JPS58170717A - B-cell separating material, separator and process therefor - Google Patents

Abstract

PURPOSE:A lymphocyte suspension is brought into contact with a separating material made of a water-insoluble solid where amino groups unevenly distributes on its surface in a flow state to effect rapid and effective separation of B-cell from the suspension. CONSTITUTION:A column 3 is filled with a separating material 5 made of a water-insoluble solid where a hydrophobic compound and a compound having amino groups, represented by the formula [R is ClH2l+1 (l=1-3); m is 1-3] unevenly distribute on the surface and equipped with a filter 4 on the outlet side 2. A lymphocyte suspension is fed from the inlet 1 and brought into contact with the separating material in a flow state to separate B-cell from the suspension. When animal serum protein is applied to the separating material, the adhesivity of lymphocytes is reduced as a whole to increase separation selectivity for T from B cell.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a separating material for removing 1 mm of adhesive from a lymphocyte suspension and sorting the tissue, and a separator using wrinkle II#, and This invention relates to a method of making mmm seven-piece thickeners using shunting materials.

Isolating a single target vesicle from the extremely complex *m mixture in a living body, as pure as possible and without impairing its function, is not only essential for basic research in immunology, but also for immunological diseases. , 41 for the diagnosis and treatment of diseases, cancer, etc.
This is useful in my opinion.

For example, lymphocytes, which are immunocompetent cells, have different origins, antigenicity, cell membrane characteristics, and T cells (
There are two subpopulations: thymus-derived lymphocytes) and B cells (bone marrow-derived lymphocytes).

If these immunocompetent cell groups can be selectively separated and understood quantitatively and qualitatively, useful knowledge can be obtained for the diagnosis of RT-induced immunodeficiency or lymphoid lymphoid tumors. In this separation of the two,
Recently, a method of staining cells using antiserum 4I labeled with a fluorochrome dye and sorting cells using a fluorescein-adjusted cell sorter has recently attracted attention. However, this method has major drawbacks, such as the monovalent device and the stimulation and damage to cells due to antiserum treatment. Cell adhesion chromatographer fever, which is a method to separate cells by utilizing the difference in their adhesion to molecular materials, has the advantage of being relatively easy to operate. A method of separating cells from T cells is known. However, in the cases reported so far, the specificity of 1 minute T cells is low, and only a few highly pure T cells can be obtained. It also has the disadvantage that the function of the collected cells is difficult to maintain because the lymphocytes are brought into contact with the fibers for a period of time.

The term B@cell here refers to a lymphocyte that has immunoglobulin on its surface, and contains fluorescently labeled anti(immunoglobulin).
Fluorescent staining is performed with the antibody. The 1973 cells are said to be lymphocytes that are not stained by the antibody.

The present inventors, based on the current state of lymphocyte separation, B.
In order to efficiently remove cells and collect T cells with purity and without giving immunoreactive stimulation to the cells, we have detailed the adhesion phenomenon of B cells and T cells to the surface of foreign substances. As a result of further investigation, we found that, to our surprise, amino groups that have a strong interaction with lymphocytes can be unevenly distributed on the surface of solids and can be quickly and selectively removed, such as B cells. After discovering that the structure is resistant to structural changes and repeating various experiments, the present invention was completed.

That is, the present invention provides a BII cell line separation material and a mosquito separation material that adhesively removes Bwi cells from a lymphocyte suspension, which are made of a water-insoluble material on the surface of which 7J groups are unevenly distributed. BIl cells filled in a cathode having a liquid inlet and outlet and at least a filter at the liquid outlet
A method for isolating B111 cells K1, characterized in that a lymphocyte suspension is agitated with a vessel and the separation material KR in a moving state.
111-.

In the present invention, the solid material used as a solid having amino groups on its surface can be used regardless of its properties and shape as long as it is made of a substance that is linked to water. However, it is desirable that a so-called hydrophobic compound, which has a low affinity for water, and a compound having an ami-7 group exist in a seabird-like or lamellar-like solid form, respectively, and that the acetic groups are held nonuniformly on the surface. In order to achieve this, mixing of a bolyanometallic compound and a hydrophobic polymer, graft polymerization of an amino compound onto a hydrophobic polymer, co-polymerization of a monomer containing a polymeric amino compound with an aX monomer, etc. A graft polymer in which an amino compound is bound to a hydrophobic polymer by a covalent bond, although this can be achieved by means such as polymerization.

Copolymers are more preferably used.

The amino compound is suitably used in a range of 1 to 2J weight, more preferably 2 to 20 weight weight, relative to the hydrophobic polymer. Too much or too little amino compound is undesirable because it causes strong adhesion of lymphocytes and reduces the difference in adhesion behavior between TitBM and B cells.

The amino compound has a molecular weight of 7000~/0,00θ, more preferably 0,000~! , 0θ0 is preferably used. If the molecular weight is too small, island structures of sufficient size will not exist, and if the molecular weight is too large, the number of island structures will decrease, which is not preferable.

Examples of the amine compound used in the present invention include alkyleneimine and its derivatives, polyaddition compounds of divinylbenyne and diamine, etc., but are not particularly limited to the chemical structure thereof, and compounds R1 having the following general formula: ,R1,R,=C,Hayashi,+,()=θ~3)m
= / ~3 is more preferably used. In particular, the molecular weight represented by the following formula is /θOO~/θ, more preferably 2. θ
A monomer K containing a 7-mino group of 0θO at θO~.

R,,R,,R,=C,H□+1 ()=θ~3)
A copolymer obtained by copolymerizing with a hydrophobic monomer using m = / ~ 3 is particularly preferably used. The hydrophobic monomers mentioned here are styrene and methylstyrene.

Included are diphenylethylene, ethylstyrene, dimethylstyrene, vinylnaphthalene, vinyl 7-enanthrene, vinylmesitylene, chlorstyrene, poomstyrene, methoxystyrene, methyl methacrylate, methyl methacrylate, cyclohexyl methacrylate, vinylpyridine, and the like.

The B111111 material of this JI@ may be made of the above-mentioned polymer alone, but it is also preferable to coat it with glass particles or other water-insoluble polymer particles or fibers.
1I11 type, and among them, those coated on spherical particles with a particle size of 0 to 40 mesh are preferred.

The IIIIIIA particulate material in this beak is preferably one in which one piece of flea white matter is attached to one water-insoluble body in which the ano groups are present in a non-uniform distribution on the separated surface.

The protein solution used during attachment is serum aldimine, r-
Globulin or serum proteins contained in animal serum, animal fetal serum, etc.
A culture solution containing 1/- or more or a slow II solution.

The method of attachment is to immerse the insoluble solid in a solution containing primary protein, leave it to stand for 7 hours or more, preferably 72 hours or more, and then remove the protein-containing solution by passing through suction V and drying.

The standing temperature may be any temperature at which the protein is not denatured, and is preferably in the range of 9°C to room temperature (/l-2j°C).

In the present invention, by attaching proteins to the B cell separation material, the adhesiveness of lymphocytes is generally reduced (2),
The selectivity of the separation of T cells and BiI cells can be improved.

Seven examples of the B cell separator used in the present invention will be explained with reference to the drawings. The pore size is large enough to prevent B cell material from flowing out. Attach a filter 4 made of polyamide or polyester, etc., and place it in the column 3.
It is filled with orchid material 5. Note that the filter 4 may also be provided on the population side.

The column of the separator has a length j1 or more, more preferably t
From ex to E, the column length/column diameter (L/D) ratio is 1
A t of 0 or more, more preferably 20 or more is suitably used.

To separate B cells in a lymphocyte suspension using a B cell separator such as the 1-type B cell separator, first, lymph nodes, human liver, or blood are separated using a method such as centrifugation or specific gravity centrifugation. The lymphocytes are suspended in a culture medium with physiological osmotic pressure, a slow liquid. The cell concentration is θ,! -2,j X/O'W
- degree is preferred. This floating spear was continuously injected into a separator in which the separation material had been brought to a humid temperature 1111 in advance with a culture solution or l11-liquid.

This is done by collecting the lymphocyte suspension flowing out from the separator outlet. The liquid for suspending lymphocytes is Cm”
, iJg" ions are more preferably not included.

The injection speed of the lymphocyte suspension into the separator is preferably set so that the retention time in the column is 7 minutes or more, more preferably 3 minutes or more.

The lymphocyte suspension discharged by the above method contains almost no B cells. In addition, the adherent B cells can be made happy during the day by mixing and stirring the two separation materials.

The temperature during the separation operation may be any temperature as long as it does not cause cell damage, but it may range from room temperature (//-uj℃) to 32℃.
A range of degrees is preferred.

The T cells obtained in this way have a high survival rate,
No changes were observed in the ability to regulate antibody production compared to before the 1-separator was created.

Hereinafter, the present invention will be explained more specifically with reference to Examples.

A glimpse of the separation material of the example: ■ Preparation of a monomer having a high molecular weight amino group In 100% of tetrahydrofuran, divinylbenzene θ, 1 mol,
7011 moles of N,N'-diethylethylenediamide and 517 moles of lithium diisopropylamide were mixed and dissolved in a nitrogen atmosphere.

By reacting at 2θ℃ for q days, further at 20℃ for 3 days, and at -73℃ for 3 days, the molecular weight of 5
Measured by perminusion chromatography.

) A monomer (abbreviated as polyamine (1)) having the following chemical structure was obtained.

■In a sample of fjA copolymer having a high molecular weight amino group, styrene, polyamine) and azobisisobutyronitrile were dissolved in benzene 10-, and then *topped and sealed, /θ~ Copolymerization was carried out by heating to 0° C. for -0 hours.

After a predetermined period of time, the reaction solution was poured into a large excess of methanol, and the precipitated copolymer (8Ax:xboria-ly containing 1
After redissolving in vt*)t benein, it was lyophilized.

(2) Water-insoluble solid aim The copolymer described in Section III was made into a tetrahydrofuran solution, and the solution was applied onto glass beads of 90 to 60 mesh, and the solvent was evaporated for coating.

Coating was performed in the same manner on the carbon-deposited collodion film, stained with osmic acid or lead acetate, and observed with a transmission electron microscope, revealing the presence of islands of polyamine in the sea of polystyrene. It was confirmed.

Creating a separator The separation material was placed on the outlet side (with a nylon net filter of two lθθ meshes attached, inside yk41-, length 10tx
Packed into a polyvinyl chloride column (Figure/Reference wax). Thereafter, a phosphoric acid** solution containing o, o, g wt- rat albumin was injected from the column inlet.

Fill the column. After standing at room temperature for 76 hours, the column is washed with Hank's solution.

A separator was created from the above operations≦2.

Separation operation: ! Hanks (Cm''-Mg''t4on) of lymphocytes collected from the mesenteric lymph nodes of age-appropriate rats.

(Not #A added) The suspension was opened (lymphocyte concentration near, 4±
0,4
).

The lymphocyte suspension was injected into a separator using a syringe-type micropump at 0.4111F), and the lymphocytes were collected.

Analysis of lymphocytes: Lymphocytes were treated with Tsubaki anti-rat IgG, which was recognized with near 0 orosein/isothiocyanate F, and superficial K IgG
The cells with this were analyzed using a fluorescent microscope.

Quantify the proportion of B cells in the total IJ cells before column injection and after column outflow, and determine the lymphocytes flowing out from the column and the I) cells adhering to the column. The change in the proportion of turtles relative to the proportion of three-legged monkeys before injection into the column was expressed in terms of α and α. That is, α・−(fB/fT) / (FB/FT)αm=((
FB - fB) / (F7 E-f D)) / (F, /
FT) Here, rBl- is the proportion of B cells in total lymphocytes before injection into the column and after flow out, and FT.

fT is that of T cells. (FT-/-FB, f
Ttsu/-fB). E is the ratio of efflux lymphocytes to total lymphocytes before column injection.

The results are shown in Table 1 and Figure 2.

(Left below) Comparative Example: Using polystyrene (PST), one separator was made in the same manner as in Example 1, and one separation experiment was conducted.
a lX10θ is 1♂-1(/-αe)X#70 is 32
It was regrettable. These results are illustrated in Figure-1@13.

Comparative Example 2 Poly(p-diethylaminoethylstyrene) (PBA8
), the amino groups were uniformly dispersed, and a separator was created from the existing separation material and a separation experiment was conducted.
-αa)×100 is 9%, (/-a@)xioo is j
It was Ochi. These results are also illustrated in FIGS. 21 and 3.

Using the separation material prepared in Example/Example/, the length is
/ 0cxa, / ! A separation experiment was conducted by creating a column of Infusion rate of lymphocyte suspension respectively.

O, ko, o, 4t, o, 4 were set as Mutsumu. The results are shown in Table 1.

Table 2 According to the present invention, as described above in detail.

B cells can be rapidly isolated from lymphocyte suspensions without immunostimulation, and are useful for basic research in immunology and cytology, as well as the diagnosis and treatment of diseases involving immune reactions such as cancer and autoimmune diseases. extremely useful.

[Brief explanation of the drawing]

FIG. 1 is a sectional view showing an example of the B cell separator of the present invention. The figure shows the selectivity of B cells adhering to the column (l-αm)
shows. Figure 3 shows the selectivity of B cells flowing out from the column (/-C1e
) is shown. 1: Liquid inlet 2: Liquid outlet 3: Column 4: Filter - 52B Cell separation material agent Patent attorney Hoshino 2 Figure 3 PSt SA9 SA1! ;5A25P
Continuation of EAS Page 1 0 Inventor: Kataki Nori 2-40-10 Kotake-cho, Nerima-ku, Tokyo. 9 Inventor Mitsuo Okano 6-12-12 Kokufudai, Ichikawa City 9 Inventor Takao Nishimura 1-16-26 Mukaioka, Bunkyo-ku, Tokyo Inventor Mizuo Maeda 6-23-8 Nogata, Nakano-ku, Tokyo Inventor Tetsuo Watanabe 1-23-14 Shimo, Kita-ku, Tokyo Inventor Atsushi Maruyama 125 Tsukimidai, Hodogaya-ku, Yokohama Procedural amendment (spontaneous) June 23, 1980 Commissioner of the Patent Office Haruki Shimada Tono 1 Case Indication 1982 Patent Application No. 32762 3 Relationship with the case of the person making the amendment Patent applicant - Applicant 4 Agent 6 Number of inventions increased by amendment

Claims (1)

[Scope of Claims] (1) An IJIJIII bulking material that adhesively removes degeneracy from a lymphocyte suspension, which is made of a water-insoluble solid having amino groups distributed unevenly on its surface. (2) Hydrophobic 2. A separation material according to claim 1-II, wherein the chemical compound and the metal compound having an amino group are present in a non-uniform distribution in a seabird shape or a lamellar shape. (3) A compound having an amino group is represented by the following formula.
” CJH, ＊＊＊ (1-/ -j )-Aki/~
J A patent claim that is a compound - Kakoroki Sen's portion @111゜ (4) A patent claim that is attached with an animal serum protein -
Separation material for fill/article positions. (S) Lymphocytes consisting of a water-insoluble solid in which a non-uniform distribution of reward groups is present in a column having an inlet and an outlet for waves and a filter at least at the outlet of the body fluid. A m1ll separator filled with a 11111 separation material that separates mm1i from a suspended liquid. (6) To separate BII from a lymphocyte suspension consisting of a water-insoluble solid with amino groups distributed non-uniformly on its surface, the lymphocyte suspension should be brought into contact with the fluidized part. A method for separating and removing 1IIIl11 from a lymphocyte suspension.