JPH04242661A - Agent for concentrating natural killer cell, concentrating device and concentrating method - Google Patents

Abstract

PURPOSE:To enhance the separation efficiency by fixing an organic substance having a low adhesive property with respect to natural killer cells and having an affinity with cells other than the natural killer cells in a solution to be tested, to a water-insoluble solid substance. CONSTITUTION:In a natural killer (hereinbelow denoted as 'NK') cell concentrating agent 5 in which an organic substance having a low adhesive property with respect to the NK cells and having an affinity with at least one kind of cells other than the NK cells in a solution to be tested is fixed to an water- soluble solid substance, sugars such as, in particular, polysaccharide or glycopeptide originated from plants are used as the organic substance since the concentration ability of the NK cells is particularly excellent. In particular, polysaccharide having a molecular weight of 200,000 to 1,000,000 and extracted from the stems of taros is preferable. In an NK cell concentrating device in which the NK cell concentrating agent 5 is charged in a column container 4 and is held and fixed by filters 6a, 6b, blood is introduced from a blood inlet port 7 and is then brought into contact with the NK cell concentrating agent 5, thereby it is possible to obtain blood in which the NK cells are concentrated.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an NK cell concentrator, a concentration device, and a concentration method for concentrating natural killer cells (hereinafter also referred to as NK cells), which are a subclass of lymphocytes.

0002

[Prior Art] The main immunocompetent cells are lymphocytes that are equipped with antigen-specific receptors and have the ability to recognize antigens and differentiate into effector cells that produce specific humoral and cellular antibodies. , immunocompetent cells (immuno
These cells are called competent cells. However, it is impossible to carry out immune responses in vivo using lymphocytes alone; dendritic cells of the macrophage subgroup are required for the ascending leg of the immune response, which processes antigens and presents them to lymphocytes. In addition, foreign body disposal, which is the final effect of the antigen-antibody reaction, and inflammatory reactions centered on vascular reactions,
This is the descending leg of the immune response, and in addition to macrophages (monocytes), granulocytes and mast cells are involved.

[0003] Various immunocompetent cells are characterized by their rapid response to the demands of complex immune responses by local recruitment and proliferation, and their proliferation and differentiation in response to stimuli is similar to that of other cell systems in the body. It has a role in regulating the functions of

[0004] Lymphocytes have the following subclasses. In other words, T lymphocytes that mature under the influence of the thymus, B lymphocytes that mature under the influence of the bursa of Fabricius or the equivalent organ in mammals (hematopoietic foci such as the liver and bone marrow), and null cells. It is broadly classified into other lymphocytes such as

[0005] In the case of B lymphocytes, after receiving antigen stimulation, they show a so-called unidirectional change of differentiation and proliferation into antibody-producing cells and production of antibodies, whereas T lymphocytes show a change in a single direction, i.e., differentiation and proliferation into antibody-producing cells and production of antibodies. Rather, a single antigen stimulus causes changes in several types of T lymphocytes with various functions, with some becoming effector cells and others functioning as regulatory cells (promoting or suppressing differentiation and proliferation).

[0006] In other words, there are functional subsets of T lymphocytes. These are subsets of so-called helper/inducer T lymphocytes (TH/I), suppressor/cytotoxic T lymphocytes (TS/C), and T lymphocytes involved in delayed-type allergies (TDTH).

[0007] Also, in B lymphocytes, classes of immunoglobulins (IgG, IgM, I
It is thought that there are subsets classified by gA, IgD, IgE).

[0008] The null cell group is a cell group that does not have a characteristic marker on its surface, and those belonging to this group include N.
K cells and K cells (Killer cells) are known.

[0009] NK cells do not depend on the presence of the thymus (they are not mature T cells), do not have immunoglobulin in the cytoplasm or on their membranes (they are not B cells), and also exhibit phagocytic function and adhesive ability. (not macrophages), but morphologically they are clearly lymphocytes and can be distinguished from differentiated cells of the hematopoietic system. It is known that NK cells have the ability to kill certain types of tumor cells without being stimulated by specific antigens.

[0010] The existence of the above-mentioned subsets and null cell groups is shown by the correlation between classification using antibodies (monoclonal antibodies) against surface antigens serving as markers and their functions.

[0011] In recent years, attempts have been made to separate and fractionate lymphocytes, which have such important functions, and to utilize them in basic immunological research and various diagnoses and treatments.
Various methods are known for separating lymphocytes, as described below.

[0012] Namely, [1] methods such as velocity sedimentation that utilize differences in cell size, [2] methods such as specific gravity centrifugation that utilizes differences in specific gravity of cells, and [3] differences in cell surface charge. [4] Removal of monocytes with carbanyl iron that utilizes oligophagic activity, [5] Nylon wool, glass wool, etc. that utilize differences in non-specific adhesion activity of cells. [6] Affinity chromatography that utilizes the specific pedestal ability of cells; [7] Rosette sedimentation that uses cell rosette formation as an indicator. [8] Fluorescence activated cell sorter (Fluore
Scence Activated Cell
Sorting, FACS) method (for example, Junichi Yada, Michio Fujiwara, eds., New Lymphocyte Function Search Method, 198)
7, see Chugai Igakusha, etc.). [9] Field-Flow Fractionation (Field-Flow Fractionation)
Fractionation, FFF) method is being considered.

[0013] However, physical cell separation methods such as those in [1] to [3] do not show any significant difference in physical properties such as specific gravity and density between T lymphocytes and B lymphocytes. Therefore, high precision cannot be required for the yield or purity of the separated and collected cells.Especially, in the specific gravity centrifugation method of [2], the cost of the medium becomes expensive, or even if it is cheap, the operation is difficult. In addition, in the cell electrophoresis method described in [3], the mobility differs depending on the level of cell maturity, the influence of the electric field on cell function has not been elucidated, and large-scale processing is difficult. It also had drawbacks.

[0014] In addition, cell separation methods using specific antigens, receptors, immunoglobulins, etc. on the cell membrane surface as indicators, such as those in [6] to [8], all require that the treated cells have strong biologically specific properties. Because of binding and stimulation, it is difficult to recover target cells in an intact state, and it is not suitable for large-scale processing. In particular, affinity chromatography in [6] requires expensive antibodies, and The FACS method of [8] has disadvantages in that pretreatment such as labeling of cells is complicated and requires expensive antibodies and equipment.

[0015] In addition, the FFF method of [9] has the equipment difficulty of continuously inflowing and outflowing the eluent via the rotating shaft, and the materials of the rotor-shaped separation cell and the flow path cause cell adhesion. Therefore, we have not reached the point where we can put cell separation into practical use.

On the other hand, the method [5] that utilizes the non-specific adhesion properties of cells has less risk of damaging the functions of treated cells, and is suitable in terms of cost, operability, and large-scale processing. However, it is insufficient in terms of selectivity and recovery rate for target cells, and in addition, the column method mentioned above requires experience and technical proficiency when performing separation operations, which is a major problem. also existed.

As separation agents and separation methods that utilize such non-specific adhesion properties, hydrophobic materials (Japanese Patent Publication No. 59
-17387, 59-36963, 62-45
206, JP-A No. 57-204454), materials with acidic functional groups (JP-B No. 59-36961, JP-A-56
-140886, 56-152740), polymers containing basic functional groups (JP-A-59-216584, JP-A-60-105490), hydroxyapatite fibers (JP-A-63-284), specific It has also been proposed to use polymers (Japanese Patent Application Laid-open Nos. 61-221123 and 64-34285), but even these methods are still insufficient in terms of selectivity and recovery rate for target cells. There wasn't.

[0018]

[Problems to be Solved by the Invention] Therefore, the present invention provides N
The object of the present invention is to provide an NK cell concentrator, a concentration device, and a concentration method that can efficiently separate K cells and recover them without impairing cell function.

[0019]

[Means for Solving the Problems] Such objects are achieved by the present invention as described in (1) to (7) below.

(1) It is characterized by immobilizing an organic substance on a water-insoluble solid substance that has low adhesion to natural killer cells and has an affinity for at least one type of other cells in the test solution. Natural killer cell concentrate.

(2) The natural killer cell concentrate according to (1) above, wherein the organic substance is a plant-derived polysaccharide or glycoprotein.

(3) The natural killer cell concentrate according to (2) above, wherein the plant-derived polysaccharide is a polysaccharide with a molecular weight of 200,000 to 1,000,000 isolated from taro stems.

(4) The natural killer cell concentrate according to (2) above, wherein the plant-derived glycoprotein is a lectin.

(5) A natural killer cell concentrator characterized by having a container filled with the natural killer cell concentrator according to any one of (1) to (4) above.

(6) A test solution containing natural killer cells is brought into contact with the natural killer cell concentrate according to any one of (1) to (4) above, and at least one of the other cells in the test solution is A method for concentrating natural killer cells, which comprises concentrating natural killer cells in a test liquid by adsorbing seeds to the natural killer cell concentrator.

(7) A test solution containing natural killer cells is passed through the container of the natural killer cell concentrator described in (5) above, and at least one of the other cells in the test solution is absorbed by the natural killer cells. A method for concentrating natural killer cells, which comprises concentrating natural killer cells in a test solution by adsorbing them to a killer cell concentrator.

[0027]

[Action] The NK cell concentrate of the present invention has low adhesion to NK cells, and has at least 1% of other cells in the test solution.
Since this NK cell concentrate is made by immobilizing (bonding) organic substances, especially plant-derived polysaccharides or glycoproteins, on a water-insoluble solid substance, a test liquid such as blood can be added to this NK cell concentrate. When brought into contact, cells other than NK cells that have an affinity for the organic substance are adsorbed to the NK cell concentrator, allowing NK cells to be concentrated with high efficiency and separated from other cells with good yield.

To explain this effect in more detail, cell membrane surface antigen receptors are composed of glycoproteins, glycolipids, proteins, and lipids, so certain organic substances, particularly plant-derived polysaccharides or glycoproteins, are It is thought that selective adsorption occurs by recognizing sugar structures on the membrane surface of specific cells. A physicochemical analysis of these specific interaction forces reveals that the organic matter that has an affinity for a specific cell and the membrane surface of a specific cell are affected by the van der Waals force (the content of this force is the orientation effect). , dielectric effect and dispersion effect), the intermolecular forces of hydrogen bond, ionic bond (Coulomb force), ion-dipole force, coordinate bond and hydrophobic bond are combined. It is assumed that the spatial structure formed by the surface molecules is sterically compatible with each other, like a key and a keyhole.

As mentioned above, lymphocytes, as immunocompetent cells, are closely involved in various immune diseases, and NK cells in particular have the effect of killing (lysing) certain tumor cells.

[0030] Therefore, if NK cells can be separated and concentrated from other cells at a high yield, it will be useful not only for immunological research but also for the diagnosis and treatment of cancer diseases.

[0031]

[Examples] <Structure of the Invention> The NK cell concentrator, concentration device, and concentration method of the present invention will be explained in detail below.

The NK cell concentrate of the present invention contains an organic substance (hereinafter simply referred to as an organic substance) that has low adhesion to NK cells and has an affinity for at least one type of other cell in the test solution. It is immobilized on a water-insoluble solid substance.

[0033] As the organic substance, saccharides are preferable, and plant-derived polysaccharides or glycoproteins are particularly preferable, since they are particularly effective in concentrating NK cells.

The plant-derived polysaccharide used in the present invention is preferably a polysaccharide isolated from taro stems with a molecular weight of 200,000 to 1,000,000, particularly 300,000 to 800,000.

[0035] Taro stems have been eaten since ancient times as a side dish or emergency food, and now as a health food, and contain a large amount of polysaccharide. Among taro varieties, those with red stems, such as Chinese sweet potatoes with less astringent stems, Yazu, and petioles of Lotus potatoes, which are specialized in Zuiki, are particularly preferred. Dried stems are often called potato stalks, but they are particularly suitable for use because they are less harsh.

The reason why a molecular weight of 200,000 to 1,000,000 is preferable is as follows.
By having such a molecular weight, the molecular chain becomes appropriately long, which facilitates adhesion with cells, and also provides an appropriate surface density of side chains and functional groups used for interaction with cells. .

[0037] Such a polysaccharide derived from taro stalks has a high affinity for T lymphocytes, which make up the majority of lymphocytes, and in particular for helper/inducer T lymphocytes, which are a subset thereof, and also has a high affinity for NK cells. Since they have low adhesion to NK cells, NK cells can be efficiently concentrated.

[0038] The polysaccharides are not limited to those isolated from the taro stems mentioned above, but may also be polysaccharides extracted from shiitake mushrooms, herbal medicines such as sandalwood, canker, and sanyaku, or polysaccharides contained in seaweed. .

[0039] Plant-derived glycoproteins in the present invention include cell wall components such as lectins, enzymes, hormones, and extensins, among which lectins are particularly preferred.

[0040] Lectin is a protein or glycoprotein that interacts with sugars, aggregates cells, or precipitates polysaccharides and complex carbohydrates, but is not an immunological product.

[0041] Representative examples of such lectins include the following.

1) L-fucose binding lectin: Lot
us tetragonolobus
, Ulex europaeus-I, rabbit serum, etc.

2) D-galactose, N-acetyl-D
- Galactosamine binding lectin: Vicia vil
losa (hairy vetch), Ricinus co
mmunis, Arachis hyp
ogea (peanut), Glycine max
(soybean), Bauhinia purpurea (
Mokwanju), Phaseolus vulgar
is (haricot bean).

3) D-mannose binding lectin: Can
avalia ensiformis (jack bean; concanavalin A), Lens culinari
s (lentil), Pisum sativum (pea), Vicia faba (fava bean).

4) Di-N-acetylchitobiose binding lectin: Triticum vulgaris (wheat germ; wheat germ agglutinin), Phytolacca ame
ricana (Pokeweed; PWM lectin)
, Solanum tuberosum (potato).

5) Sialic acid binding lectin: Limulu
s polyphemus (horseshoe crab).

In the present invention, among such lectins, the above 2) is preferred, and hemagglutinin obtained from hairy vetch or castor bean is particularly preferred.

Although the molecular weight of the lectin is not particularly determined, it is preferably about 10,000 to 500,000, particularly about 50,000 to 200,000.

[0049] Such lectins, especially those derived from hairy vetch and castor beans, have a high affinity for T lymphocytes, which make up the majority of lymphocytes, and especially for helper/inducer T lymphocytes, which are a subset thereof. ,
In addition, since it has low adhesion to NK cells, NK cells can be efficiently concentrated.

[0050] The organic substances in the present invention are not limited to the above-mentioned plant-derived polysaccharides or glycoproteins, but include, for example,
Polysaccharides or glycoproteins derived from animals or bacterial cells, or
Glycosides (eg, vitamin P), monosaccharides, oligosaccharides, etc. derived from animals and plants can also be used.

Furthermore, the organic substance in the present invention may have an affinity not only for lymphocytes but also for cells other than lymphocytes such as white blood cells, red blood cells, and platelets.

[0052] The amount of such organic substances, especially plant-derived polysaccharides or glycoproteins, supported on the water-insoluble solid substance is not particularly limited, but is 1 x 10-3 to 5 x 102 μmo.
l/g, more preferably 1×10
-3 to 1×10 2 μmol/g.

[0053] In the present invention, the water-insoluble solid substance serving as the carrier for immobilizing the organic substance may be hydrophilic or hydrophobic, and specifically, for example, , agarose-based, dextran-based,
Polysaccharides such as chitosan and cellulose, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylonitrile, styrene-divinylbenzene copolymer, polystyrene, acrylic ester, methacrylic ester, polyethylene, Organic synthetic polymers such as polypropylene, poly(4-fluoroethylene), ethylene-vinyl acetate copolymer, polyamide, polycarbonate, polyvinylidene fluoride, polyvinyl formal, polyarylate, and polyethersulfone, collagen , biologically derived natural organic polymers such as chitosan, glass-based, titanium-based, activated carbon-based, and inorganic materials such as various ceramics such as alumina, silica, and hydroxyapatite. Usually, immobilized enzyme method, Known carriers used in affinity chromatography and the like can be used without particular limitations. Among these, those with not too high cell adhesion, ie, those with relatively mild cell-carrier interaction, are particularly preferred.

The form of such a water-insoluble solid substance is not particularly limited, and any known form such as particulate, fibrous, hollow fiber, membrane, or flat plate may be used. A particulate material is preferable from the viewpoint of liquid permeability for test liquids, washing liquids, etc., and ease of handling when preparing a separating agent. In this case, the average particle size of the water-insoluble solid substance is 0
．． The thickness is preferably 0.05 to 5 mm, particularly 0.1 to 2 mm, from the viewpoint of effective surface area and liquid permeability.

That is, if the average particle diameter is less than 0.05 mm, the liquid permeability will decrease, and if it exceeds 5 mm, it will be difficult to secure a sufficient effective surface area.

[0056] The above average particle diameter is determined according to JIS-Z-88
After classification using a sieve specified in No. 01, the intermediate value between the upper limit particle size and the lower limit particle size of each class is taken as the particle size of each class, and the weight average thereof can be calculated.

Furthermore, the particle shape (especially spherical particles) is
It is preferable because it is less likely to cause physical damage to cells, less likely to cause flaking or chipping, and easier to obtain uniform particles.

Further, such a water-insoluble solid substance may be either porous or dense.

As the porous particles, for example, acrylic porous beads made of a ternary copolymer of divinylbenzene monomer, glycidyl methacrylate monomer and 2-hydroxyethyl acrylate oligomer are preferably used.

In the present invention, methods for immobilizing the organic substance on the surface of a water-insoluble solid substance include carrier bonding methods such as covalent bonding, ionic bonding, physical adsorption, hydrophobic bonding, and biochemical specific bonding, or crosslinking methods. Any known method can be used, such as the entrapment method, the combination method, etc., but from the viewpoint of stability in body fluids or cell suspensions, it is preferable to immobilize by covalent bond or hydrophobic bond.

[0061] In order to covalently bond the organic substance to the surface of the water-insoluble solid substance, various methods can be used depending on the functional groups of both substances. For example, cyanogen bromide activation method, condensation reagent method using carbodiimide reagent, acid azide derivative method, diazo method, alkylation method, carrier crosslinking method using dialdehyde, bisepoxide, diisocyanate, etc.
-glycidoxypropyltrimethoxysilane and β-(3
, 4-epoxycyclohexyl)ethyltrimethoxysilane, etc. are used.

Furthermore, if necessary, spacer molecules of arbitrary length can be introduced between the organic substance and the surface of the water-insoluble solid substance. Preferred examples of this spacer molecule include diamines such as hexamethylene diamine and diols such as hexamethylene glycol.

[0063] Further, as a method for hydrophobically bonding the organic substance to the surface of a water-insoluble solid substance, for example, a method of directly bonding it to a hydrophobic polymer such as polystyrene or polypropylene, or a method of bonding it intramolecularly to a carrier by the above-mentioned covalent bonding method. There is a method of bonding a coupling agent having a hydrophobic group and then forming a hydrophobic bond. Examples of the coupling agent having a hydrophobic group in the molecule include octadecyldimethyl[3-(trimethoxysilyl)propyl]ammonium chloride, octadecyltrichlorosilane, and phenyltrichlorosilane.

Next, the method for enriching NK cells of the present invention will be explained.

[0065] In order to concentrate NK cells using the NK cell concentrator of the present invention, which is formed by immobilizing the organic substance on a water-insoluble solid substance, these NK cell concentrators are added to a test liquid (for example, blood, blood, etc.). It is also possible to collect NK cells in batches by immersing them in body fluids such as serum, plasma, lymph, cerebrospinal fluid, ascites, or other cell suspensions, and bringing them into contact with test fluids. , the NK cells of the present invention have a container (column) filled with an NK cell concentrator, from the viewpoint of securing the absolute amount of the organic matter that acts on the adsorption of cells other than NK cells and a sufficient cell adsorption space. Preferably, the contact is carried out using a concentrator.

That is, the NK cell concentrator (hereinafter simply referred to as the concentrator) of the present invention is characterized by having a container filled with an NK cell concentrator in which the organic substance is immobilized on the water-insoluble solid substance. It is.

In this concentrator, the container can be made of synthetic resins such as polyethylene, polypropylene, polycarbonate, polystyrene, polymethyl methacrylate, glass, metals such as stainless steel, and various ceramics, but autoclave sterilization is not recommended. Particularly preferred are materials such as polypropylene and polycarbonate, which are available and easy to handle.

[0068] Furthermore, between the entrance and exit of the column, which is the container of this concentrator, and the concentrator layer filled with the NK cell concentrator, the cell components in the test liquid can pass through, but the NK cell concentrator cannot. It is preferable to include a filter member having a mesh or pores. As this filter member, for example, a mesh, a woven fabric, a non-woven fabric, etc. are suitably used, and its constituent material may be any physiologically inert and high-strength material, and it is particularly preferable to use polyester or polyamide.

FIG. 1 is a cross-sectional view schematically showing an example of the structure of the NK cell enrichment device of the present invention. Concentrator 1 shown in the figure
In this, a column container 4 having a fluid inlet 2 and a fluid outlet 3 is filled with the NK cell concentrating agent 5 preferably made of a particulate insoluble solid substance, and this separating agent 5 has a fluid inlet and a fluid outlet 3. It is held within the column container 4 by filters 6a and 6b provided near the port 2 and the fluid outlet 3, respectively.

[0070] The amount of the NK cell concentrator 5 packed into the column container 4 is not particularly limited, but it is preferably about 0.1 to 1000 g, particularly about 0.5 to 400 g.

[0071] Furthermore, in the present invention, the column container 4 is filled with two or more different organic substances immobilized in insoluble solid substances (in a state in which the various types are mixed or separated into layers). You may.

[0072] In order to perform extracorporeal circulation therapy on a patient using this concentrating device 1, it is sufficient to incorporate this concentrating device 1 into a circuit as shown in FIG. 2, for example.

To briefly explain extracorporeal circulation therapy using the extracorporeal circulation circuit shown in FIG. 2, blood removed from a patient is introduced into the circuit from the blood inlet 7 of the circuit, The NK cell concentrator 5 is introduced from the fluid inlet 2 into the concentrator 1 at a constant flow rate through a chamber 9a equipped with a pressure gauge 10a, and is housed in the column container 4 when passing through the column container 4. come into contact with.

If the NK cell concentrator 5 is capable of selectively adsorbing T lymphocytes, for example, T lymphocytes in the blood can be
Lymphocytes are selectively adsorbed to the NK cell concentrator 5, and as a result, blood enriched with NK cells can be obtained.

The blood that has been treated in this way is led out of the concentrating device 1 through the fluid outlet 3, and the blood is drawn out from the concentrator 1 through the fluid outlet 3,
After passing through the chamber 9b equipped with
3, it is returned to the patient's body.

[0076] When this extracorporeal circulation therapy is applied to, for example, a patient suffering from cancer, efficient treatment can be expected by concentrating NK cells in the patient's blood and returning the blood.

[0077] Another method is to collect the NK cells concentrated according to the present invention into a culture bag, culture them by adding cell growth factors such as cytokines, increase killer activity, and return them to the patient. As a result, even more effective therapy can be expected.

[0078] In the concentrating device 1 having the above configuration, the inside of the column container 4 (the part that comes into contact with blood) is subjected to moist heat sterilization (autoclave sterilization) or gas sterilization (for example, E
It is preferable that the sterilization process is performed by a sterilization method such as OG sterilization) or radiation sterilization (eg, γ-ray sterilization).

[0079] The concentrating device of the present invention does not deteriorate in performance even after such sterilization treatment and exhibits stable NK cell concentrating ability, so that appropriate diagnosis and safe treatment can be performed.

The container used in the concentrator of the present invention is not limited to the column container shown in the figure, but may also be, for example, a syringe outer cylinder, a tube, a bottle, a bag, or the like.

<Examples> Specific examples of the present invention will be described below.

(Example 1) As a carrier (water-insoluble solid substance), the raw material composition of divinylbenzene monomer, glycidyl methacrylate monomer, and 2-hydroxyethyl acrylate oligomer (molecular weight 230) was 22.2% by weight.
Granules of terpolymer having a ratio of 8:46.2:31.0 (
To 5 g of Fujikura Kasei Co., Ltd., average particle size 0.15 mm (hereinafter referred to as porous particles A), a polysaccharide solution extracted from taro cone (molecular weight 200,000 to 1,000,000, glucose equivalent sugar concentration 0)
．． 05M, pH 10) was added and degassed. Blood mixer (BM-10 manufactured by Kayagaki Irika Kogyo Co., Ltd.)
Type 1) and stirred at room temperature for 2 days.

Next, this was washed with 250 ml of distilled water, and then 10 ml of a 1M monoethanolamine solution adjusted to pH 8 with acetic acid was added and degassed. Then, using a blood mixer, the mixture was stirred at room temperature for one day.

[0084] The NK cell concentrate thus prepared was mixed with 400 ml of distilled water and 0.2 M carbonate buffer (pH 10).
1 dl, 0.1M acetate buffer (pH 4) 1 dl, 0.1
The plate was washed with 1 dl of M phosphate buffer (pH 7.4) in this order.

[0085] The procedure for separating polysaccharides from taro stems is as follows. First, the potato stalks were soaked in water to remove the scum until they were no longer cloudy, and then ground in a mixer. This pulverized product was soaked in water and extracted in a water bath at 85° C. for 2 hours with stirring. Next, 100 mesh filter cloth and JI
S standard No. Solid-liquid separation was performed using filter paper No. 2, and the filtrate was collected and concentrated under reduced pressure. 63% moisture from 1 kg of potato stalks (dry matter)
Approximately 340 g of extract was collected.

The NK cell concentrate thus obtained was suspended in a phosphate buffered saline solution (PBS) of pH 7.4, and a volume of 2.5 cm containing a polyester nonwoven fabric inside the tip was suspended.
0.0ml disposable syringe (manufactured by Terumo Corporation).
Filled with 4ml. Then, a needle tube and a cell collection container were attached to the tip of this syringe to form an NK cell concentrator.

[0087] Using this concentrator, the following cell separation experiment was conducted.

That is, a suspension of human peripheral blood mononuclear cells obtained using a mononuclear cell separation tube (manufactured by Becton Dickinson, trade name: Leucoprep) was obtained from the upper end opening of the syringe constituting the concentrator. Add 100 μl of PBMC (white blood cell count: 600 x 104 cells), and add 32 μl of PBS.
After the cells were submerged in the NK cell concentrator layer in the syringe, they were allowed to stand at room temperature for 30 minutes.

Next, the NK cell concentrate was rinsed with 0.8 ml of PBS, and the cells that had flowed out were collected.

Then, the number of cells in this collected solution was measured using a multi-item automatic blood analyzer (Sysmex NE manufactured by Toa Medical Electronics Co., Ltd.).
-6000), then phycoerythrin (PE)
It was stained with labeled anti-Leu-19 antibody (Becton Dickinson) and analyzed using a flow cytometer (Cyto A).
cc-150 (manufactured by JASCO Corporation), the percentages of NK cells and lymphocytes were determined. Then, the cell number and lymphocytes before contact with the NK cell concentrate, PE-labeled anti-L
The adhesion rate of each cell was calculated by comparing it with the percentage of eu-19 antibody positive cells.

The results are shown in Table 1.

(Example 2) To 10 g of the porous particles A,
2w/v% solution (pH 10) of hairy vetch (Vicia villosa) lectin (molecular weight: approximately 120,000)
) was added and degassed.

[0093] In the following, preparation of an NK cell concentrate and cell separation experiment were carried out in the same manner as in Example 1. However, N
The amount of K cell concentrate filled into the syringe was 1 ml, and the number of white blood cells added was 750 x 104.

The results are shown in Table 1.

(Example 3) Castor bean (Ricinus communis) lectin (Ricinus communis) was added to 5 g of the porous particles A.
0.45 w/v% solution (pH 10) of molecular weight approximately 120,000
10 ml was added and degassed.

[0096] In the following, preparation of an NK cell concentrate and cell separation experiment were carried out in the same manner as in Example 1. However, N
The amount of K cell concentrate filled into the syringe was 1 ml, and the number of white blood cells added was 1500 x 104.

The results are shown in Table 1.

(Example 4) To 20 g of the porous particles A,
50 ml of a 0.05M solution of vitamin P (pH 10) was added and degassed.

[0099] In the following, preparation of an NK cell concentrate and cell separation experiment were conducted in the same manner as in Example 1. However, N
The amount of K cell concentrate filled into the syringe was 1 ml, and the number of white blood cells added was 1500 x 104 cells.

[0100] The results are shown in Table 1.

(Example 5) Glycoprotein (molecular weight approximately 100,000) extracted with hot water from the corbicula was added to 5 g of the porous particles A.
10 ml of a 0.05M solution (pH 10) was added and degassed.

[0102] Hereinafter, in the same manner as in Example 1, preparation of an NK cell concentrate and cell separation experiment were conducted. However, N
The amount of K cell concentrate filled into the syringe was 1 ml, and the number of white blood cells added was 1500 x 104.

[0103] The results are shown in Table 1.

(Comparative Example) The above porous particles A were used as they were in N.
A cell separation experiment was conducted in the same manner as in Example 1, except that it was used as a K cell concentrator.

[0105] However, the amount of NK cell concentrate to be filled into the syringe is 1 ml, and the number of white blood cells to be added is 1500 x 10
There were 4 pieces.

[0106] The results are shown in Table 1.

[0107]

[Table 1]

[0108] Items A to F in Table 1 are as follows.

A: Bound organic matter B: Adhesion rate of leukocytes (%) C: Adhesion rate of lymphocytes (%) D: Adhesion rate of non-lymphocytes (%) E: Adhesion rate of NK cells (Leu19+) (%) )F:NK
Positive rate of cells (%)

[0110] As is clear from the results shown in Table 1, the NK cell concentrators of the present invention according to Examples 1 to 5, especially 1 to 3, all showed higher N
The adhesion rate of K cells is low, and NK cells are efficiently concentrated.

[0111] Furthermore, the recovered NK cells showed no decrease in function or change in morphology.

[0112]

[Effects of the Invention] As described above, according to the present invention, NK cells can be concentrated with high yield and purity without impairing cell functions, and the operation is extremely simple. It's quick.

[0113] Therefore, for example, in basic research such as functional analysis of lymphocytes related to immune function, various clinical tests, diagnosis and treatment of immune diseases, etc., more reliable results, more effective treatment, or more Efficient processing operations can be expected.

[0114] Furthermore, according to the NK cell concentrator and concentration method of the present invention, the NK cell concentrator having the above-mentioned characteristics can be brought into contact with the test liquid more efficiently. When applied to extracorporeal circulation therapy, efficient treatment can be expected by concentrating NK cells in the patient's blood and returning the blood.

Furthermore, the NK cell concentrator of the present invention does not show any deterioration in performance even when subjected to sterilization treatments such as moist heat sterilization, gas sterilization, or radiation sterilization, and exhibits stable NK cell concentration ability, making it suitable for accurate diagnosis. , it is possible to perform safe treatment.

[Brief explanation of the drawing]

FIG. 1 is a vertical cross-sectional view schematically showing a configuration example of the NK cell enrichment device of the present invention.

FIG. 2 is a schematic configuration diagram showing an example of an extracorporeal circulation circuit incorporating the NK cell concentrator of the present invention.

[Explanation of symbols]

1 NK cell concentrator 2 Fluid inlet 3 Fluid outlet 4 Column container 5 NK cell concentrate 6a, 6b filter 7 Blood introduction port 8 Blood pump 9a, 9b Chamber 10a, 10b Pressure gauge 11　　　　　Thermostatic chamber” 12 Air bubble detector 13 Blood outlet

Claims (7)

[Claims] [Claim 1] An organic substance that has low adhesion to natural killer cells and has an affinity for at least one type of other cells in the test solution is immobilized on a water-insoluble solid substance. Natural killer cell concentrate. 2. The natural killer cell concentrate according to claim 1, wherein the organic substance is a plant-derived polysaccharide or glycoprotein. 3. The natural killer cell concentrate according to claim 2, wherein the plant-derived polysaccharide is a polysaccharide separated from taro stems and having a molecular weight of 200,000 to 1,000,000. 4. The natural killer cell concentrate according to claim 2, wherein the plant-derived glycoprotein is a lectin. 5. A natural killer cell concentrator comprising a container filled with the natural killer cell concentrator according to any one of claims 1 to 4. 6. A test liquid containing natural killer cells is brought into contact with the natural killer cell concentrate according to any one of claims 1 to 4, and at least one of the other cells in the test liquid is concentrated into the natural killer cells. A method for concentrating natural killer cells, which comprises concentrating natural killer cells in a test solution by adsorbing them to a killer cell concentrator. 7. A test liquid containing natural killer cells is passed through the container of the natural killer cell concentrator according to claim 5, and at least one of the other cells in the test liquid is
A method for concentrating natural killer cells, which comprises concentrating natural killer cells in a test liquid by adsorbing seeds to the natural killer cell concentrator.