes or less, more preferably 45 minutes or less.

The present particles used in Step 1 are as described above, but are preferably organic polymer-containing magnetic particles to which a specific ligand that specifically reacts with either a target cell or a non-target cell is bound.

Specifically, Step 2 includes a step of separating the non-target cell and the target cell-present particle complex or separating the target cell and the non-target cell-present particle complex by a magnet.

The above magnetic separation does not use a column using high gradient magnetic separation (HGMS) technology as described in US Pat. No. 5,693,539 for the same reason as described above, and the gradient is as low as about 5 kilogauss. It is preferable to carry out in

Specifically, the following methods are preferable for the magnetic separation.
When separating non-target cells and target cell-target particle complexes, magnetic separation is performed from outside the reaction vessel containing the complex formed in step 1 with a magnet or the like, and the complexes are collected to obtain non-target cells other than the complexes. The sample containing cells etc. is drained, and a washing solution such as phosphate buffer is added. The magnet is then removed and the complex is dispersed and washed. This operation may be repeated several times, for example, up to about 10 times.
In addition, when the target cell and the non-target cell-the present particle complex are separated, the non-target cell complex is magnetically separated from the outside of the reaction vessel containing the complex formed in step 1 by a magnet or the like to collect and remove the complex. A sample in which target cells are removed and target cells are concentrated can be obtained.

The present separation method may include the steps of dissociating the cells and the particles from the cell-present particle complex after separating and concentrating the cells. As the method of the dissociation, a method using a dissociator such as a competitor having a different binding constant, a method using a linker cleaving by light or heat, a method using a dissociator capable of cleaving the linker by a chemical reaction or an enzyme reaction, etc. Can be mentioned.
Examples of methods using competitors with different binding constants include the methods described in Japanese Patent No. 5686098. Moreover, as a method of using a photocleavable linker, a method of applying the method described in Japanese Patent No. 4669704 can be mentioned.

«Kit»
A kit according to an embodiment of the present invention comprises the present separated particles and a member for separating the particles.
As a member for separating the main separated particles, when the main separated particles are the main particles, a magnetic field application member such as a permanent magnet or an electromagnet, more specifically, a magnetic stand or the like can be mentioned. When the particles are particles other than the present particles, a centrifugal separator, a filter member such as a filter having a predetermined pore diameter, and the like can be mentioned.

In addition, the kit may be a component other than the above, for example, a medium in which the present separated particles are dispersed, and if the present separated particles are particles having a ligand, a substance that specifically reacts with the ligand (labeled with a fluorescent substance etc. And the dissociating agent, the washing solution, and the blocking agent.

<< Device for separating or concentrating cells >>
One embodiment of the present invention may be an apparatus for separating or concentrating cells, which is an apparatus for separating or concentrating cells using the separated particles, for example, the separated particles When the particle is the present particle, a container for holding the dispersion liquid of the present separated particle, a magnetic field applying member for capturing the present particle contained in the container on the inner wall surface of the container, etc. and a substance not captured by the magnetic field applying member And a magnetic separation mechanism having a removing member.

The present application relates to the embodiments of the present invention described above, and also discloses the following embodiments.

[10 '] An organic polymer-containing particle satisfying the following requirements (1) to (3) used to separate or concentrate cells.
Requirement (1): The organic polymer-containing particles contain an organic polymer and inorganic particles Requirement (2): the content of inorganic particles in the organic polymer-containing particles is 40% by mass or more Requirement (3) The volume average particle size of the organic polymer-containing particles is 10 to 1000 nm

[11 '] The particle according to [10'], wherein the inorganic particle is a magnetic particle.

[12 '] The particle according to [10'] or [11 '], wherein the organic polymer-containing particle has a polymer layer on the surface of at least a part of a particle containing an organic polymer and an inorganic particle.
[13 '] The particle according to [12'], wherein the polymer is a hydrophilic polymer.

[14 '] The particle according to any one of [10'] to [13 '], and
Including a member for separating the particles,
kit.

Hereinafter, the present invention will be described in detail by way of examples and comparative examples, but the present invention is not limited to these examples.

The volume average particle size of the dispersoid or particles in the solution in the following synthesis example was measured using a dynamic light scattering type particle size distribution measuring apparatus (Nanotrac UPA-EX150, manufactured by Nikkiso Co., Ltd.).
Further, the content of magnetic particles in the particles obtained in the following Examples and Comparative Examples was measured at 500 ° C. using a differential type differential thermal balance (TG-8120 manufactured by Rigaku Corporation).

Synthesis Example 1
Magnetic fluid “EMG 2001” (17.0 g of magnetic particles in 27.0 g of the magnetic fluid, heptane dispersion, manufactured by Farotech) 27.0 g of styrene, 1.35 g of styrene, 0.15 g of divinylbenzene and 2, 2 0.06 g of '-azobisisobutyronitrile was added and mixed to obtain a monomer mixture. Next, 75 g of an aqueous solution in which 0.75 g of sodium dodecyl sulfate is dissolved is added to the obtained monomer mixture, and ultrasonic waves are applied under ice cooling using an ultrasonic homogenizer (US 300T, manufactured by Nippon Seiki Seisakusho Co., Ltd.) The treatment was performed (ultrasonication for 2 minutes (ultrasonic output: 150 W) and then for 2 minutes after the termination of ultrasonic irradiation). This ultrasonication was repeated 10 times to prepare an emulsion in which a monomer mixture containing magnetic particles was dispersed in water. The volume average particle size of the droplets in the obtained emulsion was 104 nm.

Next, the obtained emulsion was polymerized at 70 ° C. for 7 hours, and washed with water by magnetic separation to obtain a dispersion liquid of organic polymer-containing magnetic particles A. The volume average particle diameter of the organic polymer-containing magnetic particles A in the particle dispersion thus obtained was 103 nm, and the magnetic particle content of the organic polymer-containing magnetic particles A was 90% by mass. The value of the magnetic particle content is in agreement with the theoretical value, and it can be seen that organic polymer-containing magnetic particles (dispersion liquid) were obtained with high production efficiency. The theoretical value (% by mass) means the value when it is assumed that all the charged magnetic particles, monomers, and initiators have become organic polymer-containing magnetic particles, and the amount of magnetic particles × 100 / (magnetic particles It is calculated from “amount + amount of each monomer + amount of initiator)”. In addition, since the particle size distribution of the obtained organic polymer-containing magnetic particles became a group, it is understood that organic polymer-containing magnetic particles of a desired shape could be obtained with high production efficiency.

1.2 g of methyl methacrylate, 0.3 g of trimethylolpropane trimethacrylate, 0.3 g of methacrylic acid and 0.1 g of di (3,5,5-trimethylhexanoyl) peroxide (manufactured by NOF Corporation; Peroyl 355) 30 g of an aqueous solution in which 0.15 g of sodium dodecyl sulfate was dissolved was added to the mixed solution, and an emulsion c was prepared by an ultrasonic homogenizer (US300T).

In a separable flask, 73.5 g of the prepared dispersion liquid of organic polymer-containing magnetic particles A (containing 3.0 g of organic polymer-containing magnetic particles A) are placed, and the temperature of the emulsion c is controlled to 60 ° C. by a water bath. It dripped over 2 hours. After completion of the dropwise addition, the temperature was maintained at 60 ° C., polymerization was carried out for 1 hour, and after raising the temperature to 80 ° C., the reaction was terminated for 2 hours. The obtained dispersion liquid was washed with water by magnetic separation to prepare a dispersion liquid of coated particles C. The volume average particle diameter of the coated particles C in the obtained particle dispersion was 109 nm, and the magnetic particle content of the coated particles C was 59% by mass.

10 mg of coated particles C are dispersed in 1 mL of 100 mM MES (2-Morpholinoethanesulfonic acid, monohydrate) buffer of pH 5.0, and 1-ethyl-3-dimethylaminopropyl carbodiimide hydrochloride (WSC, manufactured by Dojindo Laboratories, Inc.) 0.1 mL of a WSC solution dissolved in 100 mM MES buffer, pH 5.0, to a concentration of 10 mg / mL was added, and the solution was rotationally stirred at room temperature for 30 minutes. Furthermore, 0.1 mL of 100 mM MES buffer of pH 5.0 in which 0.2 mg of streptavidin was dissolved was added thereto, and the mixture was rotationally stirred at room temperature for 2 hours, and then washed four times with Tris buffer to be unreacted Streptavidin was removed, and streptavidin-conjugated particles in which streptavidin was immobilized on the surface of coated particle C were prepared. The amount of bound streptavidin was determined by BCA assay to be 18 μg / mg coated particles C.

To 2 mg of anti-human CD4 antibody, 8 μl of 5.69 mg / ml NHS-modified biotin (H1759, manufactured by Sigma-Aldrich) DMSO solution was added, and allowed to stand at room temperature for 3 hours. Thereafter, unreacted biotin is removed by ultrafiltration, and the DMSO solution is replaced with a D-PBS solution (hereinafter referred to as "reaction buffer") containing 0.5 wt% BSA and 2 mM EDTA to obtain biotin-labeled anti-human CD4 antibody was obtained.

0.2 mg of streptavidin-conjugated particles and 0.4 μg of the biotin-labeled anti-human CD4 antibody obtained above were placed in a test tube, mixed by shaking at room temperature for 60 minutes, and allowed to react. Thereafter, streptavidin-conjugated particles to which a biotin-labeled anti-human CD4 antibody is bound are separated from the reaction solution using a magnetic stand, and washed twice with TBS containing 0.05% Tween 20 to obtain an anti-human CD4 antibody-conjugated organic antibody. Molecular-containing magnetic particles I (magnetic particle content: 57% by mass, volume average particle diameter: 150 nm) were obtained. The variation coefficient of the volume average particle diameter was 20%.

Synthesis Example 2
In Synthetic Example 1, by changing the amount of magnetic fluid used to 1 g, anti-human CD4 antibody-binding organic polymer-containing magnetic particles II having a magnetic particle content of 22 mass% and a volume average particle diameter of 155 nm Obtained. The variation coefficient of the volume average particle diameter was 23%.

Example 1
<Positive separation of CD4 (+) cells from PBMCs>
The anti-human CD4 antibody-binding organic polymer-containing magnetic particle I obtained above was taken into a test tube so as to have a dry weight of 0.2 mg and dispersed in 0.5 ml of a reaction buffer to obtain a particle dispersion 1. .

The purchased human PBMCs (human peripheral blood mononuclear cells, manufactured by Precision Bioservices) were thawed, and then cultured overnight in RPMI medium (manufactured by Thermo fisher scientific) containing 10% FBS. Then, the reaction buffer was washed 3 times and adjusted to 2 × 10 ⁶ cells / mL with the same buffer. After adding 50 μl each of the prepared human PBMC fluid to the particle dispersion 1 described above, the cells were allowed to react with the organic polymer-containing magnetic particles by inverting and mixing for 30 minutes at room temperature. After that, the magnetic particles containing the organic polymer are magnetically separated from the reaction solution using a magnetic stand, and washed three times with the reaction buffer to obtain a complex of the target cell-antibody-bound organic polymer containing magnetic particles. Then, CD4 (+) cells in the adjusted human PBMC fluid were separated.

Thereafter, the target cell-antibody-conjugated organic polymer-containing magnetic particle complex is suspended in 100 μl of reaction buffer, to which 10 μl of PE labeled anti-CD4 antibody (manufactured by Biolegend) is added. React with cells for 10 minutes. After the reaction, the reaction solution was washed three times with reaction buffer to remove unreacted PE-labeled anti-CD4 antibody. Thereafter, the target cell-antibody-conjugated organic polymer-containing magnetic particle complex was suspended in 2 ml of reaction buffer, and the cells in the suspension were analyzed with an Accuri C6 Flow cytometer (manufactured by Becton Dickinson & Co.) . Gating was performed with the vertical axis as forward scattered light (FSC) and the horizontal axis as side scattered light (SSC). Subsequently, with the vertical axis as FSC and the horizontal axis as FL-2, the percentage of CD4 (−) cells and the percentage of CD4 (+) cells were plotted.

The ratio of CD4 (−) cells to CD4 (+) cells in the adjusted human PBMC solution was also determined in the same manner as described above. The results are shown in FIG.

The percentage of CD4 (+) cells in human PBMC fluid was 56.7%, but could be separated and concentrated to 91.8% by magnetic separation using antibody-bound organic polymer-containing magnetic particles. The number of cells calculated from the formula (the number of CD4 (+) cells after magnetic separation / the number of CD4 (+) cells in the adjusted human PBMC liquid × 100) based on the number of cells measured by the flow cytometer The recovery rate was 95%.

Comparative Example 1
CD4 (+) cells were separated from the prepared human PBMC solution in the same manner as in Example 1 except that the anti-human CD4 antibody-binding organic polymer-containing magnetic particle II obtained above was used. Magnetic separation was able to separate and concentrate the percentage of CD4 (+) cells to 90.2%, but the cell recovery was only 38%.

Example 2
<Positive separation of CD8 (+) cells from human PBMC>
An anti-human CD8 antibody-binding organic polymer-containing magnetic particle is prepared in the same manner as in Synthesis Example 1 except that an anti-human CD4 antibody is used instead of the anti-human CD4 antibody in Synthesis Example 1, and the anti-human CD8 antibody is prepared. Separation and concentration of CD8 (+) cells from the prepared human PBMC solution were carried out in the same manner as in Example 1 except that the bound organic polymer-containing magnetic particles were used. As a result, the proportion of initial CD8 (+) cells in the adjusted human PBMC solution was 62.3%, but it was separated up to 91.4% by magnetic separation using antibody-bound organic polymer-containing magnetic particles We were able to. The recovery rate of cells was 93%.

Comparative Example 2
In Synthetic Example 2, an anti-human CD8 antibody-binding organic polymer-containing magnetic particle is prepared in the same manner as in Synthetic Example 2 except that an anti-human CD8 antibody is used instead of the anti-human CD4 antibody, and the anti-human CD8 antibody is prepared. Separation and concentration of CD8 (+) cells from the prepared human PBMC solution were performed in the same manner as in Example 2 except that the bound organic polymer-containing magnetic particles were used. As a result, separation and concentration up to 90.8% could be achieved by magnetic separation using antibody-bound organic polymer-containing magnetic particles, but the cell recovery rate was only 24%.

[Example 3]
<Percentage of cell death before and after cell separation>
The cell viability of CD4 (+) cells separated in the same manner as in Example 1 was examined using Annexi VAssay Kits (manufactured by Medical and Biological Laboratories, Inc.). As a result, the cell viability of the CD4 (+) cells in the adjusted human PBMC solution is 87.4% when a part is removed from the adjusted human PBMC solution and measured, and the CD4 after separation by magnetic particles is performed. The cell viability of (+) cells was 88.1%, and no difference was observed in cell viability.

Example 4
<Culture of cells after separation, presence or absence of activation>
The target cell-antibody-conjugated organic polymer-containing magnetic particle complex (complex before reacting with PE-labeled anti-CD4 antibody) separated in the same manner as in Example 1 is added to RPMI medium containing 10% FBS (Thermo fisher scientific) Incubate for 48 hours. Then, in the same manner as in Example 1, human PBMC before separation and target cells after separation (CD4 (+) cells) were stained with a PE-labeled anti-human CD69 antibody (manufactured by Biolegend). As a result, no cells were stained with the PE-labeled anti-human CD69 antibody, and neither human PBMC before separation nor cells after separation were activated.

[Example 5]
<Activation of cells after separation>
Dynabeads Human T-Activator CD3 / CD28 (Thermofisher) was used for the target cell-antibody-conjugated organic polymer-containing magnetic particle complex (complex before reacting with PE-labeled anti-CD4 antibody) separated in the same manner as in Example 1. Cell activation was performed using scientific). The activated cells were stained with a PE-labeled anti-human CD69 antibody (manufactured by Biolegend) in the same manner as in Example 4. As a result, 63.2% of the cells were stained, and it was confirmed that cell activation had occurred.
When human PBMC before separation were similarly activated and stained in the same manner, (64.1)% of cells were stained, and it was confirmed that cell activation had occurred.

[Example 6]
<Dethiobiotinylation of antibody>
5 mg of desthiobiotin (manufactured by MP Biomedicals) was dissolved in 0.5 ml of dimethyl sulfoxide. This solution contains 5.36 mg each of N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC hydrochloride) (1.2 for carboxy group of desthiobiotin). Equal volumes were added and reacted for 60 minutes at room temperature. From this reaction solution, 3 μl was separated, added to a solution in which 2 mg of anti-human CD4 antibody was dissolved in 1 ml of PBS (phosphate buffered saline), and further reacted at room temperature for 3.5 hours. Unreacted desthiobiotin was removed by ultrafiltration to obtain a desthiobiotin-labeled anti-CD4 antibody.

A desthiobiotin-labeled anti-human CD4 antibody-conjugated organic polymer-containing magnetic particle is prepared in the same manner as described in Example 1 using the above-described desthiobiotin-labeled anti-human CD4 antibody instead of the biotin-labeled anti-human CD4 antibody. did.

<Cell separation from human PBMC>
CD4 (+) cells were separated from human PBMC in the same manner as described in Example 1.

<Preparation of Dissociation Agent (Synthesis of Polyacrylic Acid Bonded with Biotin Derivative)>
0.1 g of polyacrylic acid having a molecular weight of 250,000 (PAA, manufactured by Wako Pure Chemical Industries, Ltd.) is dissolved in 5 ml of 10 mM phosphate buffer solution (pH 7.0), and 133 mg of EDC hydrochloride (carboxy of polyacrylic acid) Added 0.5 equivalents to the group). Subsequently, 1.67 ml of a dimethyl sulfoxide solution (10 mg / ml) of a compound (Biotin-PEO-LC-Amine; manufactured by Thermofisher scientific) represented by the following formula (7) which is a biotin derivative having an amino group is added, Furthermore, it reacted at room temperature for 3.5 hours. The unreacted biotin derivative was removed by ultrafiltration to obtain a water-soluble biotin derivative-bound PAA.
The quantification of biotin bound to PAA was performed according to the HABA method.
From the above, a dissociation agent (referred to as “PAA-Biotin 40”) to which an average of 40 molecules of biotin derivative was bound per PAA molecule was obtained.

<Dissociation of cells and organic polymer-containing magnetic particles by addition of a dissociator>
The dissociation agent prepared above is adjusted to 2 mg / ml with PBS solution, and 250 μl is added to the separated CD4 (+) cells (CD4 (+) cell-complex of antibody-containing organic polymer-containing magnetic particles), Mix gently for 20 minutes at room temperature. Thereafter, the organic polymer-containing magnetic particles were separated using a magnetic stand, and the dissociated CD4 (+) cells in the supernatant were recovered. When the number of CD4 (+) cells dissociated from the organic polymer-containing magnetic particles was counted using a flow cytometer, 95% of the cells were dissociated.

[Example 7]
The ratio of cell death was confirmed in the same manner as in Example 3 for the cells dissociated from the organic polymer-containing magnetic particles in the same manner as in Example 6. Before and after cell separation (before cell dissociation and after dissociation in Example 6) There was no difference in cell viability in.

[Example 8]
With respect to the cells dissociated from the organic polymer-containing magnetic particles in the same manner as in Example 6, the presence or absence of cell activation was confirmed in the same manner as in Example 4. As with the cells before separation, the cells were activated. Was not happening.

[Example 9]
The cells released from the organic polymer-containing magnetic particles in the same manner as in Example 6 were activated in the same manner as in Example 5. As with the cells before separation, the activation occurred. Was confirmed.

[Test example]
<Required particle amount comparison>
Anti-human CD4 antibody in the same manner as in Synthesis Example 1 except that in Example 1, the coated particles C were changed to organic polymer-containing magnetic particles (Magnosphere MS300 / Carboxyl, manufactured by JSR Life Science Co., Ltd.) having a particle diameter of 3 μm. The bonded organic polymer-containing magnetic particles III were produced. With the antibody-bound particles I and antibody-bound particles III prepared, isolation of the cell line SUPT1 cells, which are CD4 (+), was attempted. As a result, the amount of particles required for recovering 80% or more of 1 × 10 ⁷ cells was 0.25 mg for antibody-bound particles I, and 3 mg for antibody-bound particles III.

Claims (14)

Including the following steps 1 and 2,
The following organic polymer-containing magnetic particles contain organic polymer and magnetic particles, and the content of magnetic particles in the organic polymer-containing magnetic particles is 40% by mass or more, and the volume average particle size of the organic polymer-containing magnetic particles 10 to 1000 nm in diameter,
How to isolate target cells or non-target cells:
Step 1; contacting the sample containing the target cells with the organic polymer-containing magnetic particles,
Step 2: Magnetic separation of the complex of the organic polymer-containing magnetic particle and the target cell or the non-target cell generated in the step 1; The method according to claim 1, wherein the organic polymer-containing magnetic particles have a polymer layer on the surface of at least a part of particles containing the organic polymer and the magnetic particles. The method of claim 2, wherein the polymer is a hydrophilic polymer. The hydrophilic polymer is (meth) acrylic acid, methyl (meth) acrylic acid, diacetone (meth) acrylamide, (meth) acrylamide, dimethyl (meth) acrylamide, dimethylaminopropyl (meth) acrylamide, dimethylaminopropyl (meth) Acrylamide hydrochloride, N, N-diethyl (meth) acrylamide, N-isopropyl (meth) acrylamide, glycerol (meth) acrylamide, (meth) acrylamide-N-glycolic acid, hydroxyethyl (meth) acrylate, hydroxybutyl (meth) Acrylate, ethylene glycol mono (meth) acrylate, 2-sulfoethyl (meth) acrylate, phosphorus-containing (meth) acrylic acid ester, dimethylaminoethyl (meth) acrylate, diethylamino ether (Meth) acrylate, t-butylaminoethyl (meth) acrylate, glycidyl (meth) acrylate, glycerol (meth) acrylate, vinyl sulfone, styrene sulfonic acid, 2- (meth) acrylamido-2-methylpropane sulfonic acid, Meta) acryloyl morpholine, N-vinyl pyrrolidone, N-vinyl caprolactam, N-vinyl oxazolidone, N-vinyl succinimide, vinyl pyridine, vinyl acetate, itaconic acid, crotonic acid, N-vinyl imidazole, vinyl benzyl ammonium salt, vinyl benzyl The method according to claim 3, which is a polymer comprising a structural unit derived from at least one monomer selected from the group consisting of alcohol and hydroxystyrene. The method according to any one of claims 1 to 4, wherein the coefficient of variation of the volume average particle diameter of the organic polymer-containing magnetic particles is 25% or less. The method according to any one of claims 1 to 5, wherein the organic polymer is crosslinked. The method according to any one of claims 1 to 6, wherein the volume average particle size of the magnetic particles is 5 to 25 nm. The method according to any one of claims 1 to 7, wherein at least one ligand is physically adsorbed or chemically bonded to the organic polymer-containing magnetic particle. The ligand is at least one selected from the group consisting of an antibody, an antigen, a nucleic acid, a nucleotide, a nucleoside, a protein, a peptide, an amino acid, a polysaccharide, a sugar, a lipid, a vitamin, a drug, a substrate, a hormone and a neurotransmitter. The method according to item 8. Organic polymer-containing particles comprising an organic polymer and inorganic particles, wherein
The content of inorganic particles in the organic polymer-containing particles is 40% by mass or more,
The volume average particle size of the organic polymer-containing particles is 10 to 1000 nm,
Particles for cell separation or concentration. 11. The particles of claim 10, wherein the inorganic particles are magnetic particles. The particle according to claim 10, wherein the organic polymer-containing particle has a polymer layer on the surface of at least a part of the particle containing the organic polymer and the inorganic particle. 13. The particles of claim 12, wherein the polymer is a hydrophilic polymer. The particles according to any one of claims 10 to 13, and
Including a member for separating the particles,
kit.

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