JP2501500B2 - Granulocyte adsorption carrier and granulocyte removal device - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a carrier for adsorbing granulocytes for selectively removing granulocytes from blood and a device for removing granulocytes using the carrier for adsorbing granulocytes.

[0002]

2. Description of the Related Art Conventionally, as a method for separating granulocytes in blood, a centrifugation method utilizing a difference in specific gravity or a substance for selectively adhering granulocytes, for example, cotton, nylon fiber, polyester fiber or silicone-treated glass wool And the like have been proposed (for example, JP-A-54-4681).
No. 2, JP-A-57-11920).

However, the method utilizing the difference in specific gravity requires a great deal of labor and a long operation for separating the granulocytes, and the lymphocytes tend to be mixed. It is considered that the method using a substance that selectively attaches granulocytes, as compared with the method using the difference in specific gravity, can efficiently adsorb granulocytes by a simple operation. In the method for selectively adsorbing granulocytes as described above, generally, the surface area of the adsorbent is made as large as possible, and thereby the adsorbing ability is enhanced. Therefore, fibers are mainly used as the adsorbent.

However, it is not possible to more selectively adsorb granulocytes by simply increasing the adsorbing area of the adsorbent. In fact, in Japanese Patent Publication No. 58-54126, not only granulocytes but also lymphocytes are adsorbed. Adsorption is described. On the other hand, as disclosed in Japanese Patent Laid-Open No. 1993069/1990, the ratio of the number of granulocytes (G) to the number of lymphocytes (L) in cancer patients, G / L, is a host marker and is used for clinical treatment of cancer. It is considered to be a powerful tool for judging the pathological changes of cancer patients in the field. Further, by selectively removing granulocytes from the blood of cancer patients, the above G /
It has also been suggested that reducing the L ratio has a positive impact on cancer treatment.

In the above-mentioned JP-A-2-193069, a carrier having a higher affinity for granulocytes than lymphocytes as a carrier for adsorbing granulocytes, which can be used for the determination of pathological changes in cancer patients and the treatment of cancer. , Polystyrene, cellulose acetate, nylon, polytrifluoroethylene, polyethylene terephthalate, and the like are mentioned, and it is disclosed that by using these carriers, granulocytes can be selectively adsorbed. . However, for use in the above-mentioned cancer treatment applications, it is desired to introduce a carrier capable of selectively adsorbing granulocytes and a granulocyte removal device.

[0006]

DISCLOSURE OF THE INVENTION The present invention has been made to meet the above-mentioned needs, and an object of the present invention is to provide a granule capable of selectively removing granulocytes from blood more effectively. An object of the present invention is to provide a carrier for adsorbing spheres and an apparatus for removing granulocytes.

[0007]

Means for Solving the Problems The carrier for adsorbing granulocytes of the present invention has a center line average roughness Ra of 0.2 μm to 10 μm on the surface.
μm, and the uneven average spacing Sm value is 5 μm to 2
It is a carrier for adsorbing granulocytes, which has irregularities of 00 μm. Further, the granulocyte removing apparatus of the present invention comprises a granulocyte adsorbing part containing the carrier for adsorbing granulocyte, a blood inflow part for allowing blood to flow into the adsorbing part for granulocyte, and an inflowing part into the adsorbing part for granulocyte. And a blood outflow part for outflowing the collected blood to the outside of the granulocyte adsorption part.

The details of the carrier for adsorbing granulocytes and the apparatus for removing granulocytes according to the present invention will be described below. Progress of the Invention The present inventors have paid attention to the difference in characteristics between lymphocytes and granulocytes in order to adsorb granulocytes more selectively.
That is, unlike lymphocytes, granulocytes are called phagocytic cells or adherent cells and have a specific reaction to foreign substances. Therefore, in order to adsorb more granulocytes, not only increase the contact area but also granules. It was considered that the surface shape of the part in contact with the sphere, that is, the surface state, had a great influence on the adsorption of granulocytes.

Regarding the adsorptivity of neutrophils, which are the main constituents of granulocytes to various materials, it has been reported that the adhesion of neutrophils is remarkable in materials having a contact angle of water of about 70 degrees. (Abstracts of the 19th Symposium on Medical Polymers, pp. 51-52, Society of Polymer Science, 199)
June 11, 0). Therefore, the inventors of the present application have about 60 to 1
Films having four different contact angles within a range of 10 degrees, namely, a cellulose acetate film (hereinafter abbreviated as CA film), a polyethylene terephthalate film (hereinafter abbreviated as PET film), and a polystyrene film (hereinafter referred to as PSt film). Abbreviated) and a perfluoroethylene-propylene copolymer film (hereinafter abbreviated as FEP film) were used to measure the amount of adsorbed leukocytes adsorbed on the film according to the film adsorption test method described below. As a result, as shown in FIG. 2, the same results as those reported in the above-mentioned prior art documents were obtained. That is, the contact angle is 70
From around 0, the adsorbed amount of leukocytes decreased as the contact angle increased.

However, in parallel with the above adsorption test, an adsorption test was carried out on CA films, PET films, and FEP films that were polished to various roughnesses at the same time. , Found to adsorb much more leukocytes. That is, in the case of polishing the above three types of films with sandpaper having a roughness of 1200 mesh or less, that is, in the case of a film polished so that the Ra value described later has a surface roughness of 0.2 μm or more, unpolished It was found that the leukocyte adsorbability was about 4 to 11 times higher than that of the corresponding film.
And, it should be noted that regardless of the contact angle, that is, even in the FEP film that hardly adsorbs leukocytes in a smooth state, the above-mentioned polishing has the same adsorptivity as the polishing film of the CA film or the PET film. Found to show. This means that in a material having an appropriate surface roughness, the increase in leukocyte adsorption capacity is not simply due to the increase in surface area, but the unevenness of the surface is the driving force for leukocyte adsorption. This means that while the size of white blood cells is 10 μm, the Ra value of each film polished by 1200 mesh sandpaper, which has a high white blood cell adsorption capacity, is in the range of 0.6 to 0.7 μm. Since the Ra value is very small with respect to the size of, it is supported by the fact that the contact area is not considered to increase so much in the polishing film.

In order to enhance the leukocyte adsorption property, the inventors of the present invention have a Ra value, which will be described later, in the range of 0.2 to 10 μm, and an uneven average spacing Sm value of 5 μm to 2.
It was found that it is necessary to have unevenness in the range of 00 μm, and the present invention has been completed. What is the Ra value?
It is the center line average roughness according to JIS B0601-1982. Further, the uneven average spacing Sm value is a value defined as follows.

Uneven average spacing Sm value In the current JIS standard, information about the surface roughness in the height direction is specified, but information about the surface direction is not specified. However, the unevenness in the present invention is
The spacing in the surface direction of the unevenness also limits the distance, as will be apparent from the examples described later. Therefore, in the present invention, the range in the surface direction of the concavities and convexities is defined by using the mean value Sm of the spacing.

The average value of the irregular intervals Sm is determined as follows. First, with respect to the center line B of the roughness curve A shown in FIG. 3, the upper count level and the lower count level are drawn at positions of constant height and depth, respectively. Next, between the two points where the lower count level and the roughness curve A intersect, when there is one or more points where the upper count level and the roughness curve intersect, there is a "mountain" as one mountain. Is defined.

The uneven average spacing Sm value is shown in FIG.
, The distance between the peaks between the reference lengths L is Smi.
Is a value defined by the following formula.

[0015]

[Equation 1]

That is, the uneven average spacing Sm value is
The average value of the intervals between the mountains between the reference lengths L is shown. In this way, the condition of the uneven surface direction is defined by the uneven average spacing Sm value. Granule sphere adsorption carrier The inventors of the present invention polished the PET film having an Ra value of 0.2 μm or more with 1200 mesh sandpaper while changing the polishing time to give an Sm value of 47.
As a result of producing films of 4, 374, 213, 101, 56 and 31 μm and conducting a film adsorption test described later, it was found that the total leukocyte adsorption amount sharply increases when the Sm value becomes 200 μm or less. Therefore, in order to enhance the leukocyte adsorption ability, it is necessary that the centerline average roughness Ra value is 0.2 μm or more and the uneven average spacing Sm value is 200 μm or less.

However, although the adsorbability of the total number of white blood cells can be confirmed by the above film adsorption test method, it is difficult to distinguish whether the white blood cells are granulocytes or lymphocytes. Therefore, in order to confirm whether or not the change in the total white blood cell count was due to granulocytes, a bead adsorption test described below was performed. As a result, the beads having a surface roughness within the range of the present invention by polishing markedly increase the adsorptivity of granulocytes as compared with the non-abrasive beads, and the adsorptivity of lymphocytes hardly changes. It was confirmed. From this, in the adsorption carrier having a surface roughness within the scope of the present invention, the adherent cell granulocytes are more effectively adsorbed or adhered to the carrier due to the adhesive property, and the non-adherent cell lymphoid cells are absorbed. Since the surface area of spheres does not increase so much, it is considered that the adsorptivity by polishing did not change so much, and as a result, it is considered that the granule spheres can be selectively adsorbed by using the adsorption carrier according to the present invention. To be

The carrier that can be used in the present invention is one that is harmful to the human body, for example, one that does not elute harmful metals and additives such as plasticizers during extracorporeal circulation.
It can be used regardless of its material. That is,
Synthetic and natural organic polymer materials and inorganic materials such as glass and alumina can be used, and the surface has an unevenness with a centerline average roughness Ra value of 0.2 μm to 10 μm and a bumpy average interval Sm value of 5 μm to 200 μm. The material does not matter as long as it has As synthetic and natural organic polymer materials, cellulose acetate, polystyrene, nylon, polytetrafluoroethylene, polytrifluoroethylene, perfluoroethylene-propylene copolymer,
Examples thereof include polyethylene terephthalate, polyethylene, polyvinyl chloride, acrylic resin, ethyl cellulose and the like.

The shape and size of the carrier for adsorbing granulocytes are also arbitrary, and materials of any shape such as films, fibers and beads can be used, but they are easy to fill in the extracorporeal circulation column and Considering the ease of polishing, a bead-shaped carrier is preferable. The beaded carrier can be manufactured by obtaining a spherical body by a general manufacturing method such as an injection molding method and a suspension polymerization method. The particle diameter of the beads is not particularly limited, but in general, a diameter that increases the contact area, for example, a diameter of about 0.1 to 10 mm can be used, and more preferably 0.2 to It has a diameter of 5 mm.

Further, a polishing method is generally used as a method for imparting surface roughness, but a method having the above specific surface roughness can be prepared by other methods. For example, a method of physically or chemically fixing fine particles on the surface of a carrier or a method of making the surface porous can be used. As a method of fixing the fine particles on the surface of the carrier, for example, 0.1 μm to
Examples include a method of coating fine particles of 20 μm,
Thereby, the granulocytes can be effectively adsorbed. The fine particles used here can be prepared, for example, by emulsion polymerization or suspension polymerization of a vinyl monomer such as styrene or acrylate, or a mixture of two or more components. Desirably, these fine particles are made of, for example, a copolymer with a bifunctional or higher polyfunctional monomer such as divinylbenzene.

Next, a method of coating fine particles will be described. First, 0.1 to 5% by weight of a synthetic polymer or a natural polymer as a binder for coating these fine particles.
Suspend in a solution that has been dissolved. Next, the carrier for adsorbing granule spheres of the present invention can be prepared by immersing a bead, a fiber or a film carrier made of an organic or inorganic material, which has been molded in advance, in this suspension and then drying. it can. In this case, the Ra value can be adjusted by the size of the fine particles, and the Sm value can be adjusted by the concentration of the suspended particles.

Also, in the case of a porous carrier, the shape thereof may be any of film, fiber or bead, and only the surface may be porous, or the entire carrier may be porous. . As for the surface porous carrier, for example, a bead, a fiber or a film carrier made of an organic or inorganic material, a synthetic polymer or a natural polymer as a coating solution is dissolved in a good solvent of about 0.1 to 5% by weight. It is obtained by spraying the thus-obtained liquid into a mist by spraying and heating and drying. As a method for producing the adsorption carrier in which the whole is porous, a general method for producing a porous membrane, porous beads or the like can be used. For example, in the case of a film, a synthetic polymer is dissolved in a good solvent and cast on a glass plate to prepare a casting film,
Then, the porous membrane can be prepared by washing the film with a poor solvent for the synthetic polymer and extracting the good solvent.

The adjustment of Ra value and Sm value in the case of a porous carrier is achieved by adjusting the pore diameter and the pore amount,
The pore diameter and the pore amount can be easily adjusted by changing the type and amount of the solvent that dissolves the carrier. Granulocyte Removal Device Next, the granulocyte removal device of the present invention will be described. The granulocyte removal device of the present invention has a structure in which a blood inflow portion and a blood outflow portion are connected to a granulocyte adsorption portion containing the carrier for adsorbing granulocyte according to claim 1 described above. An example of the structure of the granulocyte removal device will be described with reference to FIG. Reference numeral 1 is a granulocyte adsorption part, in which the surface roughness Ra value is 0.2 to 1
0 μm, uneven average spacing Sm value is 5 μm to 200 μm
The carrier 2 for adsorbing granulocytes is filled. At one end of the granulocyte adsorption unit 1, blood to be treated (patient blood with a high G / L ratio)
Is connected to a blood inflow part 3 for allowing the adsorbent 1 to flow into the adsorbing part 1, and the other end is caused to flow into the granulocyte adsorbing part 1 and contact with the adsorbing carrier 2 A blood outflow portion 4 for connecting the adsorbed and removed blood to the outside of the granulocyte adsorption portion 1 is connected. In addition, the blood inflow part 3
Further, the blood outflow unit 4 may be provided with a filter for the purpose of preventing the outflow of the carrier.

The granulocyte-removing device of the present invention can be used so as to continuously remove granulocytes by extracorporeal circulation and improve the G / L ratio in accordance with ordinary plasmapheresis therapy. For example, a silicone rubber tube, a pipe 11 made of a harmless material such as polyvinyl chloride, and a blood pump 5 can be provided. In addition, the blood outflow section 4
In the circulation circuit of the delivered blood, an arterial pressure gauge 8 and a venous pressure gauge 9 for confirming that blood circulation is normally performed, a drug for preventing blood coagulation, for example, heparin for administration. A drug administration port 6 and a drug administration port 10 for injecting a drug that prevents the action of a blood anticoagulant, for example, protamine, and a warmer 7 for raising the temperature of blood that falls in the extracorporeal circulation are installed. May be. Furthermore, a normal detector for detecting a blood image of circulating blood and a blood component replenisher for compensating for the lack of blood components other than granulocytes due to the present invention can be installed.

[0025]

In the carrier for adsorbing granulocytes of the present invention, since the irregularities in the above-mentioned specific range are provided on the surface, the granulocytes are effectively and selectively adsorbed. This, as mentioned above,
In lymphocytes that are non-adhesive cells, the adsorptivity does not change much depending on the surface roughness, whereas in granulocytes, because they are adherent cells, the adsorptivity changes due to the surface irregularities, and the irregularities in the above specific range are given. This is considered to be because the adsorptivity of granulocytes is remarkably enhanced.

Further, in the granulocyte removing apparatus of the present invention, the granulocyte adsorbing section is constituted by using the granulocyte adsorbing carrier having the irregularities in the above-mentioned specific range on the surface thereof, and therefore, the granulocyte adsorbing portion from the blood is formed. Can be selectively and efficiently separated and removed.

[0027]

EXAMPLES The present invention will be clarified by giving non-limiting examples and comparative examples of the present invention. First, a method for preparing and a test method for the samples used in Examples 1 to 16 and Comparative Examples 1 to 16 described below will be described. (1) Method for producing polishing film (Examples 1 to 12, Comparative Examples 2, 3, 5, 6, 8, 8, 9, 11, 12) A film was prepared as a carrier for adsorbing granulocytes, and the surface of the film was prepared. After washing with methyl alcohol, use Struers (Denmark) -made automatic polishing machine (trade name: Planoball Pedex Max) with 220, 500, 1200, 24
Using sandpapers of 00 and 4000 mesh attached, abrasive films having various surface roughness were prepared. (2) Film adsorption test method (Examples 1 to 12, Comparative Example 1)
-12) The test apparatus 12 made of the polycarbonate resin shown in FIGS. 5 (a) and 5 (b) was used. The test apparatus 12 includes an upper plate 13 and a lower plate 14. Upper plate 13
Is a rectangular plate-shaped polycarbonate resin plate having a thickness of 10 mm, a width of 70 mm, and a length of 220 mm, and has a bottom surface of 10 m.
It has a plurality of circular openings 13a having a diameter of m and a depth of 2 mm. Each opening 13a is connected by a groove 15 having a depth of 1 mm and a width of 2 mm. 20x2 film 16 as a sample
After being cut into 0 mm square and washed with methyl alcohol, it was placed on the lower surface side of the opening 13 a of the upper plate 13.

Next, with the film 16 in contact with the opening 13a, the lower plate 14 of 10 mm thick made of polycarbonate resin is pressure-bonded and fixed through the sheet material 17 made of silicone rubber of 2 mm thick. did. Then, an isotonic phosphate buffer solution (hereinafter abbreviated as PBS. PH =) is provided in the cell formed by the opening 13a of the test apparatus 12.
7.2) was flown for 10 minutes at a flow rate of 1 ml / min, then replaced with 8 ml of heparinized blood of a healthy person, circulated at a flow rate of 1 ml / min using a peristaltic pump, and adsorbed in a water bath at 37 ° C. for 1 hour. The test was conducted.

Thereafter, the film was taken out from the test apparatus 12, washed with the same PBS as above, fixed with alcohol, stained with May-Giemsa stain to prepare a smear film, and adsorbed on the film per unit area by microscopic observation. The white blood cell count was determined. (3) Surface Roughness Measuring Method Center line average roughness Ra value (cutoff value, 0.8 mm for film, described in each Example and Comparative Example,
For beads, 0.08 mm) and the uneven average spacing Sm value are the surface roughness measuring device (trade name; manufactured by Kosaka Laboratory Ltd.).
It was measured with a surf coder SE-30D). (4) Bead Adsorption Experiment Method (Examples 13 and 14 and Comparative Examples 13 and 14) Granule sphere adsorption experiments were carried out in the following manner using beads as the adsorption carrier. First, the beads were immersed in 300 ml of a 5% by weight aqueous solution of a surfactant (trade name; SCAT 20X-N) manufactured by Daiichi Kogyo Co., Ltd. for 30 minutes. Next, the beads were washed with deionized water 5 times, further washed twice with methanol, and then air dried. Furthermore, 3 g of the above beads, 5 ml
A blood component was adsorbed to the beads by adding 2 ml of heparin-collected blood of a healthy person to the beads by shaking and mixing at 37 ° C. for 1 hour.

As a control, 2 ml of blood was similarly added to a 5 ml syringe and the mixture was similarly mixed by shaking at 37 ° C. for 1 hour. Blood after mixing by shaking was collected from a syringe, a smear was prepared, and granulocytes and lymphocytes were calculated. The total white blood cell count was measured by an automatic blood cell analyzer (trade name; Sysmex E-4000) manufactured by Toa Medical Electronics. The number of adsorbed granulocytes was determined from the total white blood cell count, granulocytes and lymphocytes.

Examples 1 to 3 and Comparative Examples 1 to 3 Using FEP films (manufactured by Daikin Industries, Ltd., perfluoroethylene-propylene copolymer film, trade name; NEOFLON (registered trademark)), the above-mentioned polishing films were prepared. According to the method, a film polished in 5 steps was prepared.
Then, after obtaining the Ra value and the Sm value for each of the unpolished film and the film polished in 5 steps,
The film adsorption test of (2) above was performed. The results are shown in Table 1 below.

[0032]

[Table 1]

As is clear from Table 1, the Ra value is 0.7.
When it was 1 μm or more, the amount of leukocytes adsorbed increased rapidly. For example, in the film of Example 1, the leukocyte adsorption amount was 8.92 times that of the unpolished film (Comparative Example 1). Moreover, Ra values are 1.67 μm and 2.94 μm.
Also in the polishing films of Examples 2 and 3,
The leukocyte adsorption amount was 8.12 times and 9.21 times that of the unpolished film, showing high leukocyte adsorption property as in Example 1.

On the other hand, the Sm value of the unpolished film was 246 μm, whereas that of the polished films (Comparative Examples 2 and 3 and Examples 1 to 3) was 20 to 100 μm. Examples 4 to 6 and Comparative Examples 4 to 6 Examples 1 to 6 except that a PET film (polyethylene terephthalate film manufactured by Unitika Ltd., trade name; Embrette S-75) was used instead of the FEP film.
The films of Examples 4 to 6 and Comparative Examples 4 to 6 were prepared in the same manner as in Example 3 and Comparative Examples 1 to 3, and evaluated in the same manner as in Example 1. The results are shown in Table 2.

[0035]

[Table 2]

As is clear from Table 2, Ra value is 0.6.
When it exceeded 1 μm, the leukocyte adsorption amount increased sharply. For example, in the film of Example 4 having a surface roughness Ra value of 0.61 μm obtained by polishing with 1200 mesh sandpaper, the leukocyte adsorption amount was 11. compared to the unpolished film (Comparative Example 4). 1 times, and in the polishing film of Example 5 (Ra value = 1.55 μm) and the polishing film of Example 6 (Ra value = 2.24 μm), the leukocyte adsorption amount of the unpolished film (Comparative Example 4) was increased. 7.
The leukocyte adsorption amount was 74 times and 9.26 times.

Regarding the Sm value, the unpolished film of Comparative Example 4 had a thickness of 475 μm, whereas the other polished films had a Sm value of 30 to 130 μm. Examples 7 to 9 and Comparative Examples 7 to 9 FEP films were CA films (manufactured by Art Plus, cellulose acetate film, trade name; acetyl film VR-).
R), and soxhlet extraction with methyl alcohol instead of washing with methyl alcohol (24 hours)
After extracting the plasticizer and taking out the film, 1
After air-drying for 5 hours and further drying at 80 ° C. for 5 hours, unpolished and polished films were prepared in the same manner as in Examples 1 to 3 and Comparative Examples 1 to 3, and evaluated in the same manner as in Example 1. did. The results are shown in Table 3.

[0038]

[Table 3]

As is apparent from Table 3, the polishing film (Ra) polished with sandpaper of 1200 mesh or less was used.
(Value = 0.58 μm or more), the amount of adsorbed leukocytes increased rapidly. For example, in each polishing film of Example 7 having an Ra value of 0.58 μm, Example 8 having an Ra value of 1.28 μm and Example 9 having an Ra value of 2.37 μm, an unpolished film (comparative example Compared with 7), the amount of leukocytes adsorbed was 3.92 times, 2.36 times, and 2.62 times, respectively.
Doubled.

The Sm value of the unpolished film (Comparative Example 7) was 250 μm, while that of the polished film was in the range of 30 to 130 μm. Examples 10 to 12 and Comparative Examples 10 to 12 Examples 4 to 6 except that 1200 mesh sandpaper was used for polishing, and the polishing time was changed.
Similarly, the surface roughness Ra value is 0.6 μm or more, Sm
Five types of polishing films having values in the range of 30 to 370 μm were prepared (see Table 4). Prepare the above-mentioned five types of polished films and unpolished PET film (Comparative Example 10),
A leukocyte film adsorption test was conducted in the same manner as in Example 1. The results are shown in Table 4 below.

[0041]

[Table 4]

As is clear from Table 4, the Sm value is 200.
It can be seen that the amount of adsorbed leukocytes is very large in Examples 10 to 12 having an Sm value of 200 μm or less, as compared with Comparative Examples 10 to 12 in which the value exceeds μm. That is, in the polished films of Comparative Examples 11 and 12 having Sm values exceeding 200 μm, the leukocyte adsorption amount was not so different from that of the unpolished PET film (Comparative Example 10), while Examples 10 to 12 were used.
The unpolished PET film (Comparative Example 1)
The amount of leukocytes adsorbed was 8 times or more compared with 0).

In Examples 1 to 12 described above, it was confirmed that an excellent leukocyte adsorbing ability was recognized. As described above, since the leukocyte adsorbing ability has a correlation with the granulocyte adsorbing ability, It can be presumed that each of the polishing films of Examples 1 to 12 similarly exhibits a remarkable effect in the adsorptive ability of granulocytes as compared with each of the films of Comparative Examples. Example 13 and Comparative Example 13 Nylon 66 pellets (trade name; manufactured by Ube Industries, Ltd .; Ube 6)
6 2020B) was injection-molded to produce spherical beads having a diameter of 2.5 mm. When the surface roughness of the beads was measured, the Ra value and the Sm value were 0.21 μm, respectively.
m and 294 μm.

Further, the polishing beads were prepared by using a pot mill (manufactured by Toyobo Engineering Co., Ltd., trade name; 5l-ceramic pot mill BP-5), 200 ml of the beads and an abrasive material WHITE ABRAX (WA) # 3 having the same volume.
4 (manufactured by Japan Abrasives Industry Co., Ltd.), and several balls for ceramic pot mills (manufactured by Toyobo Engineering Co., Ltd .; brand name: BB-13) were charged, and a ball polishing machine (pot mill manufactured by Nitto Kagaku Corp., brand name) ; AN-3S) for 5 hours. As a result, Ra value and S
Abrasive beads having m values of 9.1 μm and 124 μm, respectively, were obtained, and the above bead adsorption experiment was conducted using 3 g of each of the polished beads and unpolished beads as an adsorption carrier. The results are shown in Table 5 below.

[0045]

[Table 5]

As is clear from Table 5, the amount of lymphocytes adsorbed on the polished beads (Example 13) was unpolished (Comparative Example 1).
Although not so different from that of 3), the abrasive beads exhibited a granulocyte-adsorbing capacity 4.6 times that of the unpolished beads. Example 14 and Comparative Example 14 Cellulose acetate pellets (manufactured by Art Plus, containing 30% by weight of acetyl triethyl citrate as a plasticizer) were injection-molded to prepare beads having a diameter of 2.3 mm. 50 g of these beads are treated with 300 ml of methanol at 50 ° C. for 2 hours.
Soxhlet extraction was performed for 4 hours to extract the plasticizer. Then, the beads from which the plasticizer was extracted were taken out into a stainless steel vat, air-dried for 15 hours, and further dried at 80 ° C. for 5 hours to give an Ra value of 0.186 μm and an Sm value of 298.7.
μm beads were obtained. Next, in the same manner as in Example 13,
The beads are molded and polished to have an Ra value of 1.36 μm and S
Polished beads having an m value of 97.2 μm (Example 14) were obtained, and the above bead adsorption experiment was performed together with the unpolished beads (Comparative Example 14). The results are shown in Table 6.

[0047]

[Table 6]

As is clear from Table 6, no difference was observed in the lymphocyte adsorbing ability, but the granulocyte adsorbing ability of the polished beads (Example 14) was higher than that of the unpolished beads (Comparative Example 14). 11.5 times. Example 15 and Comparative Example 15 Styrene-divinylbenzene copolymer (copolymerization ratio is 1: 1) fine particles (Sekisui Chemical Co., Ltd.) prepared by suspension polymerization of the CA beads (unpolished beads) obtained in Comparative Example 14. Manufactured by Daicel Chemical Industries, Ltd. (trade name: Linter) in which 2% by weight of cellulose acetate (particle size: 3 μm) was suspended in a 1% by weight solution of methylene chloride-ethanol (9: 1 by weight). Air dried, thereby preparing particulate coated beads (Example 15).

It was confirmed by an electron microscope that the above-mentioned fine particles were coated with cellulose acetate on the surface of the beads. The bead adsorption test described above was performed using 3 g each of the fine particle coated beads (Example 15) and uncoated beads (Comparative Example 15) obtained as described above. The results are shown in Table 7.

[0050]

[Table 7]

As is clear from Table 7, the Ra value of the fine particle coated beads (Example 15) was 2.46 μm.
The m and Sm values were 62.5 μm, and the lymphocyte adsorptivity did not change much as compared with the uncoated beads (Comparative Example 15), but the granulocyte adsorptivity exhibited 14.1 times the performance. Example 16 and Comparative Example 16 Nylon 66 pellets (manufactured by Ube Industries, Ltd.) were injection-molded as a porous carrier used for the adsorption experiment of granulocytes to prepare beads having a particle diameter of 2.3 mm. 4.6 kg of the beads
Then, a 5% by weight methylene chloride / ethanol (9: 1 by weight ratio) solution of cellulose acetate resin (manufactured by Daicel Chemical Industries, Ltd.) was used as a coating liquid, and a flow coater (manufactured by Freund Corporation, trade name; FL0- 5) was sprayed to prepare beads coated with cellulose acetate resin. The coated beads were photographed by a scanning electron microscope and observed. As a result, it was confirmed that surface beads having a coating layer having a thickness of 200 μm were obtained. The coating conditions are as follows.

Spray air pressure; 3.5 kg / cm ² spray liquid temperature; room temperature spray liquid flow rate; 100 ml / min spray temperature; 60 ° C. spray nozzle diameter; 1.2 mm coated beads and uncoated beads obtained as described above The bead adsorption test described above was performed using 3 g. The results are shown in Table 8.

[0053]

[Table 8]

As is clear from Table 8, the coated beads (Example 16) had an Ra value of 1.18 μm and an Sm value of 5.8 μm, and the uncoated beads (Comparative Example 1).
Compared to 6), the adsorptivity of lymphocytes was not so different, but the adsorptivity of granulocytes was 7.3 times as high. Example 17 and Comparative Example 17 A polyester column (manufactured by NBC Industries, trade name; T-No. 70s) was fixed to a polycarbonate column having an inner diameter of 29 mm and a length of 90 mm, and the column was sealed with a polypropylene nut. Filled with CA beads of Example 14 and 0.9 wt% aqueous sodium chloride solution, 120
Autoclave sterilization was carried out at 20 ° C. for 20 minutes to prepare a rabbit extracorporeal circulation column (hereinafter referred to as this column).

Rabbit (Japanese white, female weight about 3 kg, 6
This column was tested for efficacy. That is,
A winged needle is inserted into the ear vein of a rabbit, blood is introduced into this column, and it is perfused to the contralateral ear vein with a peristaltic pump, and the flow rate is set to 1 ml / min. The concentration of granulocytes, lymphocytes, etc. was measured by a hemocyte counter H-1 type manufactured by Technicon. The results are shown in Fig. 6. As a result, the number of granulocytes in the blood at the column inlet was 2406 / mm ³ , and at the column outlet was 588 / m 3.
becomes m ^3, it was about 1/4. For lymphocytes, 3218 / mm ³ to 2501 /
mm ^3, and the remained reduction of approximately 20%.

Also, rabbits of the same type as above (n = 6)
1 x 10 ⁷ cells of Shope's papilloma-derived tumor cells (VX ₂ ) were transplanted subcutaneously on the back of the mouse, and the tumor area was 400 to 500 mm ^2.
When, the extracorporeal circulation was performed, the reflux time was 2 hours, the flow rate was 1 ml / min, and the extracorporeal circulation was repeated.
After the start of extracorporeal circulation, twice a week until the 4th week, then 1 week thereafter
The extracorporeal circulation was performed once, and a circulation test was conducted until 10 weeks later.
Next, the antitumor property was examined. That is, after the tumor transplantation, a rabbit (n = 4) not treated with this column was used as a control, and its progress was also observed. The tumor area (major axis / 2 × minor axis / 2 × π
(Mm ² )).

As shown in FIG. 7, the tumor area in the control group gradually increased after the tumor was transplanted, but the tumor growth inhibitory effect was clearly observed in the column-treated group.

[0058]

As described above, in the carrier for adsorbing granulocytes according to the present invention, since the irregularities in the above specific range are provided on the surface, granulocytes can be adsorbed with high efficiency. Therefore, by using the carrier for adsorbing granulocytes and the device for removing granulocytes of the present invention for cancer treatment and the like by the extracorporeal circulation method, granulocytes can be selectively and efficiently removed from blood. Therefore, by using the carrier for adsorbing granulocytes and the device for removing granulocytes of the present invention, G showing an abnormally high value in cancer patients
The / L ratio can be improved, that is, the G / L ratio can be brought close to the normal range.

The carrier for adsorbing granulocytes and the device for removing granulocytes according to the present invention can be used not only for the extracorporeal circulation method but also for promoting the removal of granulocytes from the collected blood or the reduction of the granulocyte concentration. it can.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram for explaining an example of use of a granulocyte removal device of the present invention.

FIG. 2 is a diagram showing a relationship between various films having different contact angles and leukocyte adsorbability.

FIG. 3 is a diagram for explaining a mountain defining method for obtaining an uneven average spacing Sm value.

FIG. 4 is a diagram for explaining a method for obtaining an uneven average spacing Sm value.

5A and 5B are cross-sectional views for explaining a film adsorption test method, respectively.

FIG. 6 is a graph showing changes in blood leukocyte concentrations due to extracorporeal circulation in rabbits measured in the experiment of Example 17.

FIG. 7 is a diagram showing the change over time in the tumor area measured in Example 17.

[Explanation of symbols]

 1 ... Granulocyte adsorption part 2 ... Granulocyte adsorption carrier 3 ... Blood inflow part 4 ... Blood outflow part

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Claims (2)

(57) [Claims] 1. A center line average roughness Ra value of 0.2 μm to 1
0 μm, and the uneven average spacing Sm value is 5 μm to 20
A carrier for adsorbing granulocytes, which has irregularities in the range of 0 μm on the surface. 2. A granulocyte adsorbing part containing the granulocyte adsorbing carrier according to claim 1, a blood inflow part for allowing blood to flow into the granulocyte adsorbing part, and a granulocyte adsorbing part flowing into the granulocyte adsorbing part. And a blood outflow portion for outflowing blood to the outside of the granulocyte adsorbing portion.