JP5592450B2 - Self-propelled cell trap - Google Patents

Description

  The present invention relates to a self-propelled cell trap.

  As the etiology of inflammatory diseases such as rheumatoid arthritis, Crohn's disease, multiple sclerosis, ulcerative colitis, and systemic lupus erythematosus, the involvement of humoral factors such as inflammatory cytokines is well known. Attempts have been made to treat inflammatory diseases by inactivating these humoral factors with biopharmaceuticals such as low molecular weight drugs and antibodies. However, these humoral factors do not act alone on the inflamed site, but a plurality of humoral factors act synergistically to develop and advance the disease state. For this reason, recently, attempts have been made to treat inflammatory diseases by removing activated leukocytes, which are a source of humoral factors, from the living body.

  As a method of removing activated leukocytes from a living body, a method of extracorporeally circulating blood of an inflammatory disease patient using an extracorporeal circulation column and selectively attaching and removing leukocytes is known. Specifically, a leukocyte removal filter in which a nonwoven fabric is packed and fixed in a column is disclosed (see Patent Document 1). Further, as shown in FIG. 5, a granulocyte adsorption part filled with a granulocyte adsorption carrier 34 such as cellulose acetate having a diameter of about 30 mm and filtering the blood flowing in from the inflow part 27 and outflowing from the outflow part 29 There is disclosed a granulocyte removing device 50 having 45 (see Patent Document 2). Furthermore, an extracorporeal circulation column is disclosed that is capable of simultaneously removing leukocytes and toxins, which is filled with a nonwoven fabric having amino groups on the surface as an adsorbent (see Patent Document 3).

Japanese Patent Publication No. 2-13587 Japanese Patent No. 2501500 JP 2002-113097 A

  When using an extracorporeal circulation column or the like that removes leukocytes from a living body as disclosed in Patent Documents 1 to 3, etc., blood is passed (filtered) through the filter structure of the column using a powerful blood pump. There is a need. Specifically, a large leukocyte removal apparatus including a blood pump installed at the bedside, which is used for hemodialysis of patients with renal failure, is used. For this reason, the activity of the patient is suppressed while the leukocyte removal device is driven, and the patient must spend a long time on the bed. The patient's pain and economic loss due to such activity suppression have not been resolved to date.

  On the other hand, techniques such as filtration and centrifugation have been adopted to separate or concentrate target self-running cells from a treated fluid containing self-running cells such as cultured cells. However, such a method for separating or concentrating self-propelled cells has not always been simple because the apparatus is large-scale or the labor required for operation is great.

  The present invention has been made in view of such problems of the prior art, and the problem is that the pressure loss is small even when a fluid to be treated such as blood or culture fluid is circulated. Without using a powerful large pump or just using a small portable pump, it is possible to easily process the fluid to be processed to separate, concentrate, or remove free-running cells. It is to provide a self-propelled cell trap.

  As a result of intensive studies to achieve the above-mentioned problems, the present inventors fixed at least one end of a self-running cell trapping part having irregularities on the surface thereof inside the container, and the minimum flow path diameter is a predetermined size or more. It was found that the above problem can be achieved by forming the flow path in the container, and the present invention has been completed.

That is, according to the present invention, the following self-propelled cell trap is provided.
[1] A container, and a self-propelled cell trapping part having irregularities on the surface thereof disposed in the container, wherein at least one end of the self-propelled cell trapping part is fixed inside the container. cage, the said container, the self cell that can contact with the fluid to be treated the self cell trapping portion comprising the minimum flow path diameter is formed is 100μm or more channels for circulating therein, said self A self-propelled cell capturing device, wherein the migratory cell capturing unit includes at least one of a fiber having a single fiber degree of 1.0 dtex or less and a particle having a diameter of 10 μm or less .
[2] The self-propelled cell trap according to [1], wherein the minimum flow path diameter of the flow path is 1000 μm or more .
[3] The self-propelled cell trap according to [1] or [2], wherein the shape of the self-propelled cell trapping portion is a thread shape, a rod shape, a sponge shape, a strip shape, a paper piece shape, or a hollow roll shape.
[4] The self-propelled cell capturing device according to any one of [1] to [3], wherein the material of the self-propelled cell capturing unit is a synthetic polymer material or a natural polymer material.
[5] The self-propelled cell trap according to any one of [1] to [4], wherein the overall shape is a tubular shape having an inner diameter of 1 to 20 mm and a total length of 10 cm to 10 m.
[6] The self-propelled cell trap according to any one of [1] to [4], which is a hermetic container having one or more blood outlets.
[7] In any one of the above [1] to [6], the self-running cell is at least one selected from the group consisting of fibroblasts, cultured cells, bone marrow cells, amoeba cells, nerve cells, and leukocytes. The self-propelled cell capturing device described.
[8] The self-propelled cell trap according to any one of [1] to [7], which is used for leukocyte removal therapy or leukocyte removal from collected blood.
[9] The self-propelled cell capturing device according to [8], wherein at least a part of the self-propelled cell capturing unit has antithrombotic properties.

  The self-propelled cell trap of the present invention has a small pressure loss even when a fluid to be treated such as blood or a culture solution is circulated. For this reason, it is possible to easily process the fluid to be processed and separate, concentrate, or remove the free-running cells without using a conventional powerful large pump or using a small pump that is portable. can do. Therefore, the self-propelled cell capture device of the present invention is suitable as a leukocyte removing means incorporated in a portable (portable) leukocyte removing apparatus, or a simple separating / concentrating means such as fibroblasts and cultured cells.

It is sectional drawing which shows typically one Embodiment of the self-propelled cell trap of this invention. It is the elements on larger scale of FIG. It is sectional drawing which shows typically other embodiment of the self-propelled cell trap of this invention. It is a perspective view which shows typically other embodiment of the self-propelled cell trap of this invention. It is sectional drawing which shows typically an example of the conventional granulocyte removal apparatus.

  The self-propelled cell capturing device of the present invention includes a container and a self-propelled cell capturing unit disposed in the container and having irregularities on the surface thereof. And at least one end of the self-running cell trapping part is fixed inside the container. In addition, a channel having a minimum channel diameter of 100 μm or more is formed in the container so that the fluid to be processed including the free-running cells flowing through the container can come into contact with the free-running cell capturing unit. The details will be described below.

  In the self-propelled cell capture device of the present invention, a self-propelled cell capture unit is disposed in the container, and a flow path is formed in the container. For this reason, when a treated fluid such as blood is circulated through the flow path in the container, the free-running cells in the treated fluid that are in contact with the free-running cell trapping portion are selectively captured (trap) by the free-running cell trapping portion. Is done. Therefore, it is possible to easily separate the free-running cells from the treated fluid from which some or most of the free-running cells have been removed.

  In addition, the self-propelled cell capture device of the present invention has a sufficiently small minimum channel diameter of the channel formed in the container, specifically 100 μm or more, preferably 500 μm or more, more preferably 1000 μm or more, particularly preferably. It is 2000 μm or more. That is, the self-propelled cell trap of the present invention does not have a so-called filter structure in which a filtering material such as beads or nonwoven fabric is closely packed in a container. For this reason, the self-propelled cell trap of the present invention can suppress the pressure loss that occurs even when a fluid to be treated such as blood is circulated. Therefore, the self-propelled cell capture device of the present invention can easily circulate the treated fluid and effectively remove, separate, or concentrate the self-propelled cells without using a conventional powerful large pump. it can. Or even if it is a case where a pump is used, a to-be-processed fluid can be processed easily only by using the small pump of a portable grade.

  Here, capture refers to a state in which self-running cells adhere to the free-running cell trapping part and are not easily detached. The self-propelled cell capturing part refers to a part that captures self-propelled cells and does not easily desorb.

  Examples of the shape of the self-propelled cell trapping part include a thread shape, a rod shape, a sponge shape, a strip shape, a paper piece shape, and a hollow roll shape. Sponge refers to a porous structure having moderate flexibility.

  When the shape of the self-propelled cell trapping part is a thread shape, a strip shape, a paper piece shape, or a hollow roll shape, the self-propelled cell trapping part having these shapes can be formed by, for example, a woven fabric or a non-woven fabric.

  Examples of the material for the self-propelled cell trapping part include a synthetic polymer material and a natural polymer material derived from a living body. Specific examples of the synthetic polymer material include: polyurethane; segmented polyurethane; polystyrene; polyether; polyamide; polyolefin such as polyethylene, polypropylene, polypropylene-polyethylene copolymer, polybutylene-polypropylene copolymer; blend or alloy these Can be mentioned. Among these synthetic polymer materials, polystyrene is preferable from the viewpoint of ease of modification of the carrier surface. On the other hand, polypropylene and a polypropylene-polyethylene copolymer are preferred from the viewpoints of heat resistance and shape retention of the nonwoven fabric. Furthermore, when the self-propelled cell trapping part is formed of a nonwoven fabric, it is also preferable to add a skeleton material fiber made of polypropylene or the like to the nonwoven fabric in order to maintain the shape of the self-propelled cell trapping part.

  Specific examples of the natural polymer material include collagen, gelatin, chitin, chitosan, fibroin, keratin, and derivatives thereof.

  The self-propelled cell trapping part preferably includes at least one of fibers having a single fiber degree of 1.0 dtex or less and particles having a diameter of 10 μm or less, and is substantially formed of at least one of these fibers and particles. Is more preferable.

  When the self-running cell trapping part contains fibers, the single fiber degree of the fibers is more preferably 0.8 dtex or less, and particularly preferably 0.5 to 0.05 dtex. Moreover, it is preferable that the diameter (fiber diameter) of a fiber is 0.0001-20 micrometers from a viewpoint of exhibiting the capture | capture (adhesion) ability of a self-propelled cell more effectively. On the other hand, from the viewpoint of further stabilizing the capture (adhesion) performance of capturing self-running cells, it is preferably 0.1 to 20 μm. In addition, it is preferable that it is 1-20 micrometers from a viewpoint of removing a granulocyte more selectively.

  When the fiber diameter of the fiber is less than 10 μm, a fiber having a larger diameter may be mixed from the viewpoint of securing the strength of the self-propelled cell trapping part. The fiber diameter of the large diameter fiber to be mixed is preferably 10 to 50 μm. The fiber may be a short fiber or a long fiber, and may be a hollow fiber.

  The fiber diameter of the fiber is taken from 1000 to 3000 times using a scanning electron microscope for 10 randomly sampled small pieces, and the average of the fiber diameters at 10 locations (100 locations in total) for each photo. Value.

  When the self-propelled cell trapping part contains particles, the diameter of the particles is preferably 0.001 to 10 μm. In addition, it is preferable that it is 0.01-10 micrometers from a viewpoint of removing a granulocyte more selectively.

  For example, polymer particles for immunodiagnosis are preferably used as the particles. By using such polymer particles, it is possible to supplement the self-running cells more effectively. Specific examples of the polymer particles for immunodiagnosis include polystyrene particles, polyethylene particles, natural rubber latex, and the like.

  It is preferable that at least a part of the self-running cell trapping part has antithrombotic properties. The antithrombogenicity refers to a property that does not cause blood coagulation and mainly does not promote adhesion of platelets and / or fibrin. When anti-thrombogenicity is imparted to at least a part of the leukocyte capturing part, for example, when the self-propelled cell capturing device of the present invention is used to remove leukocytes in blood, the amount of anticoagulant administered to the patient is reduced. be able to. In order to provide at least a part of the self-propelled cell trapping part with antithrombogenicity, for example, the self-propelled cell trapping part may be prepared using a substance having antithrombotic properties.

  In addition, when the self-propelled cell capturing device of the present invention is used as a leukocyte removing device for capturing and removing leukocytes in blood, the self-propelled cell is recognized by a recognition molecule having specific binding affinity for leukocytes. It is preferable to modify the surface of the capturing part. Examples of such recognition molecules include E-selectin, antibody, peptide, protein, nucleic acid, sugar chain, glycoprotein, and low molecular weight compound that do not have any functional group of acetyl group, ester bond, and hydroxyl group. Can be mentioned.

  The surface of the free-running cell trapping part can be modified by binding the recognition molecule to the surface of the free-running cell trapping part directly or through an active group. The recognition molecule can be bound to the surface of the self-propelled cell trapping part by a covalent bond, an ionic bond, a van der Waals bond, a hydrogen bond, a hydrophobic bond, a bond by a resultant force thereof, or the like. Among these bonds, a covalent bond or a hydrophobic bond is preferable, and a covalent bond is more preferable from the viewpoint of suppressing peeling of the recognition molecule.

  The container of the self-propelled cell capturing device of the present invention is made of, for example, a general material constituting a medical instrument. Specific examples of the material constituting the container include polymer materials such as vinyl chloride, fluororesin, polyester, polyethylene, polypropylene, polystyrene, acrylic resin, AS resin, polyamide, polycarbonate, ABS resin, and polylactic acid. it can.

  The overall shape of the self-propelled cell capturing device of the present invention is not particularly limited, and examples thereof include a tube shape (tubular shape) and a bag shape. When the overall shape of the self-propelled cell trap is tubular, the inner diameter is preferably 1 to 20 mm, and more preferably 3 to 10 mm. Further, the length is preferably 10 cm to 10 m, and more preferably 50 cm to 5 m. In addition, the capture efficiency of self-propelled cells can be increased as the thickness is reduced and / or lengthened. For this reason, what is necessary is just to change the length and thickness of a self-propelled cell capture tool suitably according to a use aspect. On the other hand, when the overall shape is a bag shape, the container can be a hermetic container such as a blood bag having one or more blood outlets.

  By processing the to-be-processed fluid containing a self-propelled cell using the self-propelled cell capture tool of this invention, a self-propelled cell can be isolate | separated, concentrated, or removed. Examples of self-running cells include fibroblasts, cultured cells, bone marrow cells, amoeba cells, neurons, leukocytes, hepatocytes, endothelial cells, mesothelial cells, cardiomyocytes, smooth muscle cells, testis cells, nerve sheath cells, Examples include glial cells, periosteal cells, osteoblasts, keratinocytes, T cells, intestinal epithelial cells, iPS cells, olfactory cells, retinal cells, oral mucosal cells, islet cells, transgenic cells, stem cells, and hepatocytes. . And as a to-be-processed fluid containing these self-propelled cells, a culture solution, a bone marrow fluid, the feces dilution liquid of an amoeba dysentery patient, blood, etc. can be mentioned, for example.

  For example, the case where the culture solution containing a cultured cell is processed using the self-propelled cell capturing tool of the present invention is assumed. In this case, since the cultured cells contained in the culture solution are captured by the free-running cell trapping part of the free-running cell trapping device, the cultured cell concentrated by peeling and separating the free-running cell trapping part after processing from the container. Cells can be easily removed. Moreover, the case where the feces dilution liquid of an amoeba dysentery patient is processed is assumed. In this case, the amoeba cells contained in the stool dilution are captured by the free-running cell trapping part, and thus the concentrated amoebial cells can be easily taken out by separating and separating the free-running cell trapping part after the treatment from the container. be able to.

  If the self-propelled cell capturing device of the present invention is used, the self-propelled cells in the fluid to be treated circulated in the flow path can be efficiently removed without using a powerful large pump or the like. Moreover, the case where the self-propelled cell capturing device of the present invention is used as a leukocyte removing device for removing leukocytes in blood is assumed. In this case, by circulating blood through the flow path, the number of leukocytes in the flowing out blood is usually 5% or more, preferably 10% or more, more preferably 50% or more, compared with the number of leukocytes in the blood that has flowed in. Particularly preferably, it can be reduced by 90% or more.

  The self-propelled cell capturing device of the present invention can be suitably used for leukocyte removal therapy or leukocyte removal from collected blood. Leukocyte removal therapy refers to a treatment method that treats a disease by temporarily guiding the blood flowing through the patient's body to the outside of the body and then removing the leukocyte and returning the blood to the patient's body. The removal of leukocytes from collected blood refers to a treatment method for removing leukocytes from blood collected for the purpose of blood transfusion or the like.

  In addition, clinical confirmation that the self-propelled cell capturing device of the present invention is actually used for leukocyte removal therapy and the like and exhibits an excellent effect must be performed after obtaining consent from the patient. It is often difficult because it does not become. For this reason, with respect to the leukocyte removal performance of the self-propelled cell capturing device of the present invention, for example, blood guided outside the body of an animal (excluding humans) is processed with the self-propelled cell capturing device of the present invention or collected for blood transfusion. The treated blood can be confirmed by processing with the self-propelled cell capturing device of the present invention.

  Hereinafter, specific embodiments of the self-propelled cell trap of the present invention will be described with reference to the drawings. FIG. 1 is a cross-sectional view schematically showing an embodiment of the self-propelled cell trap of the present invention. FIG. 2 is a partially enlarged view of FIG. As shown in FIGS. 1 and 2, the self-propelled cell capturing device 10 of the present embodiment includes a tubular (tubular) container 2 and a self-propelled cell capturing unit 4 disposed in the container 2. The self-propelled cell trap 4 is made of a non-woven fabric made of ultrafine fibers, for example. For this reason, the surface of the self-running cell trapping part 4 has irregularities. One end of the self-propelled cell trap 4 is fixed inside the container 2. Further, in the container 2, a flow path 6 is formed in which a fluid to be processed flowing through the container 2 can come into contact with the self-propelled cell capturing unit 4. And the minimum flow path diameter of this flow path 6 is 100 micrometers or more. In FIG. 2, a state in which the self-running cells 15 are captured by the self-running cell capturing unit 4 is schematically illustrated.

  In order to fix the one end of the self-propelled cell capturing unit 4 to the inside of the container 2, for example, when the container 2 is formed of a synthetic polymer material such as vinyl chloride, the synthetic polymer material is appropriately dissolved. An organic solvent capable of being applied may be applied to the inside of the container 2, and the self-propelled cell trapping portion 4 formed of a nonwoven fabric may be pressure-bonded to a portion where the organic solvent is applied.

  FIG. 3 is a cross-sectional view schematically showing another embodiment of the self-propelled cell trap of the present invention. The self-propelled cell capturing device 20 of the embodiment shown in FIG. 3 includes a tube-shaped (column-shaped) container 12 and a plurality of thread-shaped (string-shaped) self-propelled cell-capturing units 14 disposed in the container 12. Is provided. The self-propelled cell capturing unit 14 is made of, for example, a nonwoven fabric made of ultrafine fibers. One end portion and the other end portion of the self-propelled cell capturing portion 14 are respectively fixed inside the lid portions 25 and 35 constituting the container 12. The lid portion 25 is formed with an inflow portion 7 for allowing the fluid to be processed to flow into the container 12. Further, the lid portion 35 is formed with an outflow portion 9 through which the treated fluid flows out from the container 12. Further, since the plurality of thread-like (string-like) self-propelled cell trapping portions 14 are stored in the container 12 in a state of keeping an appropriate distance from each other, the fluid to be treated is contained in the container 12 in the self-propelled cells. A flow path 16 that can contact the capturing section 14 is formed.

  FIG. 4 is a perspective view schematically showing still another embodiment of the self-propelled cell trap of the present invention. The self-propelled cell capturing device 30 of the embodiment shown in FIG. 4 includes a tube-shaped (column-shaped) container 22 and a plurality of self-propelled cell-capturing units 24 arranged in the container 22 in a hollow roll shape. . The self-propelled cell trap 24 is made of a nonwoven fabric made of ultrafine fibers, for example. One end and the other end of the self-propelled cell trap 24 are fixed to the inside of the upper lid (not shown) constituting the container 22 and the inside of the lower lid 37, respectively. Note that an inflow portion for allowing the fluid to be processed to flow into the container 22 is formed in the upper lid portion (not shown). Further, the lid portion 37 is formed with an outflow portion 19 through which the processed fluid flows out from the container 22. And the flow path 26 in which a to-be-processed fluid can contact the self-propelled cell capture part 24 is formed in the inside and outside of the several self-propelled cell capture part 24 of a hollow roll shape.

  EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to these Examples. The following “parts” and “%” are based on mass unless otherwise specified.

Example 1
An artificial blood vessel having a thickness of 400 μm made of polyester fiber having a single fiber degree of 0.1 dtex was inserted into a soft vinyl chloride tube having an inner diameter of 5 mm and a length of 1 m, which was coated with tetrahydrofuran (THF) on the inner peripheral surface thereof. Furthermore, a fluororesin rod was inserted into the artificial blood vessel, the artificial blood vessel was pressed against the inner peripheral surface of the soft vinyl chloride tube, and the artificial blood vessel was fixed in the soft vinyl chloride tube to produce a self-propelled cell trap. . In addition, the minimum flow path diameter of the flow path in the produced self-propelled cell trap was 4200 μm. The produced self-propelled cell trap was sterilized with ethylene oxide gas and used for animal experiments. The right femoral artery and left femoral vein of a dog under general anesthesia were exposed, and an arteriovenous shunt was formed using the produced self-propelled cell trap. At this time, no blood pump was used. The dogs used were intravenously administered with 100 U of heparin per 10 kg of body weight to prevent blood clotting. When the shunt experiment was performed continuously for 4 hours, the amount of blood flowing through the inter-arteriovenous shunt was about 20 mL / min. Further, the leukocyte count of blood flowing into the arteriovenous shunt was 9200 / μL, and the leukocyte count of blood flowing out was 4700 / μL. From the above, it was found that about 50% of leukocytes were captured while flowing through the inter-arteriovenous shunt.

After the physiological saline was gently poured into the self-propelled cell trap after completion of the shunt experiment, 10% formalin solution was poured to fix the deposits. Next, a section for an optical microscope was prepared using an artificial blood vessel peeled from the inner peripheral surface of the soft vinyl chloride tube. The prepared sections were stained with hematoxylin and eosin and then observed at 400 times magnification using an optical microscope. As a result, it was found that an average of 160 leukocytes were captured in one visual field (0.15 mm ² ) of the artificial blood vessel wall.

(Example 2)
A cellulose acetate bead with a diameter of 1.5 μm is attached and fixed over the entire inner peripheral surface of a soft vinyl chloride tube having an inner diameter of 5 mm and a length of 1 m, which is coated with THF on the inner peripheral surface, thereby capturing self-propelled cells. A tool was prepared. In addition, the minimum channel diameter of the channel in the produced self-propelled cell trap was about 4800 μm. The produced self-propelled cell trap was sterilized with ethylene oxide gas and used for animal experiments. The right femoral artery and left femoral vein of a dog under general anesthesia were exposed, and an arteriovenous shunt was formed using the produced self-propelled cell trap. At this time, no blood pump was used. The dogs used were intravenously administered with 100 U of heparin per 10 kg of body weight to prevent blood clotting. When the shunt experiment was performed continuously for 4 hours, the amount of blood flowing through the inter-arteriovenous shunt was about 20 mL / min. Further, the leukocyte count of blood flowing into the arteriovenous shunt was 9500 / μL, and the leukocyte count of blood flowing out was 6700 / μL. From the above, it was found that about 30% of white blood cells were captured while flowing through the inter-arteriovenous shunt.

  After the physiological saline was gently poured into the self-propelled cell trap after completion of the shunt experiment, 10% formalin solution was poured to fix the deposits. Subsequently, the internal peripheral surface of the self-propelled cell trap was observed at a magnification of 100 to 3000 using a scanning electron microscope. As a result, it was found that a large number of beads were adhered to the inner peripheral surface of the soft vinyl chloride tube, and countless leukocytes were attached to both the front and back surfaces of each bead. Moreover, using a scanning electron microscope and observing at a magnification of 3000, it was found that an average of 20 leukocytes were attached and captured per bead.

(Example 3)
An artificial blood vessel in which nanofibers of chitosan are sprayed on an artificial blood vessel (thickness 400 μm) made of polyester fiber having a single fiber degree of 1.5 dtex using electrospinning technology, and the nanofibers are entangled between the fibers. Was made. When nanofibers were sprayed, suction was performed using a suction device so that the nanofibers reached the lumen of the artificial blood vessel through the fiber gaps of the polyester fibers. Moreover, when observed and measured with the scanning electron microscope, the fiber diameter of the nanofiber was about 20-200 nm. An artificial blood vessel in which the prepared nanofilaments were entangled between the fibers was inserted into a soft vinyl chloride tube having an inner diameter of 5 mm and a length of 1 m, to which THF was applied on the inner peripheral surface thereof. Furthermore, a fluororesin rod was inserted into the artificial blood vessel, the artificial blood vessel was pressed against the inner peripheral surface of the soft vinyl chloride tube, and the artificial blood vessel was fixed in the soft vinyl chloride tube to produce a self-propelled cell trap. . In addition, the minimum flow path diameter of the flow path in the produced self-propelled cell trap was 4200 μm. The produced self-propelled cell trap was sterilized with ethylene oxide gas and used for animal experiments. The right femoral artery and left femoral vein of a dog under general anesthesia were exposed, and an arteriovenous shunt was formed using the produced self-propelled cell trap. At this time, no blood pump was used. The dogs used were intravenously administered with 100 U of heparin per 10 kg of body weight to prevent blood clotting. When the shunt experiment was performed continuously for 4 hours, the amount of blood flowing through the inter-arteriovenous shunt was about 20 mL / min. In addition, the white blood cell count of blood flowing into the arteriovenous shunt was 11000 / μL, and the white blood cell count of the outflow blood was 3200 / μL. From the above, it was found that about 70% of leukocytes were captured while flowing through the inter-arteriovenous shunt.

After the physiological saline was gently poured into the self-propelled cell trap after completion of the shunt experiment, 10% formalin solution was poured to fix the deposits. Next, a section for an optical microscope was prepared using an artificial blood vessel peeled from the inner peripheral surface of the soft vinyl chloride tube. The prepared sections were stained with hematoxylin and eosin and then observed at 400 times magnification using an optical microscope. As a result, it was found that an average of 180 leukocytes were captured in one visual field (0.15 mm ² ) of the artificial blood vessel wall.

After the physiological saline was gently poured into the self-propelled cell trap after completion of the shunt experiment, 10% formalin solution was poured to fix the deposits. Subsequently, the artificial blood vessel peeled off from the inner peripheral surface of the soft vinyl chloride tube was observed using a scanning electron microscope. As a result, it was found that nanofibers were attached to the polyester fibers, and countless leukocytes were attached to the nanofibers. Moreover, using a scanning electron microscope and observing at a magnification of 3000, it was found that an average of 23 leukocytes adhered and captured in one field of view (1000 μm ² ). Further, when the magnification was further increased and further observation was performed, it was found that a part of the nanofibers had engulfed the leukocytes, that is, the leukocytes phagocytosed the nanofibers. From this, it was proved that the captured white blood cells are less likely to come off than when they are simply attached.

Example 4
Polymer particles with a diameter of 2 μm used for immunodiagnosis using an electrostatic adsorption method are attached to an artificial blood vessel (thickness: 400 μm) with rough stitches produced using a polyester fiber having a single fiber degree of 1.5 dtex. An artificial blood vessel with particles attached was prepared. An artificial blood vessel having the prepared polymer particles attached thereto was inserted into a soft vinyl chloride tube having an inner diameter of 5 mm and a length of 1 m. Furthermore, a fluororesin rod was inserted into the artificial blood vessel, the artificial blood vessel was pressed against the inner peripheral surface of the soft vinyl chloride tube, and the artificial blood vessel was fixed in the soft vinyl chloride tube to produce a self-propelled cell trap. . In addition, the minimum flow path diameter of the flow path in the produced self-propelled cell trap was 4200 μm. The produced self-propelled cell trap was sterilized with ethylene oxide gas and used for animal experiments. The right femoral artery and left femoral vein of a dog under general anesthesia were exposed, and an arteriovenous shunt was formed using the produced self-propelled cell trap. At this time, no blood pump was used. The dogs used were intravenously administered with 100 U of heparin per 10 kg of body weight to prevent blood clotting. When the shunt experiment was performed continuously for 4 hours, the amount of blood flowing through the inter-arteriovenous shunt was about 20 mL / min. Further, the leukocyte count of blood flowing into the arteriovenous shunt was 9500 / μL, and the leukocyte count of blood flowing out was 6200 / μL. From the above, it was found that about 35% of leukocytes were captured while flowing through the arteriovenous shunt.

After the physiological saline was gently poured into the self-propelled cell trap after completion of the shunt experiment, 10% formalin solution was poured to fix the deposits. Next, a section for an optical microscope was prepared using an artificial blood vessel peeled from the inner peripheral surface of the soft vinyl chloride tube. The prepared sections were stained with hematoxylin and eosin and then observed at 400 times magnification using an optical microscope. As a result, it was found that an average of 70 leukocytes were captured in one visual field (0.15 mm ² ) of the artificial blood vessel wall.

After the physiological saline was gently poured into the self-propelled cell trap after completion of the shunt experiment, 10% formalin solution was poured to fix the deposits. Subsequently, the artificial blood vessel peeled off from the inner peripheral surface of the soft vinyl chloride tube was observed using a scanning electron microscope. As a result, it was found that a large number of fine polymer particles were attached to the polyester fiber, and countless leukocytes were attached on the polymer particles. Moreover, using a scanning electron microscope and observing at a magnification of 3000, it was found that an average of 13 leukocytes were attached and captured in one visual field (1000 μm ² ). Further, when the magnification was increased and further observation was performed, it was found that leukocytes were covered with particles, that is, leukocytes were phagocytosing nanofibers. From this, it was proved that the captured white blood cells are less likely to come off than when they are simply attached.

(Example 5)
4,4′-diphenylmethane diisocyanate (MDI), polytetramethylene glycol (PTMG) having a number average molecular weight of 1000, and 1,4-butanediol (1,4-BD) (molar ratio: MDI / PTMG / 1,4- BD = 2/1/1) is polymerized by a prepolymer method in a mixed solvent of N, N-dimethylacetamide (DMAc) and 1,4-dioxane (mass ratio: DMAc / dioxane = 7/3), A segmented polyurethane was obtained. The segmented polyurethane obtained using ethanol was purified by Soxhlet extraction washing for 3 hours. The purified segmented polyurethane was dissolved in 510 parts of a mixed solvent of DMAc and 1,4-dioxane (mass ratio: DMAc / dioxane = 5/5). To this, 2.6 times the mass of segmented polyurethane (mass ratio) and about 50 μm of NaCl crystal as a pore-forming agent were added and mixed well to prepare a dope for artificial blood vessel production (polyurethane concentration: 15%). Prepared. A glass rod having an outer diameter of 4 mm was immersed in the prepared dope and then pulled up, and the glass rod was coated with the dope. The glass rod coated with the dope was immersed in purified water to solidify and precipitate the segmented polyurethane. After standing overnight, the glass rod was pulled out to obtain a segmented polyurethane tubular body. The obtained segmented polyurethane tubular body was thoroughly washed with purified water to remove NaCl and residual solvent. The obtained segmented polyurethane tubular body was a tubular body having an inner diameter of 4 mm, an outer diameter of 5 mm, and a thickness of 400 μm, and a porous structure in which a large number of communication holes communicating from the inner surface to the outer surface were formed.

  The obtained segmented polyurethane tubular body was inserted into a soft vinyl chloride tube having an inner diameter of 5 mm and a length of 1 m, which was coated with THF on the inner peripheral surface thereof. Furthermore, a fluororesin rod is inserted into the artificial blood vessel, the segmented polyurethane tubular body is pressed against the inner peripheral surface of the soft vinyl chloride tube, the artificial blood vessel is fixed in the soft vinyl chloride tube, and a self-propelled cell trap Was made. In addition, the minimum flow path diameter of the flow path in the produced self-propelled cell trap was 4200 μm. The produced self-propelled cell trap was sterilized with ethylene oxide gas and used for animal experiments. The right femoral artery and left femoral vein of a dog under general anesthesia were exposed, and an arteriovenous shunt was formed using the produced self-propelled cell trap. At this time, no blood pump was used. The dogs used were intravenously administered with 100 U of heparin per 10 kg of body weight to prevent blood clotting. When the shunt experiment was performed continuously for 4 hours, the amount of blood flowing through the inter-arteriovenous shunt was about 20 mL / min. Further, the white blood cell count of blood flowing into the arteriovenous shunt was 12000 / μL, and the white blood cell count of the outflow blood was 8200 / μL. From the above, it was found that about 30% of white blood cells were captured while flowing through the inter-arteriovenous shunt.

After the physiological saline was gently poured into the self-propelled cell trap after completion of the shunt experiment, 10% formalin solution was poured to fix the deposits. Subsequently, the segmented polyurethane tubular body peeled off from the inner peripheral surface of the soft vinyl chloride tube was observed using a scanning electron microscope. As a result, it was found that many leukocytes were attached to the segmented polyurethane tubular body. Moreover, using a scanning electron microscope and observing at a magnification of 3000, it was found that an average of 18 leukocytes were attached and captured in one field of view (1000 μm ² ).

(Example 6)
A mixture of 95 parts of the purified segmented polyurethane obtained in Example 5 and 5 parts of dipyridamole was dissolved in 510 parts of a mixed solvent of DMAc and 1,4-dioxane (mass ratio: DMAc / dioxane = 5/5). It was. To this, 2.6 times the mass of segmented polyurethane (mass ratio) and about 50 μm of NaCl crystal as a pore-forming agent were added and mixed well to prepare a dope for artificial blood vessel production (polyurethane concentration: 15%). Prepared. A self-propelled cell trap was produced in the same manner as in Example 5 except that the artificial blood vessel dope thus prepared was used. In addition, the minimum flow path diameter of the flow path in the produced self-propelled cell trap was 4200 μm. The produced self-propelled cell trap has a function of gradually releasing dipyridamole, which is an antiplatelet agent. The produced self-propelled cell trap was sterilized with ethylene oxide gas and used for animal experiments. The right femoral artery and left femoral vein of a dog under general anesthesia were exposed, and an arteriovenous shunt was formed using the produced self-propelled cell trap. At this time, no blood pump was used. The dogs used were intravenously administered with 50 U heparin per 10 kg body weight to prevent blood clotting. When the shunt experiment was continued for 4 hours, no thrombus adhesion to the self-propelled cell trap was observed. From this result, it can be seen that the use of anticoagulant (heparin) can be reduced by imparting antithrombogenicity to the self-propelled cell trap.

(Example 7)
An artificial blood vessel having a thickness of 400 μm made of polyester fiber having a single fiber degree of 1.5 dtex was inserted into a soft vinyl chloride tube having an inner diameter of 5 mm and a length of 1 m, to which THF was applied on the inner peripheral surface. Furthermore, a fluororesin rod was inserted into the artificial blood vessel, the artificial blood vessel was pressed against the inner peripheral surface of the soft vinyl chloride tube, and the artificial blood vessel was fixed in the soft vinyl chloride tube to produce a self-propelled cell trap. . In addition, the minimum flow path diameter of the flow path in the produced self-propelled cell trap was 4200 μm. The produced self-propelled cell trap was sterilized with ethylene oxide gas and used for animal experiments. The right femoral artery and left femoral vein of a dog under general anesthesia were exposed, and an arteriovenous shunt was formed using the produced self-propelled cell trap. At this time, no blood pump was used. The dogs used were intravenously administered with 100 U of heparin per 10 kg of body weight to prevent blood clotting. When the shunt experiment was performed continuously for 4 hours, the amount of blood flowing through the inter-arteriovenous shunt was about 20 mL / min. Further, the leukocyte count of blood flowing into the arteriovenous shunt was 8900 / μL, and the leukocyte count of blood flowing out was 6000 / μL. From the above, it was found that about 30% of white blood cells were captured while flowing through the inter-arteriovenous shunt.

(Reference Example 1)
A blood bag from which 200 mL of dog blood was collected and another blood bag were connected via the self-propelled cell trap produced in Example 1. Blood was introduced into another blood bag through this self-propelled cell trap. The white blood cell count before introduction into the other blood bag was 9200 / μL, and the blood white blood cell count after introduction into the other blood bag was 100 / μL. From the above results, it was found that almost 100% of leukocytes were removed.

(Comparative Example 1)
An arteriovenous shunt was formed in the same manner as in Example 1 except that a soft polyvinyl chloride tube having an inner diameter of 5 mm and a length of 1 m was used as it was as a self-propelled cell trap. At this time, no blood pump was used. The dogs used were intravenously administered with 100 U of heparin per 10 kg of body weight to prevent blood clotting. When the shunt experiment was performed continuously for 4 hours, the amount of blood flowing through the inter-arteriovenous shunt was about 20 mL / min. In addition, the leukocyte count of blood flowing into the arteriovenous shunt was 8900 / μL, and the leukocyte count of blood flowing out was 8700 / μL.

  After the physiological saline was gently poured into the self-propelled cell trap after completion of the shunt experiment, 10% formalin solution was poured to fix the deposits. Subsequently, the inner peripheral surface of the soft vinyl chloride tube was observed at a magnification of 100 to 4000 using a scanning electron microscope. As a result, it was found that most leukocytes were not captured.

  The self-propelled cell capturing device of the present invention is suitable as a leukocyte removing means incorporated into, for example, a portable (portable) leukocyte removing device because the pressure loss is small even when a fluid to be treated such as blood is circulated.

2, 12, 22: Container 4, 14, 24: Self-propelled cell trapping part 6, 16, 26: Channel 7, 27: Inflow part 9, 19, 29: Outflow part 10, 20, 30: Self-propelled cell capture Tool 15: Autonomous cells 25, 35, 37: Lid 34: Granulocyte adsorption carrier 45: Granulocyte adsorption unit 50: Granulocyte removal device

Claims (9)

A container, and a self-propelled cell trapping part having irregularities on the surface thereof disposed in the container,
At least one end of the self-propelled cell trapping part is fixed inside the container,
In the container, a flow path having a minimum flow path diameter of 100 μm or more, in which a fluid to be processed including self-propelled cells flowing through the container can come into contact with the self-propelled cell capturing unit, is formed .
The self-propelled cell trapping unit, wherein the self-propelled cell trapping unit includes at least one of a fiber having a single fiber degree of 1.0 dtex or less and a particle having a diameter of 10 µm or less . The self-propelled cell trap according to claim 1, wherein a minimum flow path diameter of the flow path is 1000 µm or more.   The self-propelled cell trapping device according to claim 1 or 2, wherein the shape of the self-propelled cell trapping part is a thread shape, a rod shape, a sponge shape, a strip shape, a paper piece shape, or a hollow roll shape.   The material of the said self-propelled cell capture part is a synthetic polymer material or a natural polymer material, The self-propelled cell capture device as described in any one of Claims 1-3.   The self-propelled cell trap according to any one of claims 1 to 4, wherein the overall shape is a tubular shape having an inner diameter of 1 to 20 mm and a total length of 10 cm to 10 m.   The self-propelled cell trap according to any one of claims 1 to 4, which is a hermetic container having one or more blood outlets.   The self-propelled cell according to any one of claims 1 to 6, wherein the self-propelled cell is at least one selected from the group consisting of fibroblasts, cultured cells, bone marrow cells, amoeba cells, nerve cells, and leukocytes. Cell trap.   The self-propelled cell trap according to any one of claims 1 to 7, which is used for leukocyte removal therapy or leukocyte removal from collected blood.   The self-propelled cell trap according to claim 8, wherein at least a part of the self-propelled cell trap has antithrombotic properties.

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