JPH02167071A - Separating material for leukocytic cell of nonhuman animal origin, separator therefor and separation thereof - Google Patents

Abstract

PURPOSE:To obtain a material capable of readily, simply, quickly separating the title cells in high yield and purity by immobilizing on the surface of a water-insoluble solid matter such a carbohydrate as to preferably adsorb the B lymphocyte fraction in the lymphocytes of nonhuman animal origin rather than the other lymphocyte fractions. CONSTITUTION:A separating material 2 for lenkocytic cells of nonhuman animal origin is prepared by immobilizing on the surface of a water-insoluble solid matter (e.g. oxirane group-contg. acrylic beads) such a carbohydrate (e.g. alginic acid) as to preferably adsorb the B lymphocyte fraction in the lymphocytes of nonhuman animal origin rather than the other lymphocyte fractions. This material 2 is then filled between filters 3 within the real diameter part 6 of a column 1 having said real diameter part 6 and a contracted diameter part 7, and a suspension of leukocytic cells of nonhuman animal origin is passed through said material 2 to selectively scavenge the granular spheres, single spheres and B lymphocytes present in this suspension to effect recovery of a T lymphocyte-rich non-scavenged liquid fraction via an outlet 5 into a test tube 8 through a stopcock 4 operation.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to an H1 separator and a method for separating leukocyte cells derived from non-human animals for separating and purifying lymphocytes and subgroups thereof. be.

Specifically, the present invention involves selectively capturing B lymphocytes (as well as monocytes and granulocytes) from a leukocyte cell suspension obtained from lymphoid tissues of animals such as mice, and then
The present invention describes a separator, a separator, and a method for separating leukocyte cells derived from non-human animals, which collect a lymphocyte-rich fraction and easily, rapidly, and highly accurately separate and purify lymphocyte subpopulations.

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 B lymphocytes that produce antibodies and play a role in humoral immunity, T lymphocytes that produce immunoregulatory lymphokines and inflammatory lymphokines and play a role in cellular immunity, and null cells.
In addition, T and B lymphocytes have functionally different subsera!・Exists. In recent years, technology to purely separate, purify, and concentrate subclasses such as T and B lymphocytes with different immunological characteristics while minimizing functional damage has been strongly desired in a wide range of social fields. There is. For example, in the field of basic research such as cytology and immunology, we are trying to elucidate the role (interaction) of valleys in the hyperdifferentiation of T and B lymphocytes, and to understand the role of each in the immune function of the in vivo immune system. For purposes such as in vitro elucidation or identification of the surface markers (surface antigens) of each cell, it is necessary to separate at least T and B lymphocytes, and this is the final step in research. It is important not only for humans as a target but also for other animals such as mice as a model system.

(Conventional technology) As a method for separating T lymphocytes and B lymphocytes, first, mononuclear cell fractions are obtained from peripheral blood by specific gravity centrifugation using a high-density isotonic solution such as a sodium metrizoate ficoll mixture, and by the cardiac method. Conventionally, the conventional method is to directly obtain a mononuclear cell fraction from a living tissue, etc., and then perform a separation process to separate it into T lymphocytes and B lymphocytes. The subsequent separation processing methods are broadly divided into (1) methods using specific antigens, receptors, and immunoglobulins on the cell membrane surface as indicators;
It can be divided into three parts: 2) physical cell separation and (3) adsorption chromatography. Methods for (1) include rosette formation, affinity talomadography (conjugated with antibodies or immune complexes), cell lysis using complement, removal of mitogen-activated lymphocytes, and fluorescein-activated cell sorter ( F, A, C, S, )
However, with these methods, it is difficult to collect T lymphocytes or B lymphocytes in an intact state because they are subject to strong binding and stimulation with high biological specificity, and it is difficult to collect them in large quantities. There are many methods that are not suitable for processing. Also,
Density gradient centrifugation and cell electrophoresis are known methods for (2), but there are significant differences in physical properties such as specific gravity and density between TIJ lymphocytes and B lymphocytes. Therefore, high precision cannot be required for the yield or purity of 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 B lymphocytes to foreign substances such as nylon, Tetoron, and cotton fibers. In order to obtain this, it is necessary to treat the adsorption carrier with fetal bovine serum (Fe2), etc., it is difficult to set the implementation conditions such as flow rate, and there are many contaminations with impurities such as macrophages. 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 separation material, separation device, and separation method for leukocyte cells derived from non-human animals. The present invention also provides a separator for non-human animal-derived leukocyte cells that easily and rapidly separates and purifies non-human animal-derived leukocyte cells with high yield and purity without affecting various cell activities. , a separator and a separation method.

A further object of the present invention is to provide a separation material, a separator, and a separation method for non-human animal-derived leukocyte cells that are suitable for large-scale processing.

(Means for solving the problem) The above-mentioned solution is to use a water-insoluble solid to absorb carbohydrates that preferentially adsorb the B lymphocyte fraction in lymphocytes derived from non-human animals than other lymphocyte fractions. This is achieved using a material for separating leukocytes derived from non-human animals, which is characterized by being immobilized on the surface of a substance.

The present invention also provides a material for separating leukocytes derived from non-human animals, in which the carbohydrate has a high affinity for monocytes and granulocytes derived from non-human animals. The present invention further provides a material for separating leukocytes derived from non-human animals, in which the carbohydrate to be immobilized is a natural sugar or a derivative thereof. The present invention further provides a separator for leukocytes derived from non-human animals, in which the natural sugar or its derivative is selected from homopolysaccharides (homoglycans) and heteropolysaccharides (heteroglycans). The present invention further provides a material for separating leukocyte cells derived from non-human animals, in which the carbohydrate is selected from the group consisting of gum arabic, inulin, starch, and alginic acid. The present invention further provides a material for separating leukocyte cells derived from non-human animals, in which the immobilized carbohydrate is gum arabic. The present invention also provides a material for separating leukocytes derived from non-human animals, in which the water-insoluble solid substance is in the form of particles with a particle size of 0.05 to 5 mm. The present invention further includes:
A carbohydrate that preferentially adsorbs the B lymphocyte fraction in lymphocytes derived from non-human animals over other lymphocyte fractions is immobilized on the surface of a water-insoluble solid substance by co-6 bonding, physical bonding, etc. ``Separation of leukocyte cells derived from non-human animals,'' which is a method of

The above objectives also aim to improve B in lymphocytes from non-human animals.
It is also possible to use a material for separating leukocytes derived from non-human animals, which is characterized by being composed of water-insoluble particles formed using carbohydrate itself as a base material, which preferentially adsorbs lymphocyte fractions over other lymphocyte fractions. achieved.

The present invention also provides a material for separating leukocytes derived from non-human animals, in which the carbohydrate has a high affinity for monocytes and granulocytes derived from non-human animals. The present invention further provides a material for separating leukocytes derived from non-human animals, in which the carbohydrate forming the water-insoluble particles is a natural sugar or a derivative thereof. The present invention further provides a material for separating leukocyte cells derived from non-human animals, which is selected from natural sugars or their derivatives, homopolysaccharides (homoglycans), and heteropolysaccharides (heteroglycans).

The present invention further provides an isolated white blood cell derived from a non-human animal, in which the carbohydrate forming the water-insoluble particles is selected from the group consisting of gum arabic, inulin, starch, and alginic acid. It is. The present invention further provides a material for separating leukocyte cells derived from non-human animals, in which the immobilized carbohydrate is gum arabic.

The object of item I-1 is further directed to immobilize a carbohydrate on the surface of a water-insoluble solid substance that preferentially adsorbs the B lymphocyte fraction in lymphocytes derived from non-human animals over other lymphocyte fractions. B in isolated H and/or lymphocytes from non-human animals
A non-human animal-derived product characterized by having a column filled with separation H consisting of water-insoluble particles formed using carbohydrate itself as a base material, which preferentially adsorbs lymphocyte fractions over other lymphocyte fractions. This can also be accomplished by a leukocyte cell separator.

The present invention also provides leukocyte cells derived from non-human animals, which is provided with a filter having a mesh between the inlet and outlet of the column and the separation material layer, through which cell components in the blood cell suspension can pass, but not the separation material. This shows a separator.

The two-legged purpose is further to be achieved by immobilizing carbohydrates on the surface of a water-insoluble solid substance to preferentially adsorb the B lymphocyte fraction in lymphocytes derived from non-human animals over other lymphocyte fractions. B in isolation material and/or lymphocytes from non-human animals
A leukocyte cell suspension derived from a non-human animal is brought into contact with a separation material consisting of water-insoluble particles formed using carbohydrate itself as a base material, which preferentially adsorbs lymphocyte fractions over other lymphocyte fractions. , the above separation material is allowed to selectively capture granulocytes, monocytes, and B lymphocytes present in a free fluid of white blood cells derived from a non-human animal, and a non-captured fraction rich in T lymphocytes is separated and collected. This can also be achieved by a method for separating leukocytes derived from non-human animals, which is characterized by the following.

The above features include a separation material and/or a separation material comprising a carbohydrate immobilized on the surface of a water-insoluble solid substance that preferentially adsorbs the B lymphocyte fraction in lymphocytes derived from non-human animals over other lymphocyte fractions; Alternatively, a non-human animal may be used as a separation material consisting of water-insoluble particles formed using carbohydrate itself as a base material, which preferentially adsorbs the B lymphocyte fraction in lymphocytes derived from non-human animals over other lymphocyte fractions. by contacting the leukocyte cell suspension derived from the
This can also be achieved by selectively capturing white blood cell fractions, mainly B lymphocytes other than 928 cells, and later detaching the captured cells from the separation phase to efficiently separate and collect the captured cell fraction. .

The present invention also provides a method for separating leukocyte cells derived from a non-human animal, which uses a leukocyte cell suspension derived from a non-human animal in which the presence rate of B lymphocytes is at least 10% of all lymphocytes. It is.

In addition, "white blood cell" used in this specification
These terms include granulocytes such as neutrophils, eosinophils, and basophils;
This refers to lymphocytes such as T lymphocytes and B lymphocytes, as well as monocytes, and these cells exist not only as blood cell components of blood but also in other tissues such as lymphoid tissues. It also includes things.

(Function) The present invention provides a non-human animal, which is made by immobilizing carbohydrates on the surface of a water-insoluble solid substance that preferentially adsorbs the B lymphocyte fraction in lymphocytes derived from non-human animals over other lymphocyte fractions. In order to use an animal-derived leukocyte cell isolation material or a non-human animal-derived leukocyte cell isolation material consisting of water-insoluble particles formed using the carbohydrate itself as a base material, it is necessary to use a material obtained from the lymphatic tissue of a non-human animal. By simply bringing a white blood cell suspension, such as a lymphocyte suspension, into direct contact with the separation material, it is possible to efficiently separate and purify <T, B lymphocytes.

That is, when a leukocyte cell suspension derived from a non-human animal is brought into contact with the separation material for non-human animal-derived leukocyte cells of the present invention, B lymphocytes, which have strong adhesion to foreign substances among lymphocytes, are present on the surface of the separation material. It is preferentially adsorbed by the interaction between carbohydrates and glycoproteins in the cell membrane;
The hydrophilicity based on the hydroxyl groups in the sugar skeleton actively suppresses the adhesion of non-B lymphocyte components, so it is thought that they will not be adsorbed, and most of the lymphocytes that are adsorbed to the separation material are B lymphocytes. .

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

The separated phase of leukocyte cells derived from non-human animals of the present invention preferably contains carbohydrates that preferentially adsorb the B lymphocyte fraction in lymphocytes derived from non-human animals than other lymphocyte fractions, and preferably further non-human animal-derived leukocyte cells. It is characterized by immobilizing carbohydrates that have a high affinity for human and animal-derived granulocytes and monocytes on the surface of a water-insoluble solid substance.

Examples of carbohydrates having such properties include natural polysaccharides and derivatives thereof. Specific examples of natural polysaccharides include homopolysaccharides consisting of only one constituent monosaccharide such as starch, dextrin, glycogen, cellulose, dextran, inulin, galactan, chitin, and alginic acid, as well as pectin, gum arabic, There are heteropolysaccharides consisting of two or more constituent monosaccharides, such as hyaluronic acid, chondroitin sulfate, and heparin.

Furthermore, carbohydrates that can be used in the material for separating leukocytes derived from non-human animals of the present invention include monosaccharides such as ribose, deoxyrifose, xylose, arabinose, glucose, galactose, mannose, fructose, and uronic acid. Polymerized materials, starch,
Dextrin, glycogen, cellulose, dextran, inulin, galactan, chitin, homopolysaccharides such as alginic acid, pectin, gum arabic, hyaluronic acid,
Derivatives of natural sugars obtained by chemically modifying heteropolysaccharides such as chondroitin sulfate, heparin, and heparin can also be used to achieve suitable separation of leukocytes derived from non-human animals.

Among these carbohydrates, in particular, natural heteropolysaccharides with low carboxyl group content such as gum arabic, natural homopolysaccharides such as inulin and starch, and derivatives of these natural sugars have a high affinity for B lymphocytes. Desirable for its properties and selectivity.

That is, as mentioned above, among lymphocytes, B lymphocytes, which have a strong adhesion to foreign substances, are preferentially adsorbed by the interaction between carbohydrates present on the surface of the separated phase and glycoproteins of the cell membrane. It is thought that the hydrophilicity based on the hydroxyl group actively suppresses the adhesion of non-B lymphocyte components, making them less likely to be adsorbed. Furthermore, the lymphocyte fraction obtained from non-human animals such as rats, which is the target of the adsorption H of the present invention, generally has a higher gold-white ratio of B IJ lymphocytes than that obtained from human peripheral blood. Gum arabic, which is a type of carbohydrate that more actively adsorbs lymphocytes, works well and is desirable for increasing the ratio of T lymphocytes to B lymphocytes in the non-capturing fraction.

Conventionally, various lectins, which are sugar-binding proteins, have been used to separate cells at different stages of differentiation and cells with different functions because the cell membrane complex carbohydrates that serve as surface markers differ. However, the carbohydrates used in the present invention are different from lectins that exhibit such biologically highly specific binding properties (see, for example, Clinical Immunology 16 (1): 1-11, 1984). It is thought that they adsorb to cells with completely different effects.

In the material for separating leukocytes derived from non-human animals of the present invention, the water-insoluble solid substances used as carriers for immobilizing carbohydrates having such characteristics include agarose-based, dextran-based, cellulose-based, polyacrylamide-based, Polyvinyl alcohol-based, polyvinylpyrrolidone-based, polyacrylonitrile-based, styrene-divinylbenzene copolymer, polystyrene-based, acrylic ester-based, methacrylic ester-based, polyethylene-based, polypropylene-based,
A polymers such as poly(4-fluoroethylene), ethylene-vinyl acetate copolymer, polyamide, polycarbonate, polyvinylidene fluoride, polyvinyl formal, polyarylate, polyethersulfone,
Examples include inorganic materials such as glass, alumina, titanium, activated carbon, and ceramics, but those that do not have too high cell adhesion, that is, those that have relatively mild interactions between cells and carriers, are preferred. Especially preferably, acrylic esters are used. Furthermore, collagen, chitosan, etc., which are natural aliphatic polymers derived from living organisms, may also be used. Forms of such water-insoluble solid substances include:
It is not particularly limited, and flat plate-L can also be used, preferably in the form of particles, especially with an average particle size of 0.05 to 5.
Preferably, it is in the form of particles of mm. The average particle size is JIS
- After classification using a sieve specified in Z-8801, the intermediate value between the upper limit particle size of grade 6 and the upper limit particle size is taken as the particle size of grade 6, and the! It is calculated as the average Tf amount.

Further, the particle shape is preferably spherical because it is less likely to cause physical damage to cells and it is easier to obtain uniform particles.

In the material for separating leukocytes derived from non-human animals of the present invention, methods for immobilizing carbohydrates having the above characteristics on the surface of the water-insoluble solid substance include covalent bonding, covalent bonding,
All known methods such as ionic bonding, physical adsorption, embedding, and insolubilization by precipitation on the carrier surface can be used, but from the viewpoint of the elution properties of immobilized carbohydrates, it is preferable to immobilize and insolubilize them using covalent bonds. is preferred. Therefore, in general, known insoluble carrier activation methods and ligand immobilization methods used in the field of immobilized enzymes, affinity chromatography may be preferably used.

Another non-human animal-derived leukocyte cell isolation material of the present invention is
Carbohydrates that preferentially adsorb the B lymphocyte fraction in lymphocytes derived from non-human animals over other lymphocyte fractions, and more preferably have high affinity for granulocytes and monocytes derived from non-human animals. It is characterized by being composed of water-insoluble particles formed using sugar itself as a base material.

In this way, even in a separator in which carbohydrates are directly pulverized to preferentially adsorb the B lymphocyte fraction in lymphocytes derived from non-human animals than other lymphocyte fractions, the aforementioned carbohydrate It acts in the same way as a separation material formed by immobilizing the molecule on the surface of a water-insoluble solid substance, and can perform good separation of leukocytes derived from non-human animals.

Carbohydrates that can be used in this separation H of leukocytes derived from non-human animals include those mentioned above, and such carbohydrates can be extracted using known methods such as the suspension evaporation method and the ion complex method. By gelling, preferably into a spherical shape, and crosslinking with a polyfunctional epoxide, aldehyde, isocyanate, etc. to form water-insoluble particles, it can be used as a separation material for leukocytes derived from non-human animals.

Even in separation H of non-human animal-derived leukocyte cells in which carbohydrates were directly made into particles, the average particle size was 0.05 to 5 mm.
is desirable.

The non-human animal-derived leukocyte cell separator of the present invention adsorbs carbohydrates that preferentially adsorb the B lymphocyte fraction in the non-human animal-derived lymphocytes as described above over other lymphocyte fractions.
A separation material immobilized on the surface of a water-insoluble solid substance and/or
Or a column filled with a separation material consisting of water-insoluble particles formed using carbohydrate itself as a base material, which preferentially adsorbs the B lymphocyte fraction in lymphocytes derived from non-human animals over other lymphocyte fractions. It is characterized by having the following.

In order to contact the non-human animal-derived leukocyte cell separation material of the present invention with a leukocyte cell suspension such as a lymphocyte suspension obtained from a lymphoid tissue of a rat, for example, a plate-shaped non-human animal-derived It is also possible to carry out this by bringing the leukocyte cell suspension into contact with the separation material for leukocyte cells, but sugars that are present on the surface of the separation material for non-human animal-derived leukocyte cells and act on cell adsorption, such as those mentioned above, may be used. From the standpoint of obtaining an absolute amount of quality and sufficient cell adsorption space, we recommend using a column filled with particulate material for separating white blood cells derived from non-human animals.
It is desirable that the contact be made using a non-human animal-derived leukocyte cell separator.

In this non-human animal-derived leukocyte cell separator that uses a column filled with particulate non-human animal-derived leukocyte cell separation material, the material constituting the column container is polyethylene, polypropylene, or polycarbonate. , synthetic resins such as polystyrene and polymethyl methacrylate, and metals such as glass and stainless steel can be used, but polypropylene and polycarbonate, which can be autoclaved and are easy to handle, are particularly preferred. A filter with a mesh that allows cell components in the leukocyte cell suspension to pass through but not to pass through the separator is installed between the inlet and outlet of the column and the separation H layer filled with a separator for non-human animal-derived leukocyte cells. It is preferable that the filter be equipped with 11
The material for forming the four components may be any physiologically inert and high-strength material, but polyester and polyamide are particularly preferred.

FIG. 1 is a schematic diagram of an embodiment of the non-human animal-derived leukocyte cell 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. Further, this tapered portion 7 is provided with a three-way stopcock 4, so that the flow path leading to the outlet 5 can be opened and closed.

The actual diameter portion 6 above the tapered portion 7 of this column 1 is filled with the separation material 2 for non-human animal-derived leukocyte cells of the present invention as described above. The separation material 2 is held within the column 1 by a filter 3 provided at the lower end of the actual diameter portion 6 of the column 1 to form a separation H layer.

Furthermore, the method for separating leukocyte cells derived from non-human animals of the present invention includes:
A separation material and/or a separation material comprising a carbohydrate immobilized on the surface of a water-insoluble solid substance that preferentially adsorbs the B lymphocyte fraction in lymphocytes derived from non-human animals over other lymphocyte fractions, as described above. Separation H consisting of water-insoluble particles formed using carbohydrate itself as a base material that preferentially adsorbs the B lymphocyte fraction in lymphocytes derived from non-human animals over other lymphocyte fractions.
is brought into contact with a leukocyte cell suspension derived from a non-human animal,
The above separation material selectively captures granulocytes, monocytes, and B lymphocytes present in a leukocyte cell suspension derived from a non-human animal, and the non-captured fraction rich in T lymphocytes is separated and recovered. This is a characteristic feature.

To explain more specifically the method of separating leukocyte cells derived from non-human animals of the present invention using the separator for leukocyte cells derived from non-human animals shown in FIG. The suspension is injected into a column 1 filled with a separation phase 2 of non-human animal-derived leukocyte cells, and the leukocyte cell suspension is brought into contact with the separation material 1 for non-human animal-derived leukocyte cells.

In the method of separating non-human animal-derived leukocyte cells of the present invention, the non-human animal-derived leukocyte cell suspension used as a treatment target includes mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, monkeys, sheep, Horse,
Lymphocyte suspension obtained by suspending lymphocyte fractions obtained from various lymphoid tissues such as lymph nodes, spleen, pulp, thymus, etc. of animals such as chickens in a balanced salt solution, or these non-human animals. This includes peripheral blood itself, blood cell suspensions in which blood cell components separated from peripheral blood are suspended in a balanced salt solution, and lymphocyte fractions or blood cell components resuspended in animal plasma. .

In particular, in the separation method of the present invention, when starch, inulin, alginic acid, gum arabic, etc. are used as carbohydrates, the presence rate of B lymphocytes is at least 10% or more and 80% or less of all lymphocytes. Using a suspension of the fractions, very good T, BIJ bulb separation can be achieved. That is, in this case, the existence rate of B IJ lymphocytes is 1
T obtained when using a suspension with a lymphocyte fraction of less than 0%
The yield of the lymphocyte fraction decreases, and if it is higher than 80%, the purity of the T lymphocyte fraction decreases, which is not preferable.

Originally, this separation material has the characteristic that it does not allow only T lymphocytes to adhere among the miscellaneous leukocyte cell population.
In some cases, mononuclear cell fractions obtained by specific gravity centrifugation and Huffycoat excluding red blood cells and platelets maintain the separation effect described, and there are no restrictions on the cell fractions to be separated. It is not accepted. In addition, animal serum such as fetal bovine serum may be added to a leukocyte cell suspension derived from a non-human animal, but the separated phase of the present invention has extremely low stimulation to cells, and such animal serum may be added to the suspension. Even without the addition of animal serum, the survival rate of the cells after the separation operation shows a high value, typically 97% or more, so it is not necessary to add animal serum.

The method of flowing the leukocyte 17 liquid is not particularly limited, and may be continuous or discontinuous as necessary, or desirably discontinuous for a certain period of time, preferably 0.5~ By adding incubation for 1 hour, leukocyte cells can be separated with higher efficiency and yield.

When incubating in this way using the non-human animal-derived leukocyte cell separator shown in Figure 1,
It is sufficient to close the three-way stopcock 4 in advance to close the flow path leading to the outflow port 5, and the injected leukocyte cell suspension is held in the column 1 in contact with the separation material 2, and kept stationary for a predetermined period of time. place As the temperature conditions for incubation,
There is no particular limitation, and for example, it may be carried out at room temperature or at a temperature of around 37°C, but the adsorption of granulocytes and monocytes to the separation material 2 of non-human animal-derived white blood cells is highly temperature dependent, especially around 37°C. In the temperature range of It is desirable to conduct this in the area.

When the non-human animal-derived leukocyte cell suspension is brought into contact with the non-human animal-derived leukocyte cell separation material 2 in this way, the granulocytes and monocytes contained in the leukocyte-derived cell suspension are separated from the non-human animal-derived leukocytes. It is thought that in response to the above-mentioned carbohydrates present on the surface of the material 2 for separating white blood cells derived from non-human animals, it results in phagocytosis-like adhesion and flattening. Ru. Furthermore, among lymphocytes, B lymphocytes, which have a strong adhesion to foreign substances, also adhere to the separation of non-human animal-derived leukocyte cells. It is thought that the B lymphocytes are adsorbed by this action, and this adhesion of B lymphocytes is in a spot-disseminated state and is reversible.

After the leukocyte cell suspension is brought into contact with the non-human animal-derived leukocyte cell separation material 2 for a predetermined period of time in column 1, a washing solution is injected into column 1 using various pumps, pipettes, etc. , the three-way stopcock 4 is opened, the cleaning liquid is allowed to drop gently from the outlet 5, and is collected in a test tube 8 or the like. As a result, it is possible to collect a non-captured fraction containing many T lymphocytes that were not adsorbed to the separation material 2 for animal-derived leukocytes. Note that when a blood cell suspension obtained from the peripheral blood of a non-human animal is used as the leukocyte cell suspension, the non-captured fraction will without exception contain many red blood cells, so for example, an ammonium chloride solution may be used. It can be easily removed by known hemolytic methods such as addition of red blood cell agglutinants, etc.

The purpose of the washing liquid used in this washing operation is to wash away cell components that have not been adsorbed to the non-human animal-derived leukocyte cell separation material 2 without eluting the cell components that have been adsorbed to the separation material 2. Therefore, a balanced salt solution, a cell culture medium, etc. are used, preferably PRM11640.Divalent cation-free balanced salt solution, such as Hank's balanced salt solution (frank's balanced salt solution)
balanced 5aIt 5solution: l
lB55) etc. are used.

By the washing operation as described above, the non-captured fraction rich in T lymphocytes can be easily and efficiently collected, but by further adding the following operation afterwards,
An eluate fraction rich in B lymphocytes can be easily obtained.

That is, first, following the washing operation as described above, in order to remove remaining non-captured cells, if necessary, a washing solution is further injected into the column 1 in the same manner as above, and this washing solution is gently poured into the outflow port 5. Add a cleaning operation to discharge from the tank. Next, the washing solution was injected into column 1 again, and this time it was shaken and
The material 2 for separating white blood cells derived from non-human animals is washed by applying physical vibration such as stirring, and then the three-way stopcock 4 is opened and the washing liquid is allowed to fall from the outlet 5 and collected in a test tube 8 or the like.

Isolation of non-human animal-derived leukocyte cells as described above
The adsorbed lymphocytes maintain their spherical shape and are in a dotted state, so when such physical vibration is applied and the washing operation is performed, they are easily detached from the separation material 2 for non-human animal-derived leukocyte cells. . This also applies to B lymphocytes, which are more adherent. On the other hand, granulocytes and monocytes undergo morphological changes and aggregation and are irreversibly adsorbed, so even when such physical vibrations are applied, they cannot be easily released from the separation material 2 for leukocytes derived from non-human animals. I almost never let go. As a result, the washing solution recovered after washing with physical vibration is a fractionated solution rich in B lymphocytes detached from the separation H2 of leukocytes derived from non-human animals. In addition, as the washing solution used at this time, the same balanced salt solution, cell culture solution, etc. as mentioned above can be used, but for example,
The efficiency of collecting B lymphocytes can be further increased by using an isotonic buffer containing sugars, amino acids, and albumin, such as a 50% by weight sucrose solution.

The B lymphocyte-rich fraction obtained in this way has a sufficiently high purity of B lymphocyte components, but if you want to obtain a product with even higher purity, this fraction should be used. It is also possible to separate and remove mixed granulocytes, monocytes, etc. by applying known techniques such as specific gravity centrifugation.

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

Example 1 Acrylic beads containing white oxirane groups (Eupergit C, Lehmfal? [R
6hm Pharma] (Average particle size 0°15
mm) to prepare a separation material for non-human animal-derived leukocyte cells immobilized with alginic acid, and the adhesion of lymphocyte subpopulations was examined.

First, a 1% by weight alginic acid solution was prepared using a 0.2M carbonate buffer (pH 10), and oxirane-acrylic beads washed with distilled water were added to the solution by 0.3% to 0% by weight. 5
It was added at a ratio of g (wet)/ml and degassed. This is 8
After heating in a 0°C water bath for 3 hours, the mixture was stirred at room temperature for about 20 hours using a blood mixer (BM-101, manufactured by Kayagaki Medical Science Co., Ltd.).

Then, the concentration L was increased to remove unreacted oxirane groups.
The immobilized piece washed with distilled water was added to an ethanolamine solution of M (pH 8) and stirred for about 20 hours. Finally, glycine hydrochloride buffer (0.1M pH2), acetate buffer (0.1M pH4), phosphate buffer (0.1M
Autoclave washing was performed at 121° C. for 30 minutes using pH 7, 4).

The separation material thus obtained was mixed with Hanks' balanced salt solution (
HBSS, pH 7.4) and packed into a polypropylene column in a wet state to a volume of 1 ml.

Next, lymphocytes collected from the lungs of mice (ICR 18 weeks of age or older) were added to RPM1164 containing 10% fetal bovine serum.
0 (Hinaga Pharmaceutical) and concentrated, 7 x 106
The entire suspension (0°3 ml) containing 500 cells was immersed. After leaving this column at room temperature for 10 minutes, 1 ml
The inside of the column was washed using the above HBSS to collect lymphocytes.

The percentage of lymphocytes or the percentage of T and B lymphocytes in the collected fraction was measured using an automatic hemocytometer (Ortho Instruments [OR1'1101 NS1'1'
The adsorption was evaluated using an automatic cell analyzer (Cyto ACE-100, manufactured by JASCO Corporation) and a fluorescently labeled anti-mouse Ig antibody (Becton Dickinson). The adhesion rate of each lymphocyte fraction to the material was investigated. The results are shown in Table 1.

Example 2 Inulin-immobilized beads were prepared according to the method of Example 1 except that the carbohydrate to be immobilized was changed to inulin, and the same lymphocyte separation experiment as in Example 1 was conducted.

The results obtained are shown in Table 1.

Example 3 Starch-immobilized beads were prepared according to the method of Example 1, except that the carbohydrate to be immobilized was changed to starch, and the same lymphocyte separation experiment as in Example 1 was conducted.

The results obtained are shown in Table 1.

Example 4 Gum arabic-immobilized beads were prepared according to the method of Example 1, except that the carbohydrate to be immobilized was changed to gum arabic, and the same lymphocyte separation experiment as in Example 1 was conducted. The results obtained are shown in Table 1.

Comparative Example 1 Acrylic beads with oxirane groups (Eupergit C, Röhmfal? [R6
hm Pharma] Co., Ltd. (・1 piece average particle size 0゜1
A material for separating leukocytes derived from non-human animals on which succinic acid was immobilized was prepared using a succinic acid (5 mm), and the adhesion of lymphocyte cells was examined.

First, a 1% by weight succinic acid solution was prepared using distilled water, and then immobilized according to the method of Example 1 to produce succinic acid-immobilized beads. Then, a lymphocyte separation experiment similar to that in Example 1 was conducted.

The results obtained are shown in Table 1.

Comparative Example 2 Acrylic beads with untreated oxirane groups (Eupergit C, Rehm Pharma [1-6 ha+ Pha
Lymphocyte separation experiment similar to that in Example 1 was carried out using ``Rma'' (average particle size: 0.1-5 mo+) as it was. The results obtained are shown in Table 1.

Example 5 4 suspended in RPM11640 without added serum
X 107 mouse lung lymphocytes were injected into a column filled with gum arabic-immobilized beads prepared in Example 4, and the ability to separate T and B lymphocytes was examined. Evaluation was performed according to the method of Example 1.

The results are shown in Table 2.

Example 6 Gum arabic-immobilized beads prepared according to Example 4 were packed in a polypropylene column in a wet state to a volume of 0.5 ml, and 1×10 beads prepared according to Example 1 were packed in a polypropylene column to a volume of 0.5 ml.
Cells/ml of mouse spleen lymphocyte fraction were injected in 10 ml and allowed to fall naturally at a flow rate of 1 ml/min. After this, 3
Rinse the inside of the column with ~5 ml of RPMI 1640 containing 10% FBS, and then rinse with RPM1 containing 10% FBS again.
1 ml of 1640 was added, and the column was rotated 10 times, overturned, and stirred to obtain detached cells. The evaluation method was based on the method of Example 1. The results are shown in Table 3.

Translation effect 0 ” 柘勺塡Ω Nori-ekiko 4 ministers 1 to ni朶Q ya→← ,-I C'l (Y'I Q;; Tatsumi ShigebeAs is clear from the results shown in Tables 1 and 2, the separation phase formed by immobilizing various carbohydrates or (Examples 1 to 5)
) has a strong affinity for B lymphocytes and moderately inhibited LT lymphocyte bone adhesion. In particular, gum arabic, a complex polysaccharide, has the best selectivity for T and B lymphocytes, and is a basic It is considered that the level of separation required for scientific research can be easily maintained.Also, as shown in Table 3,
It was also excellent in recovering adhered lymphocytes, and it was also possible to easily enrich and purify B lymphocytes, so it seems that its applicability was extremely wide.

(Effects of the Invention) As stated above, the present invention provides water-insoluble carbohydrates that preferentially adsorb the B lymphocyte fraction in lymphocytes derived from non-human animals over other lymphocyte fractions. A separated phase of leukocyte cells derived from non-human animals characterized by being immobilized on the surface of a solid substance, and a B lymphocyte fraction in lymphocytes derived from non-human animals preferentially compared to other lymphocyte fractions. This is a material for separating leukocytes derived from non-human animals, which is characterized by being composed of water-insoluble particles formed using the adsorbed carbohydrate itself as a base material. B lymphocytes, which are a subclass of the lymphocyte fraction, are preferentially adhered to by contacting with a non-human animal-derived leukocyte cell suspension, such as a suspension of leukocyte cells (at the same time, granules are present in the leukocyte cell suspension). If granulocytes and monocytes are present, it selectively adsorbs these granulocytes and monocytes), making it possible to easily and quickly isolate and concentrate T lymphocytes from non-human animal leukocyte cell suspensions. It is something. Therefore, the separation material for leukocyte cells derived from non-human animals of the present invention is useful for investigating cell-cell interactions between leukocytes related to immune function or for efficiently separating physiologically active substances derived from lymphocytes using non-human animal models. It can be suitably applied to the basic technology for obtaining the desired results. Furthermore, the non-human animal-derived leukocyte cell separator of the present invention is safe and uses a special It is possible to achieve highly accurate separation of leukocytes derived from non-human animals, as well as separation of T and B lymphocytes, without using physiologically active substances. Further improvement in accuracy can be expected if it is selected from the group consisting of:

The present invention also provides a non-human animal comprising a carbohydrate immobilized on the surface of a water-insoluble solid substance that preferentially adsorbs a B lymphocyte fraction in lymphocytes derived from a non-human animal over other lymphocyte fractions. A water-insoluble separator formed from the carbohydrate itself that preferentially adsorbs the B lymphocyte fraction in lymphocytes derived from non-human animals over other lymphocyte fractions. Since this is a separator for leukocytes derived from non-human animals characterized by having a column filled with a separation material for leukocytes derived from non-human animals consisting of particles, it is possible to The separation material for leukocyte-derived cells can be brought into contact with the non-human animal-derived leukocyte cell suspension more efficiently, and the separation operation for non-human animal-derived leukocyte cells can be carried out in a short time and in a safe manner. is possible.

Furthermore, the present invention provides a non-human animal in which a carbohydrate that preferentially adsorbs the B lymphocyte fraction in lymphocytes derived from a non-human animal than other lymphocyte fractions is immobilized on the surface of a water-insoluble solid substance. A water-insoluble substance formed from carbohydrate itself as a base material that preferentially adsorbs the M+A fraction of derived leukocyte cells and/or the B lymphocyte fraction of lymphocytes derived from non-human animals over other lymphocyte fractions. A non-human animal-derived leukocyte cell suspension is brought into contact with a non-human animal-derived leukocyte cell suspension consisting of particles, and the granulocytes and monomers in the non-human animal-derived leukocyte cell suspension are brought into contact with the non-human animal-derived leukocyte cell suspension. This is a method for isolating white blood cells derived from non-human animals, which is characterized by selectively capturing cells and B lymphocytes, and separating and recovering a non-captured fraction rich in T lymphocytes. Taking advantage of the excellent properties of animal-derived leukocyte cell isolation materials, it is possible to directly obtain T lymphocyte fractions from non-human animal-derived leukocyte cell suspensions that contain a mixture of various leukocyte cells, and using conventional isolation methods. Compared to other methods, T-lymphocyte fractions can be obtained at extremely high yields without requiring any operational skill, and it is also less expensive.
Since it does not cause any significant cell damage, it can be said to be an extremely useful technique. moreover,
Particularly good separation of T and B lymphocytes can be achieved when using a leukocyte cell suspension derived from a non-human animal in which the presence rate of B lymphocytes is at least 10% of the total lymphocytes. Therefore, the above effects become more pronounced.

In addition, the present invention provides a non-human animal in which a carbohydrate that preferentially adsorbs the B lymphocyte fraction in lymphocytes derived from non-human animals over other lymphocyte fractions is immobilized on the surface of a water-insoluble solid substance. Separation material for animal-derived leukocytes and/or water formed using carbohydrate itself as a base material that preferentially adsorbs the B lymphocyte fraction in lymphocytes derived from non-human animals over other lymphocyte fractions. A non-human animal-derived leukocyte cell suspension is brought into contact with a separation material for non-human animal-derived leukocytes consisting of insoluble particles, and the granulocytes, monocytes, and lymphocytes present in the non-human animal-derived leukocyte cell suspension are isolated. selectively capture the
Since this is a method for isolating leukocyte cells derived from non-human animals, which is characterized by subsequently detaching the captured cells from the separation material and separating and collecting the captured cell fraction, the T lymphocyte fraction can be easily and extremely high. Not only can it be obtained with a high yield, but it also has excellent separation and recovery of adherent lymphocytes, and it is also possible to easily enrich and purify the B lymphocyte fraction, so its applicability is high. The width of the area becomes even more extreme. Furthermore, in this case as well, particularly good TSBS syringe 8 cells can be obtained by using a leukocyte cell suspension derived from a non-human animal in which the presence rate of B lymphocytes is at least 10% of all lymphocytes. In this case, the above effects become even more pronounced.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram of an embodiment of the non-human animal-derived leukocyte cell separator of the present invention in use. DESCRIPTION OF SYMBOLS 1... Column, 2... Separation material for white blood cells derived from non-human animals, 3... Filter, 4... Three-way stopcock, 5... Outlet, 6... Actual diameter portion, 7 ...Reduced diameter section, 8...Test tube.

Claims (7)

[Claims] (1) A non-human material characterized by immobilizing carbohydrates on the surface of a water-insoluble solid substance that preferentially adsorbs the B lymphocyte fraction in lymphocytes derived from non-human animals over other lymphocyte fractions. Separation material for human and animal-derived leukocyte cells. (2) It is characterized by being composed of water-insoluble particles formed using carbohydrate itself as a base material, which preferentially adsorbs the B lymphocyte fraction in lymphocytes derived from non-human animals over other lymphocyte fractions. A material for separating leukocytes derived from non-human animals. (3) The material for separating leukocyte cells derived from non-human animals according to claim 1 or 2, wherein the carbohydrate has a high affinity for monocytes and granulocytes derived from non-human animals. (4) A separator for leukocyte cells derived from non-human animals, comprising a column filled with the separation material according to any one of claims 1 to 3. (5) The separation material according to any one of claims 1 to 3 is brought into contact with a leukocyte cell suspension derived from a non-human animal, and the separation material is provided with a leukocyte cell suspension derived from a non-human animal. selectively captures granulocytes, monocytes and B lymphocytes,
A method for separating leukocyte cells derived from non-human animals, which comprises separating and collecting a non-captured fraction rich in T lymphocytes. (6) The separation material according to any one of claims 1 to 3 is made to selectively capture granulocytes, monocytes, and lymphocytes present in a leukocyte cell suspension derived from a non-human animal, and the captured cells are subsequently released. A method for isolating leukocytes derived from non-human animals, characterized by detaching them from a separation material and separating and collecting a captured cell fraction. (7) The presence rate of B lymphocytes is at least 1 in all lymphocytes
7. The method for separating leukocyte cells derived from a non-human animal according to claim 5 or 6, which uses a leukocyte cell suspension derived from a non-human animal having a concentration of 0% or more.