JPH037165A - Separating material, device, and method for sub-group of lymphatic spheres - Google Patents

Abstract

PURPOSE:To segregate and refine lymphatic spheres simply, quickly, at high rate of collection, and with high purity by fixing an alkyl chain, which adsorbs T lymphatic sphere compartments in each lymphatic sphere in priority, to the surface of a non-water soluble solid substance through a spacer including hydrophilic molecular structure. CONSTITUTION:A separating material for sub-group of lymphatic spheres is formed by fixing an alkyl chain to the surface of a non-water soluble solid substance through a spacer including hydrophilic molecular structure, wherein the alkyl chain adsorbs T lymphatic sphere compartments in each lymphatic sphere in priority to other lymphatic sphere compartments. To this separating material, either human or non-human animal lymphatic sphere compartments are put in contact so as to allow the material to catch T lymphatic spheres efficiently, and thus un-caught compartment liquid with rich B lymphatic spheres is segregated and collected. Concretely the lymphatic sphere floating liquid such as lymphatic sphere liquid put afloat in an equilibrium salt solution to the lymphatic sphere liquid put afloat again in animal blood plasma is injected in a column 1 and contacted with lymphatic sphere sub- group separating material 2. A rinsing solution is injected into the column 1, and a three-way active plug 4 is opened to drop the washing solution from a flowout hole 5, and thus the uncaught compartment liquid containing many B lymphatic spheres can be collected in a test tube 8.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to a lymphocyte subgroup separation material, a separator, and a separation method for separating and purifying a specific subgroup of lymphocytes. . Specifically, the present invention provides a lymphocyte subpopulation separation material, a separator, and a method for separating and purifying highly pure lymphocyte subgroups from leukocyte fractions of human or non-human animals in a simple, rapid, and inexpensive manner. This shows that.

Lymphocytes are cells that are differentiated from blood cell lineage stem cells and are responsible for immune functions, and are divided into several subgroups in terms of function, cell membrane characteristics, etc. These are lymphocyte subclasses such as 8128 cells that produce antibodies and play a role in humoral immunity, T lymphocytes that produce immunoregulatory lymphokines and inflammatory lymphokines and play a role in cell-mediated immunity, and null cells. , 8128 spheres, there are functionally distinct subsets. In recent years, technology to purely separate, purify, and concentrate subclasses such as T and 8128 bulbs with different immunological properties while minimizing functional damage has been strongly desired in a wide range of social fields. . For example, in basic research such as cytology and immunology, it is necessary to separate at least T and B lymphocytes Rf in in vitro analysis of the in vivo immune system, and it is necessary for diagnosis, treatment, etc. in clinical medicine. In this case, it is necessary to transfer or remove a specific fraction with decreased or enhanced function, and in obtaining physiologically active substances of the lymphatic system, it is important to separate and concentrate the producing cell fraction.

(Prior art) As a method for separating T lymphocytes and 8128 cells, first, lymphocyte fractions are obtained from peripheral blood or biological tissue by specific gravity centrifugation using a high-density isotonic solution such as a sodium metrizoate ficoll mixture. The conventional method is to perform a separation process to separate the cells into T lymphocytes and 8128 cells.

The subsequent separation treatment methods can be broadly divided into three types: (1) those using specific antigens, receptors, and immunoglobulins on the cell membrane surface as indicators, (2) physical cell separation, and (3) adsorption chromatography.

Methods for (1) include the rosette formation method and the Affinity Taloma! method (combining antibodies and immune complexes).・Cytolysis using imaging, antibodies, complement, removal of mitogen-activated lymphocytes, fluorescein activated cell sorter (F, A, C).

), but with these methods, it is difficult to collect T lymphocytes or 8128 cells in an intact state because they receive strong binding and stimulation with high biological specificity. Moreover, there are many methods that are not suitable for mass processing.

In addition, as methods for (2), density gradient centrifugation, cell electrophoresis, etc. are known, but T lymphocytes and 8128
Since there is no significant difference in physical properties such as specific gravity and surface charge between the cells and the spheres, high precision cannot be required in the yield or purity of the separated and collected cells. Furthermore, as for method (3), there is a known method that utilizes the difference in the non-specific adhesion ability of T lymphocytes and 8128 cells to foreign substances such as nylon, tetron, and cotton fibers, but it is possible to separate cells with high precision. In order to obtain this, treatment with an adsorption carrier such as fetal bovine serum (Fe2) is required, it is difficult to set implementation conditions such as flow rate, and impurities such as macrophages are often mixed in. In addition, these conventional methods generally require complicated operations or preparations, and require experience and technical proficiency when performing the separation operation, which is a major problem.

(Problems to be Solved by the Invention) Therefore, an object of the present invention is to provide a novel lymphocyte subpopulation separator, separator, and separation method. The present invention also provides a separation material, a separator, and a separation method for lymphocyte subpopulations that easily and rapidly separate and purify lymphocytes with high yield and high purity without affecting various cell activities. It is. A further object of the present invention is to provide a leukocyte separation material, a separator, and a separation method suitable for large-scale processing.

(Means for solving the problem) - Two objectives are to create a hydrophilic molecular structure that allows alkyl chains to preferentially adsorb the T lymphocyte fraction in lymphocytes over other lymphocyte fractions. Achieved by a lymphocyte subpopulation separation material that is immobilized on the surface of a water-insoluble solid substance via a spacer containing ◇
.

The present invention also provides a material for separating lymphocyte subpopulations in which the number of methylene repeats in the alkyl chain is 25 or less. The present invention further provides a material for separating lymphocyte subpopulations, wherein the alkyl chain is a linear alkyl chain. The present invention further provides a material for separating lymphocyte subgroups, in which the spacer has a hydrophilic part and a hydrophobic part. The present invention also provides a material for separating lymphocyte subpopulations, which uses particulate matter with a particle size of 0.05 to 5 mm as the insoluble solid substance.

The purpose is also to transfer alkyl chains that preferentially adsorb the T lymphocyte fraction in lymphocytes over other lymphocyte fractions to a water-insoluble solid material through a spacer containing a hydrophilic molecular structure. This is achieved by a lymphocyte subpopulation separator characterized by having a column filled with a lymphocyte subpopulation separation material immobilized on the surface.

The present invention also provides a filter between the inlet and outlet of the column and the separation material layer that allows cell components in the lymphocyte fraction to pass through but does not allow the separation material to pass through. This shows a separator.

Further, the purpose of the invention is to transfer the alkyl chain that preferentially adsorbs the T lymphocyte fraction in lymphocytes over other lymphocyte fractions to a water-insoluble solid material via a spacer containing a hydrophilic molecular structure. A lymphocyte fraction from humans or non-human animals is brought into contact with a material for separating lymphocyte subpopulations, which is characterized by being immobilized on the surface, and T lymphocytes are efficiently captured in the -f, x mud separation material. This shows a method for isolating lymphocyte subpopulations, which is characterized by separating and collecting a non-captured fraction rich in B lymphocytes by separating and recovering a non-captured fraction rich in B lymphocytes.

(Function) The present invention enables the alkyl chain that preferentially adsorbs the T lymphocyte fraction in lymphocytes over other lymphocyte fractions to the surface of a water-insoluble solid substance through a spacer having a hydrophilic molecular structure. In order to use a material for isolating lymphocyte subpopulations characterized by immobilization in
, T, and B lymphocytes can be separated, purified, or removed.

That is, when a lymphocyte suspension is brought into contact with the material for separating lymphocyte subpopulations of the present invention, T lymphocytes, which have a relatively high contribution of hydrophobic interaction in adhesion to foreign substances among lymphocytes, are attracted to the terminal end of the adsorbent. On the other hand, B lymphocytes, which have a lower contribution of hydrophobic interaction to foreign substances, are interposed between the water-insoluble carrier and the alkyl chain at the end. It is thought that because the spacer prevents cells from approaching and interacting with the separation material surface through its molecular movement, adhesion to the adsorbent is suppressed and selective adhesion of T lymphocytes is achieved.

Furthermore, since the separation material of the present invention combines organically synthesized substances, it is highly stable and can withstand multiple autoclave treatments, and is also excellent in terms of economy and mass productivity. Become something. Furthermore, in addition to the separation procedure using the separation material of the present invention, there is no need to particularly add fetal bovine serum or the like to the lymphocyte suspension (although it is possible to add it in some cases). , simply by contacting the lymphocyte suspension with the sterilized separation material, B.
Uncaptured fractions rich in lymphocytes can be separated and collected, and the survival rate of collected cells is always maintained at 97% or higher even without serum, suggesting that stimulation of lymphocytes is extremely low. It is.

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

The material for separating lymphocyte subpopulations of the present invention has an alkyl chain that preferentially adsorbs the T lymphocyte fraction in lymphocytes over other lymphocyte fractions, through a spacer having a hydrophilic molecular structure. It is characterized by being immobilized on the surface of a water-insoluble solid substance.

The hydrophobic alkyl chain used in the present invention is
Alkyl chains of various chain lengths can be used, and may be branched in some cases, but preferably linear ones with 25 or fewer methylene repeats, especially those with 2 to 2 methylene repeats. A value of about 14 is desirable because it can provide higher properties. Further, as raw materials thereof, various compounds such as alkylamines, fatty acids, alcohols, and simple lipids and complex lipids containing these can be used. However, the hydrophilic groups such as OH groups, NH2 groups, and COOH groups of these raw material compounds are consumed during bonding with the spacer described later, and it is necessary that the alkyl chain be exposed at the terminal side.

Furthermore, the spacer having a hydrophilic molecular structure used in the separation material of the present invention is in the form of a molecule, and can be interposed between the surface of the water-insoluble carrier and the terminal alkyl chain to have an arbitrary length. It may have any noble structure as long as it can suppress the strong intermolecular interaction between the hydrophobic alkyl chains present at the ends. For example, any one of a dissociative functional group such as a carboxyl group, a sulfone group, and various amines, a hydroxyl group, or an ether bond may be present in the spacer molecule alone or in combination. . When such a spacer is interposed between the water-insoluble carrier and the alkyl chain, due to the molecular mobility of the spacer,
It is thought that the cells are prevented from approaching and interacting with the surface of the separation material, and as a result, the contact of B lymphocytes, which have a weak interaction with the separation material, is preferentially suppressed.

Such a spacer is produced by reacting known reactive functional groups such as epoxy groups and isothiocyanate groups at both ends of the molecular chain with the surface of the water-insoluble carrier and the compound forming the terminal alkyl chain as described above. One end of the molecular chain is covalently bonded to the water-insoluble carrier surface, and the other end is covalently bonded to the alkyl chain. Furthermore, the same effect can be maintained even when diamine-based or dicarboxylic acid-based spacer molecules are immobilized with a coupling agent such as bisepoxide, diisocyanate, or dialdehyde.

Among these, especially linear alkyl diols with 2 to 8 methylene repeats and 2 to 8 repeats of ethylene oxide.
25, or the amine content is 85
% or less, linear amine-containing compounds such as diethylenetriamine and triethylenetetramine are easy to handle and are preferably used. It is also possible to use these in combination, and rather better characteristics can often be obtained.

Note that when a more hydrophobic water-insoluble solid substance is used as a carrier, molecular mobility is restricted by hydrophobic bonds with alkyl chains, and the effect of inhibiting B lymphocyte adhesion may be lost. It is necessary to select a suitable spacer.

Examples of water-insoluble solid substances used as carriers in the present invention include agarose-based, dextran-based, cellulose-based, polyacrylamide-based, polyvinyl alcohol-based, polyvinylpyrrolidone-based, polyacrylonitrile-based,
Styrene-divinylbenzene copolymer, polystyrene, acrylic ester, methacrylic ester, polyethylene, polypropylene, poly(4-fluoroethylene), ethylene-vinyl acetate copolymer, polyamide, polycarbonate, polyfluoride Examples include h'' organic polymers such as vinylidene chloride, polyvinyl formal, polyarylate, and polyethersulfone, and inorganic materials such as glass, alumina, titanium, activated carbon, and ceramics, but especially for cell adhesion. It is preferable to use a carrier that is not too strong, that is, a carrier that has a relatively gentle interaction between cells and carriers.Also, natural organic polymers derived from living organisms such as collagen and chitosan can also be used. The form of the insoluble solid substance is not particularly limited,
Although a flat plate can be used, it is preferably in the form of particles, particularly particles with an average particle size of 0.05 to 5 mm. In addition, the average particle size was calculated as the weight average of the particle size of each grade, which was determined by classifying using a sieve specified in J15-Z-8801, and the intermediate value between the upper limit particle size and the lower limit particle size of each grade. It is something. Furthermore, the particle shape is preferably spherical because it is less likely to cause physical damage to cells and it is easier to maintain uniform particles.

The lymphocyte subpopulation separator of the present invention uses a spacer containing an alkyl chain with a hydrophilic molecular structure that preferentially adsorbs the T lymphocyte fraction in lymphocytes over other lymphocyte fractions. It is characterized by having a column filled with a separation material characterized by being immobilized on the surface of a water-insoluble solid substance via a column.

In order to bring the lymphocyte subpopulation separation material of the present invention into contact with a lymphocyte suspension, it is possible, for example, to contact the lymphocyte suspension with a flat lymphocyte subpopulation separation material; A hydrophobic substance that exists on the surface of separation materials such as those mentioned above and acts on cell adsorption! From the viewpoint of the absolute amount of cell adsorption and ensuring a sufficient cell adsorption space for 5i, it is desirable to use a separator having a column filled with particulate separation material for contact.

In this lymphocyte subpopulation separator that uses a column filled with particulate separation material, the material constituting the column container is synthetic resin such as polyethylene, polypropylene, polycarbonate, polystyrene, polymethyl methacrylate, etc. Glass and metals such as stainless steel can be used, but Oh! - Particularly preferred are polypropylene, polycarbonate, etc., which can be crepe sterilized and are easy to handle.

Furthermore, between the entrance and exit of the column of this lymphocyte subpopulation separator and the separation material layer filled with lymphocyte subpopulation separation material, cell components in the lymphocyte suspension pass through, but the separation material cannot pass through. It is preferable to have a filter with a mesh, and the material constituting this filter may be any physiologically inert and high-strength material, with polyester and polyamide being particularly preferred.

FIG. 1 is a schematic diagram of an embodiment of the lymphocyte subpopulation separator of the present invention in use. In the embodiment shown in FIG. 1, the column 1 has a tubular shape that is open at both ends and gradually reduces in diameter from a portion slightly above the lower end opening to the lower end opening that becomes the outlet 5. Furthermore, this tapered part 7 is equipped with a three-way stopcock 4, and an outlet 5 is provided.
The flow path leading to can be opened and closed. This column 1
A lymphocyte subpopulation separator 2 of the present invention as described in -1- is filled in the actual diameter portion 6 above the tapered portion 7 of the column 1. It is held within the column 1 by filters 3 provided on three sides of column 6 and filter 3' provided at the lower end, forming a separation material layer.

In addition, the leukocyte separation method of the present invention uses an alkyl chain that preferentially adsorbs the T lymphocyte fraction in lymphocytes over other lymphocyte fractions through a spacer having a hydrophilic molecular structure. By bringing a lymphocyte fraction from humans or non-human animals into contact with a separation material characterized by being immobilized on the surface of a water-insoluble solid substance, and allowing the separation material to efficiently capture T lymphocytes, B. This method is characterized by separating and recovering a non-captured fraction rich in lymphocytes.

To explain the method for separating lymphocyte subpopulations of the present invention in more detail using the separator shown in FIG. A lymphocyte suspension such as resuspended lymphocyte fluid is injected into a column 1 filled with lymphocyte subgroup separation material 2, and the lymphocyte suspension is brought into contact with lymphocyte subgroup separation material 2. .

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 separation material 2 in the column 1 for a predetermined period of time in this way, a rinsing solution is injected into the column 1 using various pumps or pipettes, and the three-way stopcock 4 is opened. The cleaning liquid is gently dropped from the outlet 5 and collected in a test tube 8 or the like.

Thereby, the non-captured fraction containing many B lymphocytes that were not adsorbed to the separation material 2 can be collected.

The rinsing liquid used during this cleaning operation is as follows:
Since the purpose is to wash away cell components that have not been adsorbed to the separation material 2 without eluting the cell components that have been adsorbed, a material with low activity is desired, such as a balanced salt solution, cell culture solution, etc. is used, preferably a divalent cation-free balanced salt solution, such as Hanks' balanced salt solution (Il
ank's batanccd 5alt 5olut
ion: IIBBS) etc. are used.

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

Example 1 Chitosan beads having oxirane groups [Chitopearl EP-810 (IEI)-C) as a water-insoluble solid substance
, φ300 μm, oxirane group affi0. 12mm
ol/g (Wel) manufactured by Fujibo Co., Ltd.], an alkyl chain with 2 methylene repeats was immobilized as a hydrophobic end via triethylenetetramine (TET) and butanediol (BDO) as spacers. did.

First, 0.2M carbonate buffer (pH 10) was used.
A 2M TET solution was prepared, and EP-C washed with distilled water was mixed with 0. 3-0. It was added at a rate of 5g (wcl)/ml. After heating this in a water bath at 80°C for 3 hours,
Blood mixer (made by Kayagaki Medical Science T-gyo, BM-101)
The mixture was stirred at room temperature for about 20 hours. The reaction was then washed with distilled water and added to a 5 w/w % butanediol diglycidyl ether solution prepared using 0.2 M carbonate buffer (pH 10) in a water bath at 8,0 °C. After heating for 3 hours, use a blood mixer to heat at room temperature for about 20 minutes.
Stir for hours. After this, 0.2M carbonate buffer (pH 1
The washed reactants were added to a 0,2M ethylamine solution prepared using 0) and heated in a water bath at 80 °C for 3 h, followed by approximately 2 min at room temperature using a blood mixer.
Stirred for 0 hours. Next, in order to remove unreacted oxirane groups, the immobilized beads washed with distilled water were added to a 1M (pH 8) ethanolamine solution and stirred for about 20 hours. Finally, acetate buffer (0,1M pH4)
, phosphate buffer (0.1M pH 7.4)
．． Autoclave treatment was performed at 5121°C for 130 minutes.

The separation material thus obtained was mixed with Hanks' balanced salt solution (
HBSSS pH 7.4) and packed in a polypropylene column in a wet state to a volume of 1.5 ml.

Next, lymphocytes collected from the 1 tsubo viscera of a mouse (ICR, 8M age or older) were suspended and concentrated in RPMI 1640 (manufactured by Hinaga Pharmaceutical Co., Ltd.), and the entire suspension containing 2 x 107 or more cells was separated. Soaked in wood. 1m inside this column
Lymphocytes were collected by immediate washing with one or more RPMI 1640.

The number of cells, the percentage of monocytes and lymphocytes, and the percentage of TSB lymphocytes in the collected fractions were determined using an automatic hemocytometer (Ortho Instruments).
Evaluation was performed using an automatic cell analyzer (Cyto ACE-100, manufactured by JASCO Corporation), and a fluorescently labeled anti-mouse Ig antibody (manufactured by Becton Dickinson). . Based on these measurement results, the cell adhesion rate to the separation material was calculated. The results are shown in Table 1.

Example 2 According to the method of Example 1 except that hexylamine was used instead of ethylamine, beads were prepared in which a straight alkyl chain with 6 methylene repeats was immobilized as a hydrophobic end. Similar lymphocyte separation experiments were performed.

The results obtained are shown in Table 1.

Example 3 Example 1 except that ethylamine was replaced with decylamine and the solvent was replaced with 100% dimethylformamide.
Beads were prepared in which a linear alkyl chain having 10 methylene repeats was immobilized as a hydrophobic end, and a lymphocyte separation experiment similar to that in Example 1 was conducted. The results obtained are shown in Table 1.

Example 4 Beads in which a linear alkyl chain having 14 methylene repeats was immobilized as a hydrophobic end were prepared according to the method of Example 3 except that tetradecylamine was used instead of decylamine. A lymphocyte separation experiment similar to that described above was performed. The results obtained are shown in Table 1.

Comparative Example 1 Untreated chitosan beads [Chito Pearl 3010 (C1
1), φ300 μm, manufactured by Fujibo Co., Ltd.] itself,
A lymphocyte separation experiment similar to that in Example 1 was conducted. The results obtained are shown in Table 1.

Comparative Example 2 Ki! having an oxirane group!・Sun beads and chito pearl
Mortar)-810 (IEP-C), φ300 μm, oxirane group 3 amount 0. 12 mmol/g (wet) manufactured by Fujibo Co., Ltd.] was added to an ethanolamine solution having a concentration of LM (pH 8), and the mixture was stirred at room temperature for about 20 hours. After this, acetate buffer (0.1M pH 4), phosphate buffer (0°LM
pH 7, 4) were used sequentially and autoclaved at 121° C. for 20 minutes. A lymphocyte separation experiment similar to that in Example 1 was conducted using the thus obtained monoethanolamine-immobilized beads having -CH2CH20H at the ends. The results obtained are shown in Table 1.

Comparative Example 3 Chitosan beads having oxirane groups as insoluble carrier [Chitopal El)-810(El)-C), φ30
0pm, oxirane base ff1O, 12o+ll1ol
/g (wet) manufactured by Fujibo Co., Ltd.], a separation material in which only the spacer triethylenetetramine was bound was prepared according to the method of Example 1, and a lymphocyte separation experiment similar to that of Example 1 was conducted. Ta. The results obtained are shown in Table 1.

Comparative Example 4 The reactant prepared in Comparative Example 3 was added to a 5 w/v % butanediol glycidyl ether solution prepared using 0.2 M carbonate buffer (pH 10) and incubated in a water bath at 80°C. After heating for 3 hours, the mixture was stirred at room temperature for about 20 hours using a blood mixer. Next, the concentration LM (pH8
The immobilized beads washed with distilled water were added to the ethanolamine solution of ) and stirred at room temperature for about 20 hours. A lymphocyte separation experiment similar to that in Example 1 was conducted using the separation material in which the spacer having the -CH2CHtOH terminal was immobilized in this manner. The results obtained are shown in Table 1.

Comparative Example 5 Chitosan beads with oxirane groups as an insoluble carrier [Chitopearl CP-810 (IEP-C), φ300
μm, oxirane base ff1o, 12 mmol/
g (wet) manufactured by Fujibo Co., Ltd.], and a linear alkyl chain having 4 methylene repeats was directly immobilized thereon.

First, 0.2M carbonate buffer (pH 10) was used.
A 2M butylamine solution was prepared and the EP was washed with distilled water.
-C was added. This was heated in a water bath at 80° C. for 3 hours, and then stirred at room temperature for about 20 hours using a blood mixer. Next, the reaction product washed with distilled water was added to an ethanolamine solution having a concentration of LM (pH 8), and the mixture was stirred at room temperature for about 20 hours to block the remaining glycidyl groups. Finally, acetate buffer (0.1M pH 4) and phosphate buffer (0.1M pH 7.4) were used sequentially at 121°C for 3.
Autoclaved for 0 minutes.

The separation material thus obtained was subjected to a lymphocyte separation experiment similar to that in Example 1. The results obtained are shown in Table 1.

Comparative Example 6 The method of Comparative Example 5 was followed except that octylamine was used instead of butylamine and the solvent was replaced with 100% dimethylformamide. A lymphocyte separation experiment similar to that in Example 1 was conducted. The results obtained are shown in Table 1.

Comparative Example 7 According to the method of Comparative Example 6 except that aminododecane was used instead of octylamine, beads were prepared in which a linear alkyl chain having 12 methylene repeats was directly immobilized, and the same method as in Example 1 was prepared. Lymphocyte separation experiments were performed. The results obtained are shown in Table 1.

Example 5 Chitosan beads [Chitopearl 30
10 (C11), φ300 μm, manufactured by Fujibo Co., Ltd.], an alkyl chain having 4 methylene repeats was immobilized as a hydrophobic end via hexanediol (HDO) as a spacer.

First, a 1% hexanediol diglycidyl ether solution was prepared using a dimethylformamide solution containing 30 mo1% sodium hydroxide, and CH washed with distilled water was added thereto. This was heated in a water bath at 80° C. for 3 hours, and then stirred at room temperature for about 20 hours using a blood mixer. Next, 0°2M carbonate buffer (pH 10)
The washed reactant was added to a 0.2 M butylamine solution prepared using a 0.2 M butylamine solution, heated in a water bath at 80° C. for 3 hours, and then stirred at room temperature for about 20 hours using a blood mixer. Next, in order to remove unreacted oxirane groups, the immobilized beads washed with distilled water were added to an ethanolamine solution with a concentration of IM (pH 8) and stirred for about 20 hours. Finally, add acetate buffer (C1).

IM p)14), phosphate buffer (0.1M p)
Autoclave treatment was performed at 121° C. for 30 minutes using H7,4) sequentially.

The separation material thus obtained was subjected to a lymphocyte separation experiment similar to that in Example 1. The results obtained are shown in Table 1.

Example 6 A linear alkyl chain with 8 methylene repeats was immobilized as a hydrophobic end according to the method of Example 5 except that octylamine was used instead of butylamine and the solvent was replaced with 100% dimethylformamide. The beads were prepared, and the same lymphocyte separation experiment as in Example 1 was conducted. The results obtained are shown in Table 1.

Example 7 According to the method of Example 6 except that aminododecane was used instead of octylamine, beads were prepared in which a linear alkyl chain having 12 methylene repeats was immobilized as a hydrophobic end. A lymphocyte separation experiment similar to that described above was performed. The results obtained are shown in Table 1.

Example 8 Chitosan beads [Chitopearl 30
10 (C1+), φ 300 μmS made by Fujibo Co., Ltd.], an alkyl chain with 4 methylene repeats was added via hexanediol (HDO), triethylenetetramine (TET) and butanediol (BDO) as spacers. It was immobilized as a hydrophobic end.

First, it was washed with pre-distilled water of HDO treatment C prepared in Example 5, added to O, 2M TET solution prepared using 0.2M carbonate buffer (pH 10), and placed in a water bath at 80°C. After heating for 3 hours in a vacuum chamber, the mixture was stirred at room temperature for about 20 hours using a blood mixer. Furthermore, the washed reactant was added to a 5 w/v% butanediol glycidyl ether solution prepared using a 0° 2M carbonate buffer (pH 10), and heated in a water bath at 80°C for 3 hours. ,
Stirred at room temperature for about 20 hours using a blood mixer. After this, the washed reactants were added to a 0.2M butylamine solution prepared using a 0.2M carbonate buffer (pH 10) and heated in a water bath at 80°C for 3 hours.
The mixture was stirred at room temperature for about 20 hours using a blood mixer. Next, in order to remove unreacted oxirane groups, the immobilized beads washed with distilled water were added to an ethanolamine solution with a concentration of IM (pH 8) and stirred for about 20 hours. Finally, acetate buffer (0, IM pH 4) and phosphate buffer (0,1 mo! pH 7,4) were used in sequence at 121°C.
, and autoclaved for 30 minutes.

The separation material thus obtained was subjected to a lymphocyte separation experiment similar to that in Example 1. The results obtained are shown in Table 1.

Example 9 A linear alkyl chain with 8 methylene repeats was immobilized as a hydrophobic end according to the method of Example 8 except that octylamine was used instead of butylamine and the solvent was replaced with 100% dimethylformamide. The beads were prepared, and the same lymphocyte separation experiment as in Example 1 was conducted. The results obtained are shown in Table 1.

Example 10 According to the method of Example 9 except that aminododecane was used instead of octylamine, beads were prepared in which a linear alkyl chain having 12 methylene repeats was immobilized as a hydrophobic end. A lymphocyte separation experiment similar to that described above was performed. The results obtained are shown in Table 1.

Example 11 Chitosan beads [Chitopearl 30
10 (C11), φ300 μm, manufactured by Fujibo Co., Ltd.], using ethylene glycol (EDO), triethylenetetramine (TET) and butanediol (BDO) as spacers, an alkyl chain having 4 methylene repeats was used. was immobilized as a hydrophobic end.

First, CH was washed with distilled water, added to a 5v/V% ethylene glycol diglycidyl ether solution prepared using a 0.2M carbonate buffer, and heated in a water bath at 80°C for 3 hours. and stirred for about 20 hours at room temperature using a blood mixer. The following TET and BDO treatments were conducted in accordance with the method of Example 8. Beads were prepared in which linear alkyl chains with 4 methylene repeats were immobilized as hydrophobic ends, and lymphocyte separation experiments similar to those in Example 1 were carried out. I did it.

The results obtained are shown in Table 1.

Example 12 A linear alkyl chain with 8 methylene repeats was immobilized as a hydrophobic end according to the method of Example 11 except that octylamine was used instead of butylamine and the solvent was replaced with 100% dimethylformamide. The beads were prepared, and the same lymphocyte separation experiment as in Example 1 was conducted. The results obtained are shown in Table 1.

Example 13 According to the method of Example 12 except that aminododecane was used instead of octylamine, beads were prepared in which a linear alkyl chain having 12 methylene repeats was immobilized as a hydrophobic end. A lymphocyte separation experiment similar to that described above was performed. The results obtained are shown in Table 1.

Example 14 As a water-insoluble carrier, acrylic beads with white oxirane groups [Euperquid C (EC), φ1501tm,
Oxirane group content 13 pmol/g (wet)
A linear alkyl chain having 12 methylene repeats is made hydrophobic using triethylenetetramine (T It was fixed as a sex end.

First, wash the EC and add it to 0.2M carbonate buffer (pH1
0) to a 0.2M TET solution prepared in
After heating in a water bath at 80° C. for 3 hours, the mixture was stirred at room temperature for about 20 hours using a blood mixer. Next, 0.2
Low prepared using M carbonate buffer (pH 10)
/w% polyethylene glycidyl ether solution, heated in a water bath at 80° C. for 3 hours, and then stirred at room temperature for about 20 hours using a bra-sodo mixer. moreover,
After immobilizing aminododecane according to the method of Example 10, IM (p
The immobilized beads washed with distilled water were added to the ethanolamine solution of H8) and stirred for about 20 hours. Finally, acetate buffer (0,1M pH4) phosphate buffer (0,1M
pH 7, 4) were used sequentially and autoclaved at 121° C. for 30 minutes.

Using the separation material thus obtained, a ρ lymphocyte separation experiment was conducted in the same manner as in Example 1. The obtained results are the first
Shown in the table.

Example 15 Chitosan beads [Chitopearl 30
10 (C11), φ300 μm, manufactured by Fujibo Co., Ltd.], the methylene repeat is 12 through polyethylene glycol (EG) with the ethylene oxide repeat of 15.
A linear alkyl chain was immobilized as a hydrophobic end.

First, CH was washed with distilled water, and 0.2M carbonate buffer (
10 v/w% lauryl alcohol glycidyl ether ((:, j+, 6.Cl
IC11-0(ell.el120)15-(C11□
)1□e113) The mixture was added to the solution, heated in a water bath at 80°C for 3 hours, and then stirred at room temperature for about 20 hours using a blood mixer. Next, 0.2M carbonate buffer (pH
10) was added to the 10 w/w % polyethylene glycidyl ether solution prepared above, heated in a water bath at 80° C. for 3 hours, and then stirred at room temperature for about 20 hours using a blood mixer. After this, acetate buffer (0.1M
The mixture was autoclaved at 121° C. for 30 minutes using phosphate buffer (0.1 M pH 7.4) sequentially.

A lymphocyte separation experiment similar to that in Example 1 was conducted using the separation material thus obtained. The obtained results are the first
Shown in the table.

Comparative example 1 Comparative example 2 Ratio/bite example 3 Comparative example 4 Example 1 Example 2 Example 3 Example 4 Table 1 Cell attachment rate Lymphocyte T cell 77.2 74.6 77.7 77.2 71.4 68.0 74.0 72.2 41.7 49.1 53.0 61.8 44°4 55.1 60.0 73.0 Comparative example 5 Comparative example 6 100.0 Comparative example 7 100.0 100.0 100.0 ioo,.

100.0 (%) B cell 81.2 85°9 76.6 76.7 38.3 41.5 462 45.6 Too,.

100.0 100.0 Example 5 Example 6 Example 7 Example 8 Example 9 Example 10 Example 11 Example 12 Example 13 Example 14 Example 15 Lymphocytes 73.2 73.8 65.3 72.9 66.1 60.7 70.4 69.5 59.5 43.7 45.0 Table 1 (continued) Cell attachment rate (%) T cells B cells 7B, 9 64.0 7B, 0 65 .8 68.9 53.6 76.6 Go, 9 71.2 49.8 71.6 261 76゜l 57.1 74.8 57.7 6g, 6 40.3 47.5 38.4 60. 6 23.3 As is clear from the results shown in Table 1, the separation material in which linear alkyl chains are immobilized via a spacer containing a hydrophilic molecular structure has a strong affinity for T lymphocytes. On the other hand, it moderately suppressed the adhesion of B lymphocytes.

(Effects of the Invention) As described above, the present invention provides a spacer with a hydrophilic molecular structure that absorbs an alkyl chain that preferentially adsorbs the T lymphocyte fraction in lymphocytes over other lymphocyte fractions. This is a material for separating lymphocyte subpopulations that is immobilized on the surface of a water-insoluble solid substance through a 3-way membrane. Because of adsorption, it has become clear that lymphocytes can be easily and quickly separated and concentrated from a lymphocyte suspension. Therefore, the lymphocyte subpopulation separator of the present invention can be used to analyze cell-cell interactions of leukocyte subgroups related to immune function, to basic technology for efficiently obtaining physiologically active substances derived from lymphocytes, and to further improve various immune systems. In addition to showing that the present invention can be applied to the treatment of diseases, it was also shown that the separation material of the present invention can achieve highly accurate separation of T and B lymphocytes without using any special physiologically active substances.

Further, in the lymphocyte subpopulation separator of the present invention, the methylene repeat in the alkyl chain is 25 or less, the alkyl chain is a linear alkyl chain, and the insoluble solid substance has a particle size of 0.05 When particles of ~5 mm are used, more efficient lymphocyte subgroup separation operation becomes possible.

The present invention also immobilizes an alkyl chain that preferentially adsorbs the T lymphocyte fraction in lymphocytes over other lymphocyte fractions onto the surface of a water-insoluble solid substance via a spacer containing a hydrophilic molecular structure. This is a lymphocyte subpopulation separator characterized by having a column filled with a lymphocyte subpopulation separation material characterized by ``-2. Contact between the separating material and the blood cell suspension can be carried out more efficiently, and the operation for separating lymphocyte subpopulations can be carried out in a short time and in a controlled manner.

Furthermore, the present invention immobilizes an alkyl chain that preferentially adsorbs the T lymphocyte fraction in lymphocytes over the song lymphocyte fraction onto the surface of a water-insoluble solid material via a spacer containing a hydrophilic molecular structure. By contacting lymphocyte fractions from humans or non-human animals with a material for separating lymphocyte subpopulations characterized by the fact that they are Since this is a method for isolating lymphocyte subpopulations that is characterized by separating and collecting non-captured fractions rich in lymphocytes, it takes advantage of the excellent properties of the above-mentioned lymphocyte subpopulation isolation material to separate and collect non-captured fractions rich in lymphocytes. Since it is possible to separate and recover T lymphocyte fraction and B lymphocyte, it is possible to obtain fractions of lymphocyte subpopulations at extremely high yields without requiring any operational skill compared to conventional separation methods. can be done, and even more cheaply.
Since it does not cause any significant cell damage, it can be said to be an extremely suitable technique for G.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram of an embodiment of the lymphocyte subpopulation separator of the present invention in use. 1... Column, 2... Separation of lymphocyte subgroups) rA
, 3.3'... Filter, 4... Three-way stopcock, 5
...Outlet port, 6.Actual diameter part, 7.Reduced diameter part,
8...Test tube.

Claims (7)

[Claims] (1) An alkyl chain that preferentially adsorbs the T lymphocyte fraction in lymphocytes over other lymphocyte fractions was immobilized on the surface of a water-insoluble solid material via a spacer containing a hydrophilic molecular structure. A material for isolating lymphocyte subpopulations characterized by: (2) Methylene repeats in the alkyl chain are 25
The lymphocyte subpopulation separation material according to claim 1, which is as follows. (3) The material for separating lymphocyte subpopulations according to claim 1 or 2, wherein the alkyl chain is a straight-chain alkyl chain. (4) The material for separating lymphocyte subgroups according to any one of claims 1 to 3, wherein the spacer has a hydrophilic part and a hydrophobic part. (5) The material for separating lymphocyte subpopulations according to any one of claims 1 to 4, wherein particulate matter with a particle size of 0.05 to 5 mm is used as the insoluble solid substance. (6) A lymphocyte subgroup separator comprising a column filled with the lymphocyte subgroup separation material according to any one of claims 1 to 5. (7) A lymphocyte fraction from humans or non-human animals is brought into contact with the material for separating lymphocyte subgroups according to any one of claims 1 to 5, and T lymphocytes are efficiently captured by the separating material. A leukocyte separation method characterized in that a non-captured fraction rich in B lymphocytes is separated and collected by separating and collecting B lymphocytes.