JP2922565B2 - Separation material, separator and separation method for T lymphocyte subfraction - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a lymphocyte subfraction separation material, a separator and a separation method for separating and purifying a specific subfraction in lymphocytes. It is. More specifically, the present invention relates to T helpers / inducers (TH _{/ I} ) and T suppressors / cytotoxics, which are subfractions in T lymphocytes from lymphocyte suspensions obtained from human peripheral blood and lymphoid tissues. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a simple, rapid and highly accurate separation material, a separator and a separation method for lymphocyte subfraction capable of selectively capturing (T _{S / C} ).

Lymphocytes are cells that are responsible for immune functions differentiated from blood cell stem cells and are divided into several subgroups in terms of functions, cell membrane characteristics, and the like. They are lymphocyte subclasses such as B lymphocytes that produce antibodies and carry humoral immunity, T lymphocytes that produce immunomodulatory lymphokines and inflammatory lymphokines and carry cell immunity, and null cells,
In addition, there are functionally different subsets of T, B lymphocytes. In recent years, T,
Techniques for separating, purifying, and enriching subclasses such as B lymphocytes with minimal functional damage are strongly desired in a wide range of social fields. For example, in basic research such as cytology and immunology, in vitro
In vitro), it is necessary to separate at least the T and B lymphocyte groups, and in diagnosis and treatment in clinical medicine, it is necessary to transfer or remove a specific fraction with reduced or enhanced function. In obtaining a physiologically active substance in a system, it is important to separate and concentrate the production cell fraction.

(Prior Art) As a method for separating T lymphocytes and B lymphocytes, first, lymphocyte fractionation from peripheral blood or living tissue by specific gravity centrifugation using a high-density isotonic solution such as a mixture of sodium methisoate ficoll Which is then separated and processed to T
Conventionally, lymphocytes and B lymphocytes are separated. This subsequent separation processing method is largely (1)
It is classified into three categories: those using specific antigens, receptors, and immunoglobulins on the cell membrane surface as indicators, (2) physical cell separation, and (3) adsorption chromatography.

Examples of the method (1) include a rosette formation method, affinity chromatography (to which antibodies and immune complexes have been bound), a cell lysis method using antibodies and complement, removal of mitogen-activated lymphocytes, and a fluorescein-activated cell sorter ( FACS) is known, but in these methods, T lymphocytes or B lymphocytes are intact because they receive strong binding and stimulation with high biological specificity.
There are many techniques that make it difficult to collect lymphocytes and are not suitable for mass processing. As the method (2), a density gradient centrifugation method, a cell electrophoresis method, and the like are known. However, the physical properties such as specific gravity and surface charge between T lymphocytes and B lymphocytes are not suitable. Since there is no significant difference, high precision cannot be required for the yield or purity of the separated and recovered cells. Further, as the method (3), a method utilizing the difference in non-specific adhesion ability of T lymphocytes and B lymphocytes to foreign substances such as nylon, tetron, and cotton fiber is known. In order to obtain fetal bovine serum (FCS)
It is difficult to set conditions for implementation such as flow rate and the like, and there is much contamination with impurities such as macrophages. In addition, these conventional methods generally have a major problem in that complicated operations or preparations are required, and empirical technical learning when performing separation operations is essential. .

(Problems to be Solved by the Invention) Accordingly, the present invention provides a novel lymphocyte subfraction separation material,
It is an object to provide a separator and a separation method. The present invention also provides a simple, rapid and high-yield, high-purity natural killer (NK) without affecting various cell activities.
Alternatively, it shows a separation material, a separator and a separation method of a lymphocyte subfraction for separating and purifying an LGL [large granular lymphocyte] lymphocyte fraction. The invention further provides
An object of the present invention is to provide a leukocyte separation material, a separator and a separation method suitable for mass processing.

(Means for Solving the Problems) The above-mentioned objects are to dissolve a lipid which makes the T _{H / I} and T _{S / C} fractions in a lymphocyte fraction preferentially adsorb to other lymphocyte fractions. This is achieved by a separation material for lymphocyte subfraction, which is immobilized on the surface of an insoluble solid substance.

The present invention also provides a separating material for lymphocyte subfraction, wherein the lipid to be immobilized is a phospholipid having an acyl chain having 8 to 26 carbon atoms. The present invention further provides a separation material for lymphocyte subfraction, wherein a particulate material having a particle size of 0.05 to 5 mm is used as an insoluble solid substance. The present invention further provides a separation material for lymphocyte subfraction, wherein lipids are immobilized as hydrophobic parts introduced on the surface of a water-insoluble solid substance through hydrophobic bonds by linear alkyls composed of methylene repeats. It shows.

The above objects are further achieved by a lymphocyte subfraction separator characterized by having a column packed with a lymphocyte subfraction separating material as described above.

The above objects are further achieved by adhering a lymphocyte suspension to a separating material for lymphocyte subfractions as described above,
This is also achieved by a method for separating lymphocyte subfractions, characterized in that a non-captured fraction rich in NK or LGL lymphocytes is recovered by efficiently capturing the inducer and T suppressor / cytotoxic fraction. You.

(Action) In the present invention, a lipid that adsorbs the T _{H / I} and T _{S / C} fractions in a lymphocyte fraction preferentially over other lymphocyte fractions is immobilized on the surface of a water-insoluble solid substance. In order to use a lymphocyte subfraction separating material characterized by the fact that, for example, by merely injecting a lymphocyte suspension into a column filled with the separating material, the NK or LGL-based lymphocyte fraction can be efficiently separated.・ It can be concentrated. The separation material in which the predetermined lipid is immobilized on the surface of the water-insoluble solid substance has T
The detailed mechanism by which the _{H / I} and T _{S / C} fractions are preferentially adsorbed is currently unknown.

Furthermore, in the separation operation using the separation material of the present invention, it is not necessary to add fetal bovine serum or the like to the lymphocyte suspension (it is possible to add fetal bovine serum and the like in some cases), and sterilization is performed. The NK or LGL-based lymphocyte fraction can be aseptically separated and collected simply by bringing the lymphocyte suspension into contact with the treated separation material, and the viability of the recovered cells is always 98% or more without using serum. Maintenance suggests that the stimulation of lymphocytes is also extremely low.

Hereinafter, the present invention will be described in more detail based on embodiments.

The separating material for lymphocyte subfraction of the present invention comprises a lipid which causes the TH _{/ I} and Ts _{/ C} fractions in the lymphocyte fraction to be preferentially adsorbed over other lymphocyte fractions. It is characterized by being immobilized on the surface of a substance.

Examples of the lipid used in the present invention that adsorbs the T _{H / I} and T _{S / C} fractions in the lymphocyte fraction more preferentially than other lymphocyte fractions include phospholipids, particularly those having 8 to 26 carbon atoms. More preferably, those having an acyl chain having 12 to 20 carbon atoms can be used. The phosphoric acid portion of the phospholipid including the phosphoric acid ester of the hydrophilic portion may have any structure. The phospholipid may be any of natural or non-natural, specifically, for example,
Phosphatidylcholine, phosphatidylglycerol,
Phosphatidylethanolamine, phosphatidylinositol containing a sugar molecule, ganglioside and the like can be suitably used. In some cases, an unsaturated bond may be present in the acyl chain.

Such a lipid can be directly immobilized on the surface of the water-insoluble solid material, but preferably has a hydrophobic bond introduced by a linear alkyl composed of repeating methylene as a hydrophobic portion introduced on the surface of the water-insoluble solid material. It is desirable to be immobilized through the interface.

In the present invention, as the straight-chain alkyl composed of methylene repeats, various alkylamines having about 4 to 26 carbon atoms, fatty acids, alcohols, and simple lipids and complex lipids containing these can be used. It may be a linear one having a methylene repetition of 26 or less, particularly a methylene repetition of about 8 to 20 is preferable because higher characteristics can be imparted.

Such a linear alkyl can be introduced to the surface of the water-insoluble carrier using a known coupling agent such as a multi-sensitive epoxide, aldehyde, or carbodiimide. In particular, when glass beads are used as the water-insoluble carrier as described later, for example, octadecyldimethyl [3-
If a silane coupling agent such as (trimethoxysilyl) propyl] ammonium chloride is used, straight-chain alkyl can be easily introduced.

Then, the water-insoluble carrier having the linear alkyl introduced on its surface can easily immobilize the above-mentioned lipid by hydrophobic bonding.

Examples of the water-insoluble solid substance as the carrier used in the present invention include agarose, dextran, cellulose, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylonitrile, styrene-divinylbenzene copolymer, and polystyrene. Acrylate, methacrylate, polyethylene, polypropylene, poly4-fluoroethylene, ethylene-vinyl acetate copolymer, polyamide, polycarbonate, polyvinylidene fluoride, polyvinyl formal, polyarylate Organic polymers such as polyethersulfone, glass-based, alumina-based, titanium-based, activated carbon-based, and ceramics-based inorganic substances. Interaction between the carrier be relatively mild are preferred. In addition, collagen, chitosan, or the like, which is a natural organic polymer derived from a living body, can also be used. The form of such a water-insoluble solid substance is not particularly limited, and those on a flat plate can be used. However, it is preferably in the form of particles, particularly those having an average particle diameter of 0.05 to 5 mm. The average particle size was calculated using a sieve stipulated in JIS-Z-8801, then the median of the upper and lower particle sizes of each class was taken as the particle size of each class, and the weight average was calculated. It is. Further, the particle shape is preferably spherical from the viewpoint that the cells are hardly damaged physically and uniform particles are easily obtained.

The lymphocyte subfraction separator of the present invention comprises a lipid which adsorbs the _{TH / I} and Ts _{/ C} fractions in the lymphocyte fraction preferentially over other lymphocyte fractions as described above, It is characterized by having a column packed with a separation material for lymphocyte subfraction, characterized by being immobilized on the surface of a water-insoluble solid substance.

The contacting of the lymphocyte subfraction separating material of the present invention with a lymphocyte suspension can be performed, for example, by bringing the lymphocyte suspension into contact with a plate-like lymphocyte subfraction separating material. However, from the viewpoint that an absolute amount of hydrophobic sites existing on the surface of the separation material and acting on cell adsorption as described above and a sufficient cell adsorption space are obtained, a column packed with a particulate separation material is used. It is desirable to make contact using a separator having the same.

In the separator for lymphocyte subfractionation having a column filled with this particulate separating material, the material constituting the column container is a synthetic resin such as polyethylene, polypropylene, polycarbonate, polystyrene, polymethyl methacrylate, or glass. And metals such as stainless steel, etc., and polypropylene and polycarbonate, which can be autoclaved and easy to handle, are particularly preferred. In addition, between the inlet and outlet of the column of the lymphocyte subfraction separator and the separation material layer filled with the separation material of lymphocyte subfraction, the cell components in the lymphocyte suspension pass, but the separation material is It is preferable to have a filter having a mesh that cannot pass, as a material constituting this filter,
Any material may be used as long as it is physiologically inert and high in strength, but polyester and polyamide are particularly preferred.

FIG. 1 is a schematic view of an embodiment of the separator for lymphocyte subfraction of the present invention in use. In the embodiment shown in FIG. 1, the column 1 has a tubular shape whose both ends are opened and whose diameter is gradually reduced from a portion slightly above the lower end opening to the lower end opening which becomes the outlet 5. Further, the tapered part 7 is provided with a three-way cock 4 so that the flow path to the outlet 5 can be opened and closed. The column 1 above the tapered portion 7 is filled with the separating material 2 of the present invention for lymphocyte subfractionation as described above. The filter 3 provided above the part 6 and the filter 3 'provided at the lower end are held in the column 1 to form a separation material layer.

In addition, the method for separating a lymphocyte subfraction of the present invention comprises the step of removing a lipid which adsorbs the _{TH / I} and Ts _{/ C} fractions in the lymphocyte fraction preferentially over other lymphocyte fractions as described above. A lymphocyte suspension is brought into contact with a lymphocyte subfraction separating material, which is immobilized on the surface of a water-insoluble solid substance, to efficiently capture T helper / inducer and T suppressor / cytotoxic fractions By this, a non-captured fraction rich in NK or LGL lymphocytes is collected.

The method for separating a lymphocyte subfraction according to the present invention will be described in more detail by taking, as an example, the case where the separator shown in FIG. 1 is used. First, a lymphocyte liquid suspended in a peripheral blood balanced salt solution or A lymphocyte suspension such as a lymphocyte liquid resuspended in animal plasma is injected into a column 1 filled with a lymphocyte subfraction separating material 2 and allowed to stand.

Here, the method of passing the lymphocyte suspension is not particularly limited, and may be continuous or discontinuous as necessary.

After the lymphocyte suspension is brought into contact with the separating material 2 for a predetermined time in the column 1 in this manner, a rinsing solution is injected into the column 1 using various pumps or pipettes.
The three-way cock 4 is opened, and the washing solution is gently dropped from the outlet 5 and collected by the test tube 8 or the like.

NK or LG not adsorbed on the separation material 2
The non-captured fraction containing a large amount of the L lymphocyte fraction can be collected.

Note that the rinsing liquid used in this washing operation is intended to wash off the non-adsorbed cell components without eluting the cell components adsorbed on the separation material 2, and thus has a high activity. A low salt is desired, and a balanced salt solution, a cell culture solution and the like are used, and preferably a divalent cation-free balanced salt solution such as Hank's balanced salt solution (HBBS) is used.

(Examples) Hereinafter, the present invention will be described more specifically with reference to examples.

Comparative Example 1 Acrylic beads having an oxirane group as a water-insoluble carrier [Euperkid C (EC), φ150 μm, oxirane group content 200 μmol / g (wet) Rahme Pharma [Rh
m Pharma] was used to prepare hydrophobic beads having tetradecylamine immobilized thereon.

First, the EC was washed with distilled water, added to a 0.2 M tetradecylamine solution prepared using dimethylformamide (DMF), and heated in a water bath at 80 ° C for 3 hours.
It was allowed to stand in an oven at about 20 hours for about 20 hours.

The prepared reaction product is washed with DMF, dimethyl sulfoxide (DMSO) and water in this order, and autoclaved with DMSO and phosphate buffer (0.1 M pH 7.4) at 121 ° C. for 30 minutes. did.

The separating material thus obtained was dispersed in Hanks' balanced salt solution (HBSS, pH 7.4) and packed in a polypropylene column so as to be 0.1 ml in a wet state.

Next, 2 ml of fresh blood collected from a normal person was injected into the column and continuously flowed out. The column was immediately washed with 1 ml or more of Hanks' balanced salt solution or the like to collect lymphocytes.

The number of cells in the collected fraction, the percentage of monocytes and lymphocytes,
And the percentages of T and B lymphocytes were measured using an automatic blood cell counter (ELT Instruments, ORTHO INSTRUMENTS), ELT
-8) and an automatic cell analyzer (Cyto A, manufactured by JASCO Corporation)
(CE-100) and a fluorescently labeled anti-leu antibody (manufactured by Becton Dickinson). The cell adhesion rate to the separation material was calculated based on these measurement results. First result
It is shown in the table.

Example 1 Using the hydrophobic beads prepared in Comparative Example 1, phospholipid-immobilized beads were prepared.

First, tetradecylamine-bonded beads were phosphatidylglycerol (manufactured by Nippon Seika Co., Ltd.) using a chloroform solution (having 16 acyl chains and one unsaturated bond).
Contained. ) Solution (25 mg / ml), stirred for 1 hour and dried under reduced pressure. After this, 1 per 15g of beads
Was washed with water, stored in water for 1 day, and used for the experiment.

The lymphocyte separation experiment and the evaluation of the separation ability were performed according to the procedure of Comparative Example 1. Table 1 shows the results.

Example 2 Example 1 was repeated except that the phospholipid to be immobilized was changed to phosphatidylcholine (manufactured by Sigma) (having 16 carbon atoms in the acyl chain).
According to the method described above, phospholipid-immobilized beads were prepared and subjected to the same experiment. The results are shown in Table 1.

Example 3 A phospholipid to be immobilized was ganglioside (manufactured by Sigma)
Phospholipid-immobilized beads were prepared according to the method of Example 1 except that (derived from bovine brain) was used, and subjected to the same experiment. The results are shown in Table 1.

Example 4 Example 1 except that the phospholipid to be immobilized was changed to phosphatidylinositol (manufactured by Sigma) (from soybean).
According to the method described above, phospholipid-immobilized beads were prepared and subjected to the same experiment. The results are shown in Table 1.

(Effect of the Invention) As described above, the present invention provides a method for preparing TH _{/ I} in lymphocyte fractions.
And a lipid that allows the T _{S / C} fraction to be preferentially adsorbed over other lymphocyte fractions is immobilized on the surface of a water-insoluble solid substance. T _{H / I} (CD4 positive) and T selectively from lymphocyte suspension
It was revealed that the _{S / C} (CD8-positive) fraction adsorbed enables simple and rapid separation and concentration of NK or LGL lymphocyte fractions from lymphocyte suspensions. Therefore, the separation material of the lymphocyte subfraction of the present invention can be used for analysis of cell-cell interaction of leukocyte subfraction related to immune function, basic technology for efficiently obtaining lymphocyte-derived physiologically active substance, It was shown that the present invention can be applied to the treatment of various immune diseases, and that the separation material of the present invention can achieve high-precision T lymphocyte subfractionation without using a special physiologically active substance.

Furthermore, the present invention also provides that the lipid to be immobilized has 8 carbon atoms.
A phospholipid having an acyl chain of from 26 to 26, a particle having a particle size of 0.05 to 5 mm as an insoluble solid substance, and a hydrophobic part introduced on the surface of the water-insoluble solid substance, which is composed of methylene repetition. When the lipid is immobilized through a hydrophobic bond by a chain alkyl, a more efficient lymphocyte subfraction separation operation can be performed.

The present invention also relates to T cells in the lymphocyte fraction as described above.
A separation material for lymphocyte subfraction, characterized in that lipids that adsorb _{H / I} and T _{S / C} fractions preferentially over other lymphocyte fractions are immobilized on the surface of a water-insoluble solid substance. Since it is a lymphocyte subfraction separator characterized by having a packed column, the separation of the lymphocyte subfraction separating material having excellent characteristics as described above and the blood cell suspension are more efficiently performed. Thus, the operation of separating lymphocyte subfractions can be performed in a short time and in a defensive manner.

The invention further relates to T _{H / I} and T _{S / C} in the lymphocyte fraction.
Lipids that preferentially adsorb fractions over other lymphocyte fractions, a separation material for lymphocyte subfractions characterized by being immobilized on the surface of a water-insoluble solid substance, contacting the lymphocyte suspension, Efficient capture of T helper / inducer and T suppressor / cytotoxic fractions
Alternatively, since the method for separating lymphocyte subfractions is characterized by collecting a non-captured fraction enriched in LGL-type lymphocytes, it does not require operation skill compared to conventional separation methods.
It can be said to be an extremely useful technique because it can separate and concentrate NK or LGL lymphocytes, is inexpensive and does not cause any significant cell damage.

[Brief description of the drawings]

FIG. 1 is a schematic view of an embodiment of the separator for lymphocyte subfraction of the present invention in use. 1 ... column, 2 ... separation material for lymphocyte subfraction, 3, 3 '
... filter, 4 ... stopcock, 5 ... outlet, 6 ...
... actual diameter part, 7 ... reduced diameter part, 8 ... test tube.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Akira Takahashi 1500 Inoguchi, Nakai-machi, Ashigara-gun, Kanagawa Prefecture Inside Terumo Corporation (72) Inventor Toshihiro Akaike 4-15-23 Shimotani, Hoya-shi, Tokyo (56) References JP-A-60-231613 (JP, A) JP-A-3-23680 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) A61M 1/36 G01N 33/49 CA (STN)

Claims (6)

(57) [Claims] (1) A lipid which causes a T helper / inducer and a T suppressor / cytotoxic fraction in a lymphocyte fraction to be preferentially adsorbed over other lymphocyte fractions is immobilized on the surface of a water-insoluble solid substance. A separating material for lymphocyte subfraction, characterized by that: 2. The lipid to be immobilized is a phospholipid having an acyl chain having 8 to 26 carbon atoms.
2. The separating material for lymphocyte subfraction described in 1. above. 3. The separating material for lymphocyte subfraction according to claim 1, wherein a particulate material having a particle size of 0.05 to 5 mm is used as the insoluble solid substance. 4. The lipid according to claim 1, wherein the lipid is immobilized as a hydrophobic portion introduced on the surface of the water-insoluble solid substance through a hydrophobic bond by linear alkyl composed of methylene repeats. 2. The separating material for lymphocyte subfraction described in 1. above. 5. A separator for lymphocyte subfraction, comprising a column packed with the separation material for lymphocyte subfraction according to any one of claims 1 to 4. 6. A lymphocyte suspension is brought into contact with the lymphocyte subfraction separating material according to any one of claims 1 to 4, to efficiently perform T helper / inducer and T suppressor / cytotoxic fractionation. NK or LG by capturing
A method for separating a lymphocyte subfraction, which comprises collecting a non-captured fraction rich in L-type lymphocytes.