JP2007307280A - Apparatus for specific cell adsorption - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus for specific cell adsorption having a high adsorption selectivity of a specific cell even if drawn blood flow rate is low by improving cell adsorption selectivity of a specific cell adsorptive material allowing a water insoluble carrier to retain ligand having affinity to the specific cell. <P>SOLUTION: This specific cell adsorption apparatus is provided with a specific cell adsorber 4 which has a container with a blood inlet and a blood outlet filled with the specific cell adsorptive material allowing the water insoluble carrier to retain ligand having the affinity to the specific cell. The apparatus is used for improving the adsorption selectivity in a method for adsorbing the specific cell. This apparatus is further provided with a feedback conduit 3 and a feedback pump B for feeding back the blood in the blood outlet side of the specific cell adsorber to the inlet side of the specific cell adsorber to increase the blood flow rate flowing the specific cell adsorber more than the flow rate of a blood pump A. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a specific cell adsorption device that specifically or selectively adsorbs specific cells in blood.

  In recent years, research on techniques for adsorbing and removing specific cells in blood as a treatment method for neuroimmune diseases such as multiple sclerosis, myasthenia gravis and Guillain-Barre syndrome has been conducted. Is being developed. This specific cell adsorber is filled with a material (hereinafter referred to as a specific cell adsorbent) in which a ligand having affinity for specific cells is immobilized on the surface of a water-insoluble carrier, and specific cells are selected from various cells. The purpose is to selectively adsorb.

  However, due to the fact that some cells have high adhesiveness and the physicochemical properties of the specific cell adsorbent, the specific cell adsorbent adsorbs cells other than the intended cells, making the specific cell specific. Or it was difficult to selectively adsorb.

Until now, several attempts have been made to selectively adsorb specific cells. For example, Patent Document 1 discloses a method for selectively separating lymphocytes. In Patent Document 1, monocytes and granulocytes are captured from a blood cell suspension using a monocyte and granulocyte capturing filter, and the lymphocytes are collected to collect the lymphocytes remaining in the monocyte and granulocyte capturing filter. It is described that lymphocytes can be obtained with high purity and high recovery rate by a lymphocyte collection method characterized by using a liquid containing at least γ-globulin as a washing liquid for washing off spheres. Similarly, Patent Document 1 connects a blood cell suspension bag, a monocyte / granulocyte capture filter, and a leukocyte capture filter with a conduit, and the blood cell suspension is used as a monocyte / granulocyte capture filter and a leukocyte capture filter. In this order, in the device that discharges the blood cell suspension that has passed through both filters from the discharge tube, a feedback circuit that connects the blood cell suspension discharge tube and the blood cell suspension bag or the blood cell suspension bag side conduit A method of collecting lymphocytes using autologous blood from which at least leukocytes have been removed as a washing solution is described.
However, in the invention of Patent Document 1, cells other than the purpose of collection (monocytes and granulocytes that tend to adhere to fibers) are removed in advance with another filter, and the cells to be collected (monocytes, granulocytes) remaining in this filter Lymphocytes that are less likely to adhere to fibers compared to) are washed with a washing solution, sent to a filter that captures lymphocytes, and this filter captures lymphocytes and collects them to increase the collection rate of target cells. Although it is a technology, a specific cell adsorbent in which a ligand having affinity for specific cells is held on a water-insoluble carrier is used to reduce the number of cells that are non-selectively adsorbed when adsorbing specific cells. It is not a technique that tries to increase the adsorption selectivity. Further, although the device of Patent Document 1 has a feedback circuit, the method of use is that after blood has been flowed through the two types of filters, the blood from which the white blood cells have been removed by the two types of filters is again used for washing. It is used for the purpose of moving the lymphocytes remaining on the monocyte and granulocyte capture filter to the leukocyte capture filter after flowing through the filter of the type, and making the flow rate of blood flowing into the filter larger than the blood flow rate. Thus, there is no purpose to improve the adsorption selectivity of specific cells.

  In Non-Patent Document 1, when blood is flowed to a leukocyte removal column by extracorporeal circulation of blood to remove leukocytes, a part of the blood flowing out from the outlet of the leukocyte removal column is recirculated to the leukocyte removal column, Although a method for suppressing the pressure increase in the inlet side circuit of the leukocyte removal column is disclosed, the leukocyte removal column used in Non-Patent Document 1 is a specification in which a water-insoluble carrier holds a ligand having affinity for specific cells. It is not a column packed with a cell adsorbent, but a column that adsorbs leukocytes to a material that does not hold a ligand by using the adhesion of leukocytes to the material, and suppresses the pressure increase in the inlet side circuit of the leukocyte removal column Although the effect is mentioned, it is intended to adsorb specific cells specifically or selectively, and to further improve the adsorption selectivity. Effects are also listed.

  A specific cell adsorber filled with a specific cell adsorbent containing a ligand having affinity for specific cells on a water-insoluble carrier, one end of the blood collection side conduit connected to the blood inlet of the specific cell adsorber, and one end of the specific cell There has never been an attempt to develop a device for improving adsorption selectivity in a specific cell adsorption device using a device consisting of a blood return side conduit connected to the blood outlet of the adsorber and a blood pump on the blood collection side conduit. It was.

Although the viewpoint is different from the prior art described so far, patients with diseases that require adsorption removal of specific cells are patients who have not undergone superficial vein arterialization (shunt) surgery, such as hemodialysis patients. In most cases, there is a major restriction in the treatment implementation that the blood flow rate cannot be increased. In the case of a hemodialysis patient, a blood flow rate of 200 ml / min or more can be taken, but in the case of a patient with a disease that requires adsorption removal of specific cells, the blood flow rate from the vein is 30-50 ml / min. This is the normal blood flow. Even if it is taken, the limit is 70 ml / min, and it is actually difficult to maintain this for a long time.
JP 55-15448 A Abstracts of 2005 Annual Meeting of the Japanese Society for Apheresis, 196 page “Examination of Recirculating LCAP Method”

  An object of the present invention is to increase the cell adsorption selectivity of a specific cell adsorbent in which a ligand having affinity for specific cells is held on a water-insoluble carrier, and the adsorption selectivity of specific cells is high even when the blood flow rate is low. It is an object to provide a specific cell adsorption device.

  In order to solve the above problems, the present inventors have intensively studied for the purpose of selectively adsorbing specific cells without increasing the blood flow rate, and as a result, the specific cells are used as a water-insoluble carrier in a container having a blood inlet and a blood outlet. Provided with a conduit and a feedback pump that feeds back blood from the blood outlet of the specific cell adsorber filled with a specific cell adsorbent that holds a ligand having affinity to the blood inlet, blood flow in the specific cell adsorber is collected By increasing the flow rate above the flow rate and increasing the linear velocity of blood in the specific cell adsorber, it was surprisingly found that non-selective adsorption is reduced and only specific cells can be selectively adsorbed, and the present invention has been completed. It was.

That is, the present invention includes the following.
(1) A specific cell adsorber in which a container having a blood inlet and a blood outlet is filled with a specific cell adsorbent in which a ligand having affinity for specific cells is held in a water-insoluble carrier, and one end of the blood inlet of the specific cell adsorber A blood collecting side conduit connected to the blood outlet, a blood return side conduit having one end connected to the blood outlet of the specific cell adsorber, and a blood pump on the blood collecting side conduit. Adsorption selection in a method for adsorbing specific cells, flowing into a cell adsorber, adsorbing the specific cells in the blood to the specific cell adsorber, and returning blood flowing out of the specific cell adsorber through the return side conduit A specific cell adsorbing device used for improving the performance, wherein blood on the blood outlet side of the specific cell adsorber is further fed back to the inlet side of the specific cell adsorber. A specific cell adsorbing device comprising a feedback conduit and a feedback pump for making the flow rate of blood flowing into the flow rate larger than that of a blood pump.
(2) Flow rate setting for setting the flow rate of the blood pump and the feedback pump so that the linear velocity of blood in the specific cell adsorber is 1.1 ml / min / cm ² or more and 3.3 ml / min / cm ² or less. The specific cell adsorption apparatus according to (1), comprising means.
(3) The specific cell adsorption device according to (1) or (2), wherein the specific cell is a mononuclear cell.
(4) The specific cell adsorption device according to any one of (1) to (3), wherein an effective filtration length of the specific cell adsorber is 1.0 mm or more and 20.0 mm or less.

  By using the specific cell adsorption apparatus according to the present invention, the cell adsorption selectivity of a specific cell adsorber filled with a specific cell adsorbent in which a ligand having affinity for specific cells is held in a water-insoluble carrier can be significantly increased. it can. In addition, even in the case of a patient whose blood flow rate can only be set low, the specific cell adsorption that can set the linear velocity of the blood in the specific cell adsorber to an optimal value and, as a result, increase the adsorption selectivity of specific cells An apparatus can be provided.

The present invention will be specifically described below.
The specific cell adsorber referred to in the present invention is obtained by filling a water-insoluble carrier with a specific cell adsorbent in which a ligand having affinity for specific cells is held in a container having an inlet and an outlet. By introducing blood from the inlet of the container and bringing it into contact with the specific cell-adsorbing material and collecting the processed blood from the outlet, processed blood from which specific cells have been adsorbed and removed is obtained. The present invention is also useful when blood is introduced into a container and contacted with the specific cell adsorbent, and then the specific cells adsorbed on the specific cell adsorbent are separated and recovered to obtain only specific cells from the blood. Used for. Furthermore, it is also effectively used as a treatment method for selectively adsorbing and removing disease-causing substances in extracorporeal circulation.

The specific cell as used in the present invention refers to a cell population that expresses a specific cell surface antigen and can be identified by the cell surface antigen, and may be any cell as long as it is such a cell. Among them, the immune system mononuclear cells are preferable. Examples of immune system mononuclear cells in blood include blood cells such as lymphocytes, monocytes and hematopoietic stem cells, and helper T cells can be particularly exemplified in the case of treatment of immune diseases caused by excessive immune responses. The type of specific cell useful for disease treatment is selected depending on the disease, and there may be multiple cases. Specific cells useful for disease treatment are, for example, type 1 (Th1 type) among helper T cells such as multiple sclerosis, Crohn's disease and type I diabetes
It is Th1 cells in immune diseases predominating in Th2 cells, and Th2 cells in immune diseases predominate in helper T cell type 2 (Th2 type) such as ulcerative colitis.

  The water-insoluble carrier referred to in the present invention is not particularly limited as long as it is insoluble in water (insoluble in blood) and can stably fix a ligand having affinity for specific cells. Specific examples of the water-insoluble carrier include porous bodies, flat membranes, non-woven fabrics, woven fabrics, and particles. From the viewpoint of selective removal of cells, porous bodies, non-woven fabrics, and particles are preferable. However, non-woven fabric is most preferred from the viewpoint of ligand immobilization technology.

  The material of the water-insoluble carrier is not particularly limited as long as it is a material that can stably fix a ligand and can easily maintain a structure capable of efficiently contacting cells. Any solid can be used as long as it can be processed into various shapes, can directly or indirectly chemically bond a ligand, and can be safely used as a medical material. For example, inorganic materials such as glass, kaolinite, bentonite, natural polymers such as cellulose, dextran, chitin, chitosan, starch, agarose, protein, natural rubber, polystyrene, polyamide, polyester, polyethylene, polyurethane, polyvinyl alcohol, ethylene vinyl alcohol Examples thereof include synthetic polymers such as copolymers, polymethacrylic acid esters, polyvinyl chloride, and polyamino acids, activated carbon, and the like, and materials suitable for the shapes described below may be appropriately selected.

  The shape of the water-insoluble carrier may be any shape as long as it has a large surface area in order to increase the frequency of contact with specific cells, for example, fibrous, cotton-like, cloth-like, thread-like, hollow fiber-like, thread-bundle-like, Examples thereof include fiber structures such as non-woven fabrics, polymer porous bodies such as sponges, beads, and gels. In particular, when blood cells are separated or adsorbed, a woven fabric or a non-woven fabric is preferably used from the viewpoint of adsorption efficiency, and the non-woven fabric is most preferable because the structure of the carrier can be easily controlled.

  Here, when it shows concretely about a nonwoven fabric, as a material of the fiber which comprises a nonwoven fabric, fibers, such as a cellulose, polyester, a polypropylene, are preferably used from the point of intensity | strength, workability, and safety | security. The structure of the nonwoven fabric carrier can be controlled by, for example, appropriately selecting the average fiber diameter of the constituent fibers. For example, a nonwoven fabric composed of fibers having an average fiber diameter of 2.5 μm or more and less than 50 μm is suitable. If the average fiber diameter is less than 2.5 μm, the nonwoven fabric tends to be finer, and nonspecific cell adsorption that physically traps cells other than specific cells tends to occur. On the other hand, when the thickness is 50 μm or more, the eyes tend to be rough, and the contact frequency with a specific cell is remarkably lowered, so that the adsorption efficiency tends to be lowered.

  The ligand as used in the present invention refers to a substance that specifically or selectively binds to a specific cell, and there is no particular limitation on the structure as long as it has affinity for the specific cell. Examples of the type of ligand include antibodies such as monoclonal antibodies and polyclonal antibodies, recombinant antibodies using fragments of these antibodies or antibody fragments, and low molecular compounds, peptides, and aptamers other than antibodies. Antibodies are preferred because of their excellent affinity, and low fraction compounds are preferred because of their low immunogenicity even if they are mixed into the body during extracorporeal circulation from the viewpoint of safety. Further preferred is a recombinant antibody using a monoclonal antibody having a very high selectivity for specific cells or a part thereof.

  The above antibodies are exemplified by their antigen specificity. Anti-CD4 antibody, anti-CD8 antibody (hereinafter the same), anti-CD3, anti-CD2, anti-TCR-Vβ, anti-CXCR3, anti-CCR2, anti-CCR5, anti-CD1a, anti-CD1b , Anti-CD5, anti-CD3R, anti-CD6, anti-CD7, anti-CD9, anti-CD10, anti-CD11a, anti-CD18, anti-CD19, anti-CD20, anti-CD21, anti-CD22, anti-CD23, anti-CD24, anti-CD28, anti-CD37, anti-CD37 CD40, anti-CD72, anti-CD77, anti-CD16, anti-CD32, anti-CD33, anti-CD34, anti-CD35, anti-CD45RO, anti-CD64, anti-CD65, anti-CDw65, anti-CD66b, anti-CD66e, anti-CD89, anti-CDw90, anti-CD56, Anti-CD57, anti-CD94, anti-CD105, anti-CD106, anti-CD46, anti-CD31, anti-C w36, anti-CD41, anti-CD42a, anti-CD42b, anti-CD63, anti-CD11a, anti-CD11b, anti-CD11c, anti-CD18, anti-CD29, anti-CD44, anti-CD45, anti-CD48, anti-CD49a, anti-CD49b, anti-CD49c, anti-CD49d, Anti-CD49e, anti-CD49f, anti-CD50, anti-CD51, anti-CD54, anti-CD58, anti-CD61, anti-CD62E, anti-CD62L, anti-CD62P, anti-CD103, anti-CD26, anti-CD30, anti-CD69, anti-CD70, anti-CD71, anti-CD95 , Anti-CD96, anti-CD25, anti-CD117, anti-CD122, anti-CDw124, anti-CD126, anti-CD127, anti-IL-2R, anti-CD69, anti-CD40L, anti-CD152, anti-CD178, anti-CD183, anti-CD184, anti-CD195, anti-CDw197 , Anti-CD22 6. An antibody such as an anti-HLA-DR antibody can be mentioned, but when used for the treatment of neuroimmune diseases, an anti-CD3 antibody, an anti-CXCR3 antibody, an anti-CD4 that are antibodies against cell surface antigens involved in cellular immunity An antibody is preferably used. Of these, anti-CD4 antibodies against surface antigens of helper T cells having a central function of cellular immunity are most preferably used.

Hereinafter, the case where the ligand is an antibody will be described as an example.
As the immobilized state of the antibody, the antibody may be directly bonded to the surface of the carrier, or may be bonded via a linker. The antibody immobilization site may be any site as long as the antibody can maintain cell affinity after immobilization, but it should be the Fc part of the antibody from the viewpoint of maintaining more stable cell affinity. Is preferred.

Moreover, it is preferable to use a covalent bond for antibody immobilization from the viewpoint of suppressing antibody outflow. As a binding method at the time of immobilization, any known reaction can be used as long as it can maintain the activity of the antibody. For example, it can be favorably bound using a substitution reaction, a condensation reaction, a ring-opening reaction, or the like.
The amount of antibody immobilized on the specific cell-adsorbing material in the present invention varies depending on the number of cells to be adsorbed, the amount of antigen expression on the cell surface, the specificity of the antibody, and the affinity. It is preferable that 0.7 mg / m ² or more is fixed. In addition, when a certain amount or more of the antibody is fixed, the distance between the cells adsorbed to the antibody is too close, and the antibody may not be used effectively, so the range of 0.7 to 5.0 mg / m ² Preferably, 0.7 to 3.0 mg / m ² is most preferable from the viewpoint that the antibody is most easily interacted with the specific cell.

  The linker for immobilizing the antibody on the carrier surface used in the present invention may be any known functional group as long as it has a structure capable of covalently bonding the ligand and the carrier. For example, an α-acetaminohalogen group, N-hydroxymethyldihalopropionamide, etc. in which the carrier surface is activated to be able to bind to a ligand using N-hydroxymethylhaloacetamide, N-hydroxymethyldihaloacetamide, etc. Α, β-propionaminohalogen group, N-hydroxymethyldihaloacetamide, etc. whose carrier surface is activated by using α, α-acetaminodihalogen group, N-hydroxymethyltrihaloacetamide, etc., whose surface is activated using N-hydroxymethyldihaloacetamide, etc. Linker using a haloacetoaminoalkane agent such as α, α, α-acetaminotrihalogen group, etc. whose carrier surface has been activated by using an epoxy group, epoxy group which has activated the carrier using epichlorohydrin, etc. Can be mentioned. Other examples include, but are not limited to, isocyanate groups, isothiocyanate groups, amino groups, carboxyl groups, hydroxyl groups, vinyl groups, and bromocyan linkers. In the present invention, a linker using a haloacetaminoalkane agent is favorably used because it can react under mild conditions while maintaining the function of the ligand.

The amount of linker introduced per carrier is preferably 0.6 nmol / m ² or more because it affects the amount of ligand immobilized. However, since the ligand density at which a certain amount or more is introduced increases too much, it is not effectively used. 0.6 to 250 nmol / m ² is preferable, and 50 to 250 nmol / m ² is most preferable from the viewpoint of stability of ligand fixation.

  Such a cell adsorbent is filled in a container having an inlet of a liquid to be processed and an outlet of a processed liquid by a known method to form a specific cell adsorber. Although the specific cell adsorber is assembled and sterilized, a method of dipping or wetting with a sterile protective solution is preferably used in order to maintain the function of the antibody even after sterilization.

The sterilization protective solution here is a filling solution for maintaining the ligand function of the specific cell adsorbent during sterilization, and is not particularly limited as long as the ligand can be stably retained. Things that can be trapped and antioxidants that prevent oxidation by radicals are used more effectively. Among these, ascorbate which is an antioxidant is more preferably used from the viewpoint of safety. For reasons of solubility, the optimal concentration of ascorbate is preferably 5 mM to 2M. If it is less than 5 mM, the concentration of the ascorbate present in the vicinity of the ligand covalently bonded to the surface of the carrier tends to be low, so that it is difficult to maintain the antibody function after sterilization. On the other hand, if it exceeds 2M, ascorbate tends to precipitate due to the solubility of ascorbate, which is not preferable. Further, 5 mM to 20 mM is preferable from the viewpoint of safety, and 5 mM to 10 mM is most preferably used from the viewpoint of substitution at the time of priming.
The ascorbate may be ascorbic acid or an ascorbate produced by a reaction between ascorbic acid and an alkaline substance.

  The conduit as used in the present invention is a conduit capable of delivering a solution such as blood and / or physiological solution, and any conduit can be used as long as it can be delivered safely to a living body. In addition, from the viewpoint of safety, any material can be used as long as no harmful substances are eluted by a solution such as blood and / or physiological solution. Examples thereof include soft polyvinyl chloride, silicon, polypropylene, and polyethylene. From the viewpoint of strength, soft polyvinyl chloride is preferably used.

  In addition, the pump referred to in the present invention can be used in any form as long as the solution in the conduit can be fed. However, a pump that can maintain a constant flow rate with small pulsation is preferable in order to perform stable feeding during extracorporeal circulation.

  The feedback in the present invention is to introduce blood flowing out from the blood outlet of the specific cell adsorber into the blood inlet of the specific cell adsorber again. The feedback conduit is branched from the vicinity of the blood outlet of the specific cell adsorber. This refers to a conduit connected near the blood inlet of a specific cell adsorber.

  Further, the feedback pump provided on the feedback conduit is for making the flow rate in the specific cell adsorber larger than the blood collection side flow rate, and any of the same pumps can be used. . The flow rate of the feedback pump is not particularly limited, but the flow rate of blood in the specific cell adsorber is related to the structure of the specific cell adsorber, but may be a flow rate that can be set within a preferable range from the viewpoint of adsorption selectivity. It ’s fine.

The linear velocity in the present invention refers to the flow rate per flow area of the specific cell adsorber, and the flow rate here means the sum of the flow rates of the blood pump on the blood collection side conduit and the feedback pump on the feedback conduit. To do. The channel area is defined as the void volume obtained by subtracting the volume of the specific cell adsorbent by dividing the weight of the specific cell adsorbent by the specific gravity from the occupied volume obtained by multiplying the cross-sectional area of the specific cell adsorbent by the filtration length. The value divided by the filtration length of the adsorbent. In order to specifically or selectively adsorb specific cells in blood, the contact time between the ligand having affinity for specific cells and the specific cells is important, and the linear velocity is 1.1 ml / min / cm ² or more. At this time, only specific cells can be adsorbed and removed more specifically or selectively. In addition, in extracorporeal circulation, blood that has passed through the specific cell adsorber is returned to the body, and if the linear velocity is too high, cells may be damaged, which is not preferable. Therefore, from the viewpoint of safe and selective removal of specific cells, the linear velocity is preferably 1.1 ml / min / cm ² or more and 3.3 ml / min / cm ² or less. In addition, when the specific cell adsorber is used ex vivo such as extracorporeal circulation, it is necessary to set the blood pump at a flow rate that can secure blood flow from the peripheral blood vessel, and the blood pump is preferably about 10 to 70 ml / min. In addition, 30 to 50 ml / min is preferable in order to secure a more stable and stable blood flow. Introduced into a particular cell adsorber according to the present invention by exsanguinated patient peripheral blood, through a specific cell adsorber at a linear velocity of ^{1.1ml / min / cm 2 ~3.3ml /} min / cm 2, the patient A method for adsorbing and removing specific cells from blood and then returning the blood to the patient is a method for treating patients with immune diseases such as multiple sclerosis, Crohn's disease, type I diabetes, and ulcerative colitis described above. As possible. The specific cells that are removed from the patient's blood depend on the disease.
The flow rate setting means in the present invention means a means for adjusting the flow rate in the specific cell adsorber. For example, in addition to the flow rate setting mechanism provided in the blood pump or the feedback pump, the flow rate setting means includes a clamp. Also included are conduit opening and closing means.

  The mononuclear cells referred to in the present invention are cells involved in immunity such as lymphocytes, monocytes, and macrophages in blood cells. Among the lymphocytes, helper T cells that play a major role in immune regulation are specifically identified. It is considered that it can be a useful therapeutic means for immune diseases by selective or selective adsorption removal.

  The effective filtration length of the specific cell adsorber refers to the distance of the specific cell adsorbent in the vertical direction through which blood passes from the inlet to the outlet of the specific cell adsorber. If this effective filtration length is too short, the ligand and the target cell The contact frequency and contact time are insufficient, making it difficult to adsorb and remove target cells. Therefore, in order to adsorb and remove the target cells, the effective filtration length is preferably 1.0 mm or more. Further, if the effective filtration length is too long, nonspecific adsorption in which cells other than the target cell are physically adsorbed on the water-insoluble carrier tends to occur. Therefore, it is preferable that the effective filtration length is 1.0 mm or more and 20.0 mm or less in order to specifically and selectively remove target cells in a balanced manner, and 1.0 mm in order to suppress non-specific adsorption. The thickness is most preferably 10.0 mm or less.

[Example]
Examples are shown below, but the present invention is not limited thereto.

[Creation of activated nonwoven fabric]
To a glass flask, 0.6 g of N-hydroxymethyl iodoacetamide, 5.7 ml of concentrated sulfuric acid and 7.2 ml of nitrobenzene were added and dissolved at room temperature with stirring, and 0.036 g of paraformaldehyde was further added and stirred. To this was added 0.3 g of a nonwoven fabric made of polypropylene (average fiber diameter of 3.5 μm, basis weight of 80 g / m ² , thickness of 0.6 mm) and subjected to a light-shielding reaction at room temperature for 24 hours. After reacting for 24 hours, the nonwoven fabric was taken out, washed with ethanol and pure water, and vacuum-dried to obtain an activated nonwoven fabric.
An anti-human CD4 monoclonal antibody solution (hereinafter referred to as “anti-human”) obtained by dissolving 12 sheets of this activated nonwoven fabric cut into a circle having a diameter of 0.68 cm in a phosphate buffer solution (hereinafter referred to as PBS (−)) not containing calcium or magnesium. It was soaked in 1200 μL (48 μg / 1200 μL) at room temperature for 2.5 hours, fixed with a monoclonal antibody (hereinafter referred to as “antibody”), and then washed with 6 ml of PBS (−). Next, 0.2% polyoxyethylene sorbitan monolaurate / PBS (-) solution (hereinafter referred to as "Tween 20 solution") was added and immersed for 2.5 hours at room temperature, then washed with 6 ml of PBS (-), A specific cell adsorbent was obtained.
A container of 1 ml capacity having an inlet and an outlet is filled with 12 sheets of the specific cell adsorbent and a 10 mM ascorbic acid PBS (−) solution (molecular weight 176) as a filling liquid, an effective filtration length is 7.2 mm, and a flow path area is A specific cell adsorbent of 0.3 cm ² was prepared. The calculation method of the channel area is void volume (0.22 cm ³ ) / effective filtration length (0.72 mm). Moreover, the calculation method of void volume is the cross-sectional area of a nonwoven fabric (0.36 cm < ² >) x effective filtration length (0.72 mm)-nonwoven fabric occupation volume (0.0414 cm < ³ >). This specific cell adsorbent was sterilized by irradiation with 25 kGy of γ rays.

[Evaluation of cell adsorption]
Cell adsorbability was evaluated using an apparatus as shown in FIG. When extracorporeal circulation is assumed, the specific cell adsorber is connected with the inside of the conduit filled with physiological saline or the like before the specific cell adsorber is connected to the conduit. The blood pump was set to 0.2 ml / min so that the blood collection flow rate corresponded to 50 ml / min. In FIG. 1, the clamp II provided at the blood inlet of the specific cell adsorber and the clamp III provided on the blood return side conduit are opened and the clamp I provided at the inlet to the feedback conduit is closed. The flow rate of the blood pump A on the blood collection side conduit is set to 0.2 ml / min. When the blood reaches the vicinity of the inlet of the feedback conduit 3, the clamp I is quickly opened, and the flow rate of the feedback pump B is set to 0. It was set to 2 ml / min and operated, and 12 ml of low molecular heparin-added human fresh blood (low molecular heparin 1000 IU / L) was treated with a specific cell adsorption device. Blood was introduced from the blood collection side conduit 1 connected to the blood inlet of the specific cell adsorber. The blood linear velocity in the specific cell adsorber 4 was 1.1 ml / min / cm ² , and the treated blood was collected.
Using a flow cytometer (COULTER EPICS ELITE ESP ASSYNO. 660556078), the adsorbing rate of CD4 positive (CD4 (+)) cell, which is the target cell for collection, and the adhering rate of granulocytes that are highly sticky and difficult to recover are measured using flow cytometry. When measured by the method, the adsorption rate of CD4 (+) cells was 99.2% and the granulocyte adsorption rate was 29.8%, and CD4 (+) cells could be selectively adsorbed.

[Comparative Example 1]
The blood linear velocity in the specific cell adsorber is 0.6 ml / min / cm ^{2 under} the same conditions as in Example 1 except that the feedback pump of Example 1 is not operated and the clamp I is kept closed. A blood pump was set up to process the blood. At this time, the adsorption rate of CD4 (+) cells was 83.7%, and the granulocyte adsorption rate was 98.7%, and CD4 (+) cells could not be selectively adsorbed.

The blood pump flow rate was 0.2 ml / min, the feedback pump flow rate was 0.1 ml / min, and the blood linear velocity in the specific cell adsorber was set to 1.0 ml / min / cm ² . Except that, the same experiment as in Example 1 was performed. At this time, the adsorption rate of CD4 (+) cells was 99.7% and the granulocyte adsorption rate was 45.8%, and CD4 (+) cells could be selectively adsorbed.

The blood pump flow rate was 0.2 ml / min, the feedback pump flow rate was 0.3 ml / min, and the blood linear velocity in the specific cell adsorber was set to 1.5 ml / min / cm ² . Except that, the same experiment as in Example 1 was performed. At this time, the adsorption rate of CD4 (+) cells was 98.7% and the granulocyte adsorption rate was 25.7%, and CD4 (+) cells could be selectively adsorbed.

The blood pump flow rate was 0.2 ml / min, the feedback pump flow rate was 0.8 ml / min, and the blood linear velocity in the specific cell adsorber was set to 3.3 ml / min / cm ² . Except that, the same experiment as in Example 1 was performed. At this time, the adsorption rate of CD4 (+) cells was 93.1%, and the granulocyte adsorption rate was 24.2%, and CD4 (+) cells could be selectively adsorbed.

The blood pump flow rate was 0.2 ml / min, the feedback pump flow rate was 0.9 ml / min, and the blood linear velocity in the specific cell adsorber was set to 3.6 ml / min / cm ² . Except that, the same experiment as in Example 1 was performed. At this time, the adsorption rate of CD4 (+) cells was 90.2%, and the granulocyte adsorption rate was 45.3%, and CD4 (+) cells could be selectively adsorbed.

The results are shown in Table 1.

  Instead of using an anti-human CD4 monoclonal antibody, an anti-human TCRVβ5.2 monoclonal antibody is used, four specific cell adsorbents are filled (effective filtration length is about 4.2 mm), and the treated blood is 4 ml of ACD-A-added human fresh blood (blood : ACD-A = 8: 1) The same experiment as in Example 4 was performed except that the test was performed. At this time, the adsorption rate of TCRVβ5.2 (+) cells was 69.5% and the granulocyte adsorption rate was 7.3%, and TCRVβ5.2 (+) cells could be selectively adsorbed.

  Instead of using an anti-human CD4 monoclonal antibody, an anti-human CD154 monoclonal antibody is used, four specific cell adsorbents are filled (effective filtration length is about 4.2 mm), and the treated blood is 4 ml of ACD-A-added human fresh blood (blood: ACD -A = 8: 1) The same experiment as in Example 4 was performed except that the experiment was performed. At this time, the adsorption rate of CD154 (+) cells was 68.8% and the granulocyte adsorption rate was 8.4%, and CD154 (+) cells could be selectively adsorbed.

The results are shown in Table 2.

  Since the specific cell adsorption device according to the present invention can selectively remove specific cells, it can be usefully used in a treatment for adsorbing and removing specific cells that adversely affect diseases such as autoimmune diseases. In addition, since the blood flow rate from the patient can be reduced, effective treatment can be performed under conditions where the physical burden on the patient is low.

It is a schematic diagram which shows the structural example of this invention specific cell adsorption | suction apparatus.

Explanation of symbols

1: Blood collection side conduit with one end connected to the blood inlet of the specific cell adsorber 2: Blood return side conduit with one end connected to the blood outlet of the specific cell adsorber 3: Blood on the blood outlet side of the specific cell adsorber Feedback conduit for feeding back to the inlet side of the specific cell adsorber 4: Specific cell adsorber A: Blood pump on the blood collection side conduit B: The flow rate of blood flowing into the specific cell adsorber is made larger than the flow rate of the blood pump Feedback pump for
I: Clamp at the inlet to the feedback conduit
II: Clamp provided at blood inlet of specific cell adsorber
III: Clamp provided on the blood return conduit

Claims (4)

A specific cell adsorber in which a vessel having a blood inlet and a blood outlet is filled with a specific cell adsorbent in which a ligand having affinity for specific cells is held in a water-insoluble carrier;
A blood collection side conduit with one end connected to the blood inlet of the specific cell adsorber,
A blood return side conduit with one end connected to the blood outlet of the specific cell adsorber,
And a device consisting of a blood pump on the blood collection side conduit,
The blood is allowed to flow from the blood collection side conduit to the specific cell adsorber,
Adsorb the specific cells in the blood to the specific cell adsorber;
Returning blood flowing out of the specific cell adsorber through the return side conduit;
A specific cell adsorption device used for improving adsorption selectivity in a method of adsorbing specific cells, wherein blood on the blood outlet side of the specific cell adsorber is further added to the inlet side of the specific cell adsorber. A specific cell adsorption apparatus comprising a feedback conduit and a feedback pump for feeding back a flow rate of blood flowing into a specific cell adsorber to be greater than a flow rate of a blood pump. A flow rate setting means for setting the flow rate of the blood pump and the feedback pump so that the linear velocity of blood in the specific cell adsorber is 1.1 ml / min / cm ² or more and 3.3 ml / min / cm ² or less; The specific cell adsorption apparatus according to claim 1.   The specific cell adsorption apparatus according to claim 1, wherein the specific cell is a mononuclear cell. The specific cell adsorption device according to any one of claims 1 to 3, wherein an effective filtration length of the specific cell adsorber is 1.0 mm or more and 20.0 mm or less.