JPS5874611A - Material, unit and process for separating t-cells - Google Patents

Abstract

PURPOSE:A hydrophobic and water-insoluble porous solid with a specific average pore diameter, to which an animal serum protein is adhered, is used as a T-cell separator to separate T-cells playing a role in immunological reactions such as inhibition of antibody production from a lymphocyte suspension. CONSTITUTION:An amimal serum protein, especially a serum protein from mammarian fetus, is adhered to a hydrophbic, water-insoluble porous solid with an average pore diameter of 500-5,000Angstrom to prepare a T-cell separateor. A column 3 with inlet and outlet 1, 2 of which at least outlet 2 has a filter 4 is filled with the above T-cell separator and the resultant device is brought into contact with a lymphocyte suspension in a solution containing more than 0.1g/dl of protein to separate T-cells in high purity and high yield without immunoligical stimulation by use of less amount of protein than in the conventional process rapidly, simply in sterility.

Description

Detailed Description of the Invention The present invention provides a separation material for separating and separating T cells from a leukocyte suspension, a Kucho cell separator using the separation material, and a method for separating T cells using the separation material. Ho'7=22'57!
Ill&□, i<5i=l-4'l&1M, refers to lymphocytes that form p-cells with good sheep red blood cells2, non-T cells refers to lymphocytes, monocytes, and granulocytes that are not recognized as cells.

It is said that it is impossible to obtain T cells in the 70 to 85 range of peripheral blood lymphocytes by means of step separation. Furthermore, combining various means to obtain high purity, high yield, and high survival rate is an essential technique for immunological research, and therefore there are many problems.

Conventional methods for isolating T cells from peripheral blood all involve a specific gravity centrifugation method using a high-density suspension solution such as a sodium metrizoate-weight-equal mixture. A white blood cell suspension containing lymphocytes is obtained and then subjected to separation processing. The following three main methods are known for this subsequent separation process.

1)? Cells and neuraminidase-treated sheep red blood cells having the ability to form rosettes are added to the above pretreatment solution, left in a cold place for several hours or more, and then subjected to density centrifugation again to obtain T cells.

2) Taking advantage of the fact that T cells have a lower adhesion to foreign surfaces than non-T cells, the pretreatment solution is brought into contact with nylon fibers to obtain T cells as a fraction that is less likely to adhere to the fibers.

3) T cells in the pretreatment solution are fluorescently stained using an antiserum that recognizes fluorescently labeled T cells, and T cells are obtained as a fluorescent cell fraction using a fluorescein cell sorter.

The first method has the disadvantage of stimulating lymphocytes by allowing sheep red blood cells and lymphocytes to coexist, and is difficult to apply for research purposes to elucidate the role of T cells in the immune system. .

In the second method, the majority of T cells and non-T cells are not sufficiently separated, and only a few highly pure T cells are obtained.

The method @3 is a method that can theoretically obtain T cells with the highest purity in terms of KFi, but the problem with using antiserum is that it stimulates and damages the cells one by one. Since the operation of sorting cells is essential, it has drawbacks such as difficulty in obtaining a large number of lymphocytes of about 10 to 10 cells.

The present inventors, based on the current state of such T cell isolation methods,
The aim is to obtain T#l cells with high purity and high yield without providing immune stimulation to the cells. As a result of detailed studies on the adhesion phenomenon of cells and non-T cells to foreign surfaces, we found that a water-insoluble and hydrophobic solid with a porous surface
ttry, which has applied for a patent for the selective separation of cells
Gansho 56-ssy.

and patent application 1982-A4).

The inventors! As a result of continuing intensive studies to further simplify and simplify the cell separation procedure, we used a porous urine water-soluble and hydrophobic solid to which animal serum proteins were attached and dried, as a T cell separation material. We have discovered the effects of making the separation process easier and easier, reducing the amount of protein used during separation, and facilitating the aseptic process, and completed the present invention. = In other words, the present invention provides 5
A water-insoluble and hydrophobic porous material having an average diameter of 00 A or more and less than 500 A T cell separator, which has an inlet and an outlet and is filled with a cough liquid "□" chamber with a filter on the outlet side □ and □ in the presence of 0.1 t/dJ or more of protein in the separation material. This is a T cell separation method characterized by contacting leukocytes with permeated liquid □.

The configuration of the present invention will be explained below.

The solid to which animal serum proteins are attached in the present invention is
They must be hydrophobic, and these materials include polymers such as ethylene, vinylene, vinyl chloride, vinyl acetate, styrene, divinylbenzene, acrylonitrile, methyl methacrylate, etc. Copolymers include nylon, polycarbonate, polyethylene terephthalate, and polyester copolymers.

Although the method for preparing the hydrophobic solid described above is not particularly limited, a porous polymer can be prepared relatively freely during the polymerization process. Particularly preferred is a porous copolymer obtained by copolymerizing a monovinyl monomer such as styrene and a crosslinkable monomer such as divinylbenzene. The monovinyl monomer referred to as ′ζζ is
This applies to almost all vinyl monomers. That is,
Styrene, methylstyrene, diphenylethylene, ethylstyrene, dimethylstyrene, vinylnaphthalene, vinylphenanthrene y1 vinylmesitylene, ink e 4
#6-) Hydrocarbon compounds such as dimethylstyrene and 1-vinyl-2-ethylacetylene: chlorstyrene, methoxystyrene, bromustyrene, cyanostyrene, fluorostyrene, dichlorostyrene, N,N-dimethylaminostyrene, nitro Styrene/derivatives such as styrene, chloromethylstyrene, trifluorostyrene, trifluoromethylstyrene, aminostyrene, etc.: Vinyl sulfides such as methyl vinyl sulfite, phenyl vinyl sulfide, etc. Trout conductors: acrylonitrile, methacrylaterile, α-7cetoxyacrylonitrile Acrylic acid esters such as 7-acrylonitrile derivatives niacrylic acid, methyl niacrylate, lauryl acrylate, p-methyl acrylate, ethyl a7toxyacrylate: cyclohexyl methacrylate, dimethylaminoethyl methacrylate, methacryl Methacrylic acid esters such as glycidyl acid, tetrahydrofurfuryl methacrylate, and hydroxyethyl methacrylate Diethyl dimaleate, diethyl fumarate: Vinyl ketones such as methyl vinyl ketone and ethyl isopropenyl ketone, vinylidene chloride, vinylidene bromide, vinylidene cyanide, etc. Acrylamide derivatives such as vinylidene compounds niacrylamide, methacrylamide, N-butoxymethylacrylamide, N-phenylacrylamide, diacetone acrylamide, N,N-dimethylaminoethyl acrylamide: vinyl acetate,
Fatty acid vinyl conductors such as vinyl butyrate and vinyl caprate: phenyl thiomethacrylate, methyl thioacrylate,
Thio fatty acid conductors such as vinyl thioacetate: N-vinyl succinimide, N-vinyl pyrrolidone, N-vinyl 7-thallimide, N-vinyl carbazole, vinyl furan, 2-vinyl veil, methyl vinyl imidazole, vinyl pyrazole, There are heteroarticulated vinyl compounds such as vinyl oxazolidone, vinylthiazole, vinylnato dizole, vinylpyridine, methylvinylpyridine, 2,4-dimethyl-6-vinylpyridine, and vinylquinoline.

In addition, as the cross-linked polymerizable monomer, divinylbenzene,
Divinyltoluene, divinylxylene, divinylnaphthalene, divinylethylbenzene, divinylphenanthrene, trivinylbenzene, divinyldiphenyl, divinyldiphenylmethane, divinyldibenzyl, divinylphenyl ether, divinyldiphenyl sulfide, divinyldiphenyl7mine, divinylsulfone, divinylketone, divinylfuran , divinylpyridine, divinylquinoline, di(vinylpyridinoethyl)ethylenediamine, diallyl phthalate, diallyl maleate, diallyl fumarate, diallyl succinate, diallyl carbonate, diallyl sherate, diallyl adipate, diallyl sebacate,
Lauryl tartrate, diallylamine, triallylamine, triallyl phosphate, triallyl tricarbaryl, triallyl aconitate, triallyl citrate, N,N
'-ethylene diacrylamide, N,N'-methylene diacrylamide, N,N'-methylene dimethacrylate, ethylene glycol dimethacrylate, triethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, trimethylolpropane trimethacrylate methacrylate, pentaerythritol tetramethacrylate, 1,3-butyler glycol diacrylate,
1,6-hexanediol diacrylate, trimethylpropane triacrylate, pentaerythritol tetraacrylate, triallylinocyanurate, 1. !
1.5-) Liacroylhexahydro-1,' , ,
5'', s,=triazine-diarylmelamine and the like.

When the hydrophobic solid according to the present invention has a smooth surface, non-T cells cannot sufficiently attach to it, and SOO! That's 5ooo! The average pore diameter used is preferably 600x or more and less than asooX, particularly preferably 100◎ or more and less than zsoojL.

The average pore diameter is determined by the mercury intrusion method. Stay here - Measured by tar.

In this method, mercury is injected into a porous material, and the amount of mercury pores (the amount of mercury infiltrated per unit weight) is determined from the amount of mercury infiltrated. The pore size distribution is measured based on the principle that the pressure required to perform the process is inversely proportional to the pressure required to perform the process. For more information on this method, see
Book, Fine Measurement (Fine Part)
icleMeasurement)
Junior and G.M. Gua2Abaal (C1yd
e・0ff-Jr・and J8M, Dallaval
le) Co-author, Thee Macmilla 7-ka/Panii, New
York (Te he Macmillan Company
, New York) in 1959. With this method, it is possible to measure the pore size distribution at 35 to noXt.

The average pore diameter in the present invention means the pore diameter (r) expressed on a logarithmic scale on the horizontal axis, and dV/d1wr (dV/d1wr) on the vertical axis.
In the pore size distribution map represented by V (cumulative mercury pore volume measured with a porosimeter), the closed section surrounded by the pore size distribution ta obtained by the method in the above literature and the horizontal axis is parallel to the vertical axis with equal areas on the left and right. Defined as the pore diameter value on the horizontal axis when divided.

In the present invention, pores are defined as pores with a diameter of 40X or more and communicating from the surface, and the amount of mercury pores is also a value derived from the pores.

The water chain pore volume of the hydrophobic solid is a value measured by a mercury intrusion porosimeter like the above-mentioned average pore diameter, but it is preferably 0.1 wl/f or more and less than 4-72, 0. ! ! (d/f or more and less than 3-/) is preferable.

The hydrophobic solid in the present invention needs to be granular, and is preferably spherical in order to improve separation efficiency. 70 Particle size range is 40 microns or more and less than 800 microns in water, preferably L< is 4o microns or more and 20 microns or more.
Less than 0 microns, more preferably 40 microns or more7
The T cell separation material of the present invention, which is less than 5 microns, is one in which a protein is attached to the hydrophobic solid having a porous surface as described above.

The animal serum protein solution used for adhesion removal is 1.7f/dj
A solution containing the above proteins, preferably an autologous serum of 1.7 F/dj or more, a culture solution or buffer containing fetal mammalian serum, more preferably a fetal serum of a mammal of 2.5 f/d1 or more. It is a culture solution or a buffer solution. Examples of mammals that can be used in the present invention include cows, horses, sheep, goats, horsetails, mice, rats, and guinea pigs.

The adhesion method involves suspending a hydrophobic solid having a porous surface in the above-mentioned protein-containing solution, allowing it to stand for at least 1 hour, preferably at least 0 hours, and then filtering the protein-containing solution by suction filtration. Except for 1. Perform freeze-vacuum drying.

The standing temperature may be any temperature at which the protein does not deteriorate, and is preferably in the range of 4°C to room temperature (18 to 25°C).

The leukocyte suspension referred to in the present invention refers to a suspension containing white blood cells such as lymphocytes, granulocytes, and monocytes, with red blood cell contamination within 10 times the number of white blood cells. It may be prepared by any method such as , dextrin precipitation, etc., but the specific gravity centrifugation method is more practical since it is less likely to be contaminated with granulocytes and red blood cells.

The invention! In cell separation materials, since proteins are attached to hydrophobic substances, it is necessary to treat the separation material with protein each time prior to separation in order to prevent non-specific attachment of T cells. It is convenient because it does not need to be removed and the separation operation is simple.

In addition, when the T cell separation material and leukocytes come into contact, 0.1f
/dj or more, preferably 0.1f/d
A culture solution or buffer containing one or more autologous serum, animal serum, or fetal fetal serum, more preferably ◎, 597
A culture solution or buffer solution containing fetal serum of a mammal with a serum concentration of 41 or higher may be used, and the amount of protein used during separation can be very small. Above the solution containing the protein @Fistld
Although it is possible up to position i, the effect of the present invention can be achieved up to position 297d1. A high-density protein region is formed around the separation material, and even in a solution containing a small amount of protein, there is no adverse effect on the adhesion of non-T cells, and when T cells are attached non-externally, seven It is expressed because it can be prevented.

Since the T cell separation material of the present invention is in a dry state, it can be easily sterilized when packed in a column.
Aseptic operation becomes easier.

In addition, sterile storage can be stored for a long period of time, and a large number of columns can be prepared in a sterile state at all times. Furthermore, the column filled with separation material can be easily carried in a sterile state.

In Misaki 91, mammalian fetal serum protein is used as the protein attached to the hydrophobic porous envelope and the protein in the liquid that brings the i-cell separation material into contact with leukocytes. The fetal serum protein has the effect of preventing non-specific adhesion of T cells and (4) specifically adhering non-T cells such as B cells and monocytes.

This effect is thought to be caused by some component of the fetal serum protein adhering to the separation material.The fetal serum component contributing to this effect has not been fully elucidated, and may be due to a single protein or - or more. 4 is currently unknown.

However, since there are not many individual differences in fetal serum, there is no doubt that it is a basic component of fetal serum. Based on the above, the present invention can be used in a very simple, rapid and high-yield manner using a leukocyte suspension. It is effective in isolating T cells with high efficiency and purity.

An example of the T cell separator used in the present invention will be described based on the drawings. An inlet 1 injects a leukocyte suspension and a washing solution, and an outlet 2 through which the leukocyte suspension and washing solution flow out.
A filter 4 having a pore size large enough to prevent the KT#fil cell separation material from flowing out is attached to the column 3 having an outlet, and the column 3 is filled with the above-mentioned T cell separation material 5. It is.

Note that the filter 4 may also be provided on the inlet side.

To separate T cells from white blood cells using a T cell separator such as the one described above, firstly, prepare white blood cells from blood by centrifugation or specific gravity centrifugation. Float in a solution containing protein with If/dj or higher. Add this liquid to 0.1
The separation material is injected into a separator moistened with a liquid containing protein of f/dJ or more, and when the floating liquid has completely infiltrated into the gun separation material of the present invention, the outflow is stopped and allowed to stand for a certain period of time. , Then, wash out non-adherent cells by flowing a washing solution.

#i in the cleaning solution washed out by the above method! cells will be included.

The standing time of the leukocyte suspension in the separator is
In order to ensure sufficient adhesion to the IIA cell separation material, it is preferable to leave the mixture undisturbed for about 30 to 60 minutes.

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 (18 to 25°C) to 7°C.
A range of ＠ degrees is preferable.

Regarding the 7'LT cells thus obtained, no changes were observed in any of #i, including survival rate, blastogenesis ability, and ability to regulate antibody production, compared to those before the isolation procedure.

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

' Example 1 Preparation of separation material: ■ Preparation of a porous unit □ Divinylbenzene (purity 51-1
Contains 44% vinyl ethylbenzene as an impurity, hereinafter referred to as 56-divinylbenzene) 100f, dimethyl phthalate 150t, azobisisobutyric a 2) Ryl 1f
Add to make a homogeneous solution. Furthermore, partially saponified polyvinyl alcohol 1.5F and sodium chloride 609 were dissolved, 150 tlf of nine-distilled water was added, and the mixture was heated at 60°C for 1 hour and at 70°C for 2 hours while stirring at 250 rpm.
The mixture was heated at 90°C for 2 hours and then at 90°C for 4 hours.

The produced copolymer had a good spherical shape, had a diameter in the range of sO micrometers or more and less than zoooz micrometers, had an average pore diameter of 1,400 JL, and a mercury pore volume of 1. It's hot with lsd/f.

■ Adhesion of proteins Classify the porous solid with a stainless steel plate, separate those with a size of 40 microns or more and less than 75 microns, soak them thoroughly in phosphate i buffered saline (hereinafter abbreviated as PILB), and wash with PH1. After that, you will be attracted to it.

Next, this porous solid is thoroughly immersed in 10K of low beef tallow serum (protein content: S, 4 f/dl) and left undisturbed at room temperature (25°C) for 12 hours. Thereafter, the tallow serum is removed by suction V and freeze-drying is performed.

This material is used as a T cell separation material.

Creation of separator: Wrinkle separation material 2- is attached to the bottom with a nylon net filter of 50 microns and has an inner diameter of 10 mm with a liquid inlet and outlet.
■Fill into the proverbial acrylic column (see drawing). Then, add PBS containing 200ml of fetal bovine serum to the inlet.
(protein content 0.45 f/dj) to wet the separation material.

A separator can be created using the above operations.

Separation procedure: Ibium metrizoate from heparinized human peripheral blood
Separation was performed by specific gravity centrifugation using a Ficoll mixture (specific gravity 1,077 at 20°C), washed with PBS, and a nine leukocyte fraction (containing 64.9 T cells, viability 98%) was obtained 4X.
Nine solutions O and S- are prepared by suspending them in PBS containing 10 ml of fetal bovine serum at a concentration of 10./-.

This liquid was quietly poured into the separator inlet, and when it had permeated the floating separation material, the liquid outlet was closed to stop the liquid from flowing out. Then, after standing at 57°C for 1 hour, the outlet was opened, and PBS4- was poured from the empty inlet, and it was adsorbed to the separation material at the flow rate of gravity and "τ2":::::
ding°“°“°′.

The total cell recovery rate was determined by microscopic observation using an automatic hemocytometer or a hemocytometer. In addition, i-cells were determined by rosette reaction with neuraminidase-treated sheep red blood cells.

Exclusion of dye by °ne blue Determined by test method.

The results are shown in Table 1.

PBII (protein content 2.5 f/dj) containing 75-fetal bovine serum was added to the porous solid with a particle size of 40 microns or more and less than 75 microns used in Example 2.
The cells were adhered and dried in the same manner as in Example 1, and then filled into the same column and moistened to form a cell separator.

The same leukocyte suspension as in Example 1 was used and the same separation procedure was performed, and the results are shown in Table IK.

Example 3 Into the reaction vessel used in Example 1, 100 f of 56-dipynylbenzene, ) luene SOOf, and 1 f of azobisisobutyronitrile were added to form a homogeneous solution.

Thereafter, the same operations as in Example 1 were performed to obtain a spherical copolymer. The particle size range of this product is 30 microns or more and 100 microns or more.
The average pore diameter was zsoojL, 2.9 d/f (Mercury Pore Co., Ltd.).

This 4 was subjected to wet classification in the range of 140 microns to less than 75 microns, and proteins were attached and dried in the same manner as in Example 1, and the same column was filled and moistened to form a T cell separator.

The same leukocyte suspension as in Example 1 was used and the same separation procedure was performed, and the results are shown in Table 1.

Example 4 Into the reaction vessel used in Example 1, 100 f of 56-divinylbenzene, 150 t of dioctyl adipate, and 1 f of azobisisobutyronitrile were added to form a homogeneous solution. below,
The same operation as in Example 1 was performed to obtain a spherical copolymer.

The particle size range of this material is 30 microns or more and less than 500 microns, and the average pore size is 110. Mercury porosity is 1.7
It was d/f.

This product is wet-classified in the range of 40 microns or more and less than 75 microns, coated with proteins in the same manner as in Example 1, dried, and filled into the same column, and moistened to form a T cell separator.

The same leukocyte suspension as in Example 1 was used and the same separation procedure was performed, and the results are shown in Table 1.

Example 5 The solid having good porosity prepared in Example 1 was wet classified in the range of 75 microns or more and less than 200 microns.
Proteins are attached and dried in the same manner as above, packed into a similar column, and moistened to form a T@ cell separator in Jiutun.
.

Table 1 shows the results after performing the same separation procedure using the same leukocyte suspension as in Example 1.

Comparative Example 1 A solid-class classified material having the same porosity as that used in Example 1 was thoroughly immersed in PH1, and after i-cleaning, the protein was not attached or dried. fill the tank,
It was used as a separator.

Preliminary isolation material was prepared using PI34 containing 10-ml fetal bovine serum.
After equilibration with , using the same leukocyte suspension as in Example 1,
Table 1 shows the results after performing the same separation operation.

Nylon fiber (purchased from Wako Pure Chemical Industries, Ltd.)
was loosened well with tweezers, and 00St was put into a 5-capacity 7-last syringe and filled uniformly to the 2.5 mg mark. The syringe was filled with PI3 containing 5-ml fetal bovine serum, left for 1 hour, and then the same lymphocyte suspension used in Example 1 was injected into the upper part of the syringe and heated at 37°C for 1 hour. After standing, non-adsorbed cells were flowed out using PI310- at a flow rate of 2-7 minutes. Obtain cell fractionation.

The results are shown in Table 1.

Ta-survival rate-recovery rate - Example 1? 3,1 98 8
? , 612 918 97
85.4# 3 92,4 96
76.9# 4 90,7 9
8 85.1# 5 91,0
98 91. ? Comparison @ 1 92,
0 97 4 s, b# 2 8
7,8 96 31,0' As explained above, according to the present invention, 'rg
It does not irritate cells immunologically and can be isolated with high purity and high yield.
Furthermore, during separation, the use of a very small amount of protein allows rapid,
It has the effect of allowing simple and sterile separation.

[Brief explanation of drawings]

The drawing is a sectional view showing an example of the Ti1l cell separator of the present invention. １・・・・・・・・・Liquid inlet ２・・・・・・
...Liquid outlet 3... Force 2m
4...... Filter 5...
・・T cell isolation material

Claims (1)

[Scope of Claims] 1. Animal blood - 500 or more 5oos to which proteins are attached
From a leukocyte suspension as a hydrophobic and water-insoluble porous solid with an average pore size of less than oJL! Separate cells! Cell separation material. It has an inlet and outlet for the emulsion, and a filter at least on the outlet side of the emulsion. - A leukocyte suspension containing hydrophobic, water-distortion soluble, porous cells having an average pore diameter of 7 to 500X to which animal serum proteins are attached is prepared in a nine-column column. Filled with T cell separation material that separates cells! Cell separator. 5. Animal serum protein! 500 attached! More than 56o
The separation material for separating T cells from a leukocyte suspension, which is a hydrophobic and water-insoluble porous solid having an average pore diameter of 0.0X, is a separation material for leukocytes suspended in a solution containing protein of 0.1 f/dj or more. From leukocyte suspension, which is characterized by contact with liquid! Those who separate cells, 4. 4. The method for separating T cells from a leukocyte suspension according to claim 3, wherein the animal serum protein is a mammalian fetal serum protein.