JP2001046844A - Filter for selectively removing white blood corpuscle and coating liquid therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To remove white blood corpuscles with restraining loss of blood platelets in blood by preparing a coating solution by dissolving a hydrophilic polymer in a mixing solvent comprising two or more kinds of organic solvents having different solubilities of hydrophilic polymer and making a filter by using the solution. SOLUTION: A hydrophilic polymer is dissolved in a mixing solution comprising two or more kinds of organic solvents having different solubilities of the hydrophilic polymer so as to prepare a coating solution. At this time, a polymer having a non-ionic hydrophilic group and a basic nitrogen-containing functional group is used as the hydrophilic polymer. In the coating solution, an additive for controlling the viscosity of the solution and the state of dissolution is added. Further, a filter for selective removing white blood corpuscles is made by using the coating solution. By this filter, blood platelets can effectively be removed from a cell-suspending solution represented by blood containing both blood platelets and white blood corpuscles white restraining the loss of the blood platelets.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for removing leukocytes from a cell suspension typified by blood containing both platelets and leukocytes while suppressing loss of platelets. And a coating solution for obtaining a filter for selectively removing leukocytes. Also,
The present invention relates to a filter for selectively removing leukocytes produced using the coating solution.

[0002]

2. Description of the Related Art In recent years, with the development of hematology and immunology, component transfusion, which provides only the blood components required by patients and minimizes unnecessary components, has been attracting attention instead of conventional whole blood transfusion. Is running. Component transfusion includes erythrocyte transfusion, platelet transfusion, plasma transfusion, etc.In some cases, patients who need red blood cells are administered red blood cell concentrates and patients who require platelets are administered platelet concentrates. Many.

[0003] However, various disease cases have also been confirmed in component blood transfusion. For example, patients transfused with platelet concentrate may have non-hemolytic fever, alloimmune, post-transfusion acute lung injury, GVHD (graft-versus-host), allergic, anaphylactic, viral and bacterial infections, It has been reported to exhibit a wide variety of side effects, such as modulation. Many of the causes of such transfusion side effects are thought to be derived from leukocytes mixed in blood products, and it is necessary to remove leukocytes to a level low enough to prevent these side effects.

[0004] Methods of removing leukocytes from blood and blood products are roughly classified into a centrifugal separation method in which a red blood cell and a leukocyte are separated using a specific gravity difference using a centrifugal separator, and a fiber material or a sponge-like structure. There are two types of filter methods that remove leukocytes using a filter that uses as a filter medium.The filter method that adsorbs and removes leukocytes has excellent leukocyte removal ability, is simple in operation, and has low cost. It is widely used because of its advantages.

For example, Japanese Unexamined Patent Publication (Kokai) No. 60-193468 discloses a nonwoven fabric filter which has high leukocyte removal efficiency and can increase the blood processing speed. This nonwoven fabric filter has a specific fiber diameter and bulk density. However, although this filter is excellent in leukocyte removal ability, it cannot be said that the platelet passage rate is sufficient.

On the other hand, Japanese Patent Application Laid-Open No. 55-129755 discloses a method of collecting leukocytes and lymphocytes containing little red blood cells and platelets by using a filter having a fiber surface coated with an antithrombotic material. It has been disclosed. However, although this filter has a high platelet passage rate,
Low leukocyte trapping power and low leukocyte recovery.

Japanese Unexamined Patent Publication (Kokai) No. 60-119955 discloses that a polymer having a basic nitrogen-containing functional group and having a nitrogen content of 0.05 to 3.5% by weight has very low platelet adhesion. are doing. However, the publication does not disclose any behavior of leukocytes to the polymer.

Further, Japanese Patent Publication No. 6-51060 discloses that a filter having a high platelet permeability and an excellent leukocyte removing ability has been studied. Fibers having a group and a basic nitrogen-containing functional group, despite the fact that platelets are less likely to stick
It is described that leukocytes have been found to have the property of being easily adhered, and a leukocyte selective removal filter using a fiber containing a nonionic hydrophilic group and a basic nitrogen-containing functional group in the surrounding surface portion has been provided. I have. The publication describes an experiment in which a selective leukocyte removal filter is used for treating bovine blood. However, as a result of further studies by the present inventors, the filter described in the publication has a high platelet permeability and is excellent in the ability to selectively remove leukocytes when used for treating bovine blood. However, it has been found that when used for processing human blood, there is a problem that the platelet permeability is inferior.

[0009]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for removing leukocytes from a cell suspension typified by blood containing both platelets and leukocytes while suppressing the loss of platelets while reducing the platelet passage rate and leukocyte count. An object of the present invention is to provide a leukocyte selective removal filter having excellent removal performance, and to provide a coating solution suitable for this purpose.

[0010]

The present invention has solved the above-mentioned problems. That is, the present invention provides: (1) a coating solution for a selective leukocyte removal filter, wherein a hydrophilic polymer is dissolved in a mixed solvent comprising two or more organic solvents having different solubilities of the hydrophilic polymer. (2) The coating solution for a filter for selectively removing leukocytes according to claim 1, wherein the hydrophilic polymer is a polymer having a nonionic hydrophilic group and a basic nitrogen-containing functional group. (3) The coating solution for a leukocyte selective removal filter according to claim 1 or 2, further comprising an additive for controlling a solution viscosity and a dissolution state. (4) A filter for selectively removing leukocytes produced using the coating solution according to any one of claims 1 to 3.

According to the invention described in Japanese Patent Publication No. 6-51060, a polymer having a nonionic hydrophilic group and a basic nitrogen-containing functional group is dissolved in an organic solvent when producing a filter for selectively removing leukocytes. However, the organic solvent is methanol or ethanol alone, and is not a mixture of two or more organic solvents as in the present invention. In the present invention, by mixing and using two or more kinds of organic solvents, an unexpected effect that platelet permeability is excellent even when used for processing human blood has been found, and the above-mentioned problem has been solved. is there.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The coating solution of the present invention will be described. The coating solution of the present invention suitable for obtaining a selective leukocyte removal filter having an excellent balance between the platelet passage rate and the leukocyte removal performance comprises a hydrophilic polymer and two or more organic solvents.

The hydrophilic polymer used in the present invention is not particularly limited as long as it swells in water but does not dissolve in water, and is not particularly limited, but includes a sulfonic acid group, a carboxyl group, a carbonyl group, an amino group, and an amide group. , A cyano group, a hydroxyl group, a methoxy group, a phosphoric acid group, an oxyethylene group, an imino group, an imide group, an imino ether group, a pyridine group, a pyrrolidone group, an imidazole group, a polymer having one or more kinds of quaternary ammonium groups, etc. Examples can be given.

Among them, a copolymer having a nonionic hydrophilic group and a basic nitrogen-containing functional group is preferable in terms of non-adsorption of platelets. In the present invention, examples of the nonionic hydrophilic group include a hydroxyl group and an amide group.

The basic nitrogen-containing functional groups include primary amino groups, secondary amino groups, tertiary amino groups, quaternary ammonium groups, and nitrogen-containing aromatic groups such as pyridine groups and imidazole groups. Is mentioned.

In the present invention, the basic nitrogen atom means a nitrogen atom in the basic nitrogen-containing functional group, and the content of the basic nitrogen atom is preferably 0.05% by weight to 4.0% by weight, more preferably 0.25% to 1.5% by weight. If the content of the basic nitrogen-containing atom is less than 0.05% by weight, the white blood cells are hardly adhered together with the platelets, so that there is a tendency that the selective removal of the white blood cells cannot be performed. On the other hand, if the content of the basic nitrogen atom exceeds 4.0% by weight, both platelets and leukocytes tend to adhere, and also in this case, there is a tendency that selective leukocyte removal cannot be performed.

In the present invention, the amount of the nonionic hydrophilic group is preferably at least equimolar to the amount of the basic nitrogen atom as a hydroxyl group, an amide group or an oxyethylene group, more preferably It is 3 times or more. The amount of basic nitrogen-containing functional groups and nonionic hydrophilic groups,
The content of the basic nitrogen atom can be measured by a known method such as infrared absorption spectroscopy using a multiple total reflection infrared spectrometer, elemental analysis, and nuclear magnetic resonance spectroscopy.

The weight average molecular weight of the hydrophilic polymer used is preferably in the range of 1,000 to 3,000,000, and more preferably in the range of 10,000 to 1,000,000. If the weight average molecular weight is less than 1,000, monomers and oligomers may be eluted into the blood to be filtered, and if the weight average molecular weight exceeds 3,000,000, the solubility tends to be poor.

The organic solvent used for dissolving the hydrophilic polymer is, for example, methanol, ethanol, n (normal)-, if the hydrophilic polymer is a copolymer of 2-hydroxyethyl methacrylate and dimethylaminoethyl methacrylate. Examples thereof include alcohols such as propyl alcohol, iso-propyl alcohol, n-butyl alcohol, iso-butyl alcohol, and t (tert-butyl) alcohol, and N, N-dimethylformamide.

The mixed solvent composed of two or more organic solvents is a mixture of two or more solvents selected from the above organic solvents. In the present invention, two types having different solubilities are used.
By using a mixed solvent composed of more than two kinds of organic solvents, it is possible to suppress adhesion of platelets while maintaining high leukocyte removal performance. The composition ratio of the solvent having the lowest solubility among the organic solvents constituting the mixed solvent is 0.1% by weight or more.
It is preferably 50% by weight, more preferably 1% to less than 30% by weight. If the composition ratio of the solvent having the lowest solubility is less than 0.1% by weight or more than 50% by weight, the adhesion of platelets tends to increase. Here, the solubility means the saturated concentration of the hydrophilic polymer in the solvent at 40 ° C.

The above

Among the organic solvents listed above, alcohols belong to the one having a low solubility in a copolymer composed of 2-hydroxyethyl methacrylate and dimethylaminoethyl methacrylate, and N, H-dimethylformamide has a high solubility. However, among alcohols, there is a difference in solubility depending on the type of alcohol.

The additive may be any as long as it is compatible with the solvent and does not dissolve the hydrophilic polymer, and is used for the purpose of controlling the solution viscosity and the dissolved state. For example, when the hydrophilic polymer is a copolymer composed of 2-hydroxyethyl methacrylate and dimethylaminoethyl methacrylate, water; salts such as sodium chloride, potassium chloride, and calcium chloride; hydrochloric acid, phosphoric acid, boric acid, and oxalic acid Acids such as acids; alkalis such as sodium hydroxide, potassium hydroxide and calcium hydroxide; ketones such as acetone and methyl ethyl ketone; glycerin; diethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol (weight average molecular weight 200 to 35,000) )
Glycols, etc .; and polyvinylpyrrolidone (weight average molecular weight 1,000 to 2,800,000), etc., and a plurality of kinds can be added, and the kind and the addition amount may be selected as needed.

The concentration of the hydrophilic polymer in the coating solution is not particularly limited as long as it is within the range of a coatable concentration, but is preferably from 0.1% by weight to 30% by weight, more preferably from 0.5% by weight to 20% by weight. . If it is less than 0.1% by weight, the thickness of the coat layer tends to be uneven, and
If it is higher, the coating solution tends to be difficult because the viscosity of the coating solution is too high.

The amount of the additive in the coating solution is 0.01
It is preferably from 40% by weight to 40% by weight, more preferably from 0.1% by weight to 20% by weight. The optimum concentration is determined depending on the type of additive used.

The coating solution of the present invention is prepared by putting a solvent and a hydrophilic polymer in a temperature-controllable container and dissolving the mixture with a mixer such as a stirrer. When the additive is mixed, a method of dissolving the hydrophilic polymer after dissolving the solvent and the additive in advance, or dissolving the additive after dissolving the hydrophilic polymer in the solvent is considered, Either is acceptable.

Hereinafter, an example of a method for manufacturing a leukocyte selective removal filter will be described. The leukocyte selective removal filter is obtained by coating the coating solution of the present invention on a filter substrate, or immersing the filter substrate in the coating solution, and then mechanically compressing, gravity, centrifuging, blowing off by gas, or vacuum suction. It is manufactured by removing the excess solution from the filter substrate by, for example, drying and drying, or by immersing in a non-solvent to remove the solvent and then drying. Further, after the filter base material is filled in a container in advance, coating can be performed by passing or dipping the coating solution in the container.
The leukocyte selective removal filter thus obtained is
For example, when the filter substrate is made of fibers, the surface of each fiber is covered with a hydrophilic polymer, and each fiber has a structure in which the coating layer made of the hydrophilic polymer is bonded to each fiber.

Before coating, the surface of the filter substrate is irradiated with γ (gamma) rays, corona discharge, plasma treatment, or a chemical to improve the adhesion between the hydrophilic polymer and the filter substrate. Oxidation is also effective. In addition, heat treatment in gas or liquid after coating enhances the adhesion between the filter substrate and the hydrophilic polymer, or causes a cross-linking reaction in the polymer material to stabilize the coating layer. You can also. The coating may be performed at the same time as the filter base material is formed, or may be performed after the formation. Also, spinning the coating solution of the present invention, or
By casting, a woven fabric, a nonwoven fabric, a porous body or the like can be formed.

As a method for coating the coating solution on the filter substrate, there are two methods: a coating method for coating the filter substrate in excess of a desired coating amount, and then reducing the coating amount to a specified coating amount; Before coating, there is a pre-measurement method of transferring a coating solution that has been measured to a desired coating amount in advance to a filter substrate,
Either is acceptable. It is also possible to combine a plurality of coating methods exemplified below as the post-metering method and the pre-metering method and a plurality of coating heads used in these coating methods.

As the coating method of the post-metering method, an air doctor coater (air doctor coater), a blade coater (b
lade coater), rod coater (rod coater), squeeze coater (sqeeze coater), knife coater (knife coate)
r), impregnation coater and the like.

The coating method of the pre-measurement method includes a reverse roll coater, a transfer roll coater, a gravure coater, and a kiss roll coater (kis roll coater).
sroll coater), cast coater, spray coater, curtain coater
ater), a calendar coater, an extrusion coater and the like.

As a drying method after coating, a method of drying in a dry gas or a reduced-pressure atmosphere at room temperature or while heating is used. The drying temperature is
0 ° C to 200 ° C, preferably 10 ° C to 150 ° C, more preferably 20 ° C to 80 ° C. If the drying temperature is lower than 0 ° C., it takes a long time for the drying, and it is not preferable.

Examples of the non-solvent used for desolvation include, for example,
If the hydrophilic polymer is a copolymer comprising 2-hydroxyethyl methacrylate and dimethylaminoethyl methacrylate, water; ethers; n-hexane, n-hexane
A liquid that does not dissolve a polymer such as an aliphatic hydrocarbon such as heptane is used, but water is preferably used.
It is also possible to control the desolvation rate by adding the good solvent to the non-solvent.

The temperature of the non-solvent is from -30 ° C to 90 ° C, preferably from 0 ° C to 90 ° C, more preferably from 0 ° C to 80 ° C. When the temperature of the non-solvent exceeds 90 ° C. or is lower than −30 ° C., the shape of the coating surface in the non-solvent is difficult to stabilize.

The filter substrate used in the production of the filter for selectively removing leukocytes may be a nonwoven fabric prepared by a melt blow method, a flash spinning method, a papermaking method, or the like, as well as paper, woven fabric, mesh and porous material. Although any form of a known filter material may be used, a nonwoven fabric is a particularly preferable form. Here, the nonwoven fabric refers to a cloth-like material in which an aggregate of fibers or yarns is chemically, thermally, or mechanically connected irrespective of knitting or weaving.

Examples of the fiber material include synthetic fibers such as polyamide, aromatic polyamide, polyester, polyacrylonitrile, polytrifluorochloroethylene, polystyrene, polymethyl methacrylate, polyethylene, and polypropylene, and regenerated fibers such as cellulose and cellulose acetate. Can be exemplified.

In the case of a filter substrate composed of a nonwoven fabric and a woven fabric, the average fiber diameter is 0.3 μm to 10 μm, preferably 0.3 μm to 3 μm, more preferably 0.5 μm to 1.
8 μm. If the average fiber diameter is less than 0.3 μm, the pressure loss when filtering whole blood etc. may be too high and impractical.If it exceeds 10 μm, the probability of contact between the fiber and leukocytes is too low. This is because the effect of the present invention may not be sufficiently exhibited.

Here, the average fiber diameter is an average diameter obtained by sampling a part of a filter element from a non-woven fabric or a woven fabric constituting a filter and measuring it by an electron micrograph.

The porosity of the filter substrate made of a nonwoven fabric and a woven fabric is preferably 50% or more and less than 95%, more preferably 70% or more and less than 90%. If it is less than 50%, the flow of the leukocyte-containing liquid such as blood is poor, and if it is more than 95%, the mechanical strength of the filter base material is weak, which is not suitable.
The porosity is measured by measuring the dry weight (W1) of the filter substrate cut into a predetermined area, and further measuring the thickness to calculate the volume (V). The filter substrate is immersed in pure water, degassed, and the weight (W2) of the hydrated filter substrate is measured. From these values, the porosity is determined by the following calculation formula. Note that ρ in the following formula is the density of pure water. Porosity (%) = (W2-W1) × ρ × 100 / V

Examples of the polymer porous material include polyvinyl formal, polyacrylonitrile, polysulfone, cellulose, cellulose acetate, polyurethane, polyvinyl acetal, polyester, polyamide, polyimide, polyetherimide, polymethacrylate, polyvinylidene fluoride and the like. be able to.

The porous polymer has an average pore diameter of 1 μm or less.
60 μm, preferably 1 μm to 30 μm, more preferably 1 μm
μm to 20 μm. If it is less than 1 μm, the flow of a leukocyte-containing liquid such as blood is poor. The average pore diameter here means POROFIL (COULTER ELECTRONICS LT) according to the air flow method described in ASTM F316-86.
D.) indicates an average pore diameter measured in a liquid.

The thickness of the coating layer made of a hydrophilic polymer from the surface of the filter substrate is preferably from 10 to 1,000, more preferably from 40 to 400. For example,
When the filter substrate is made of fiber, if the average thickness of the coating layer is less than 10 mm, it is difficult to completely cover the surrounding surface of the fiber with the hydrophilic polymer, and it is necessary to suppress the loss of platelets and selectively remove leukocytes. Becomes difficult. 1,000
If the average thickness of the coating layer exceeds Å, it is not preferable if the mechanical strength of the hydrophilic polymer is low because the peripheral portion of the coating layer may peel off and enter the blood to be filtered.

The filter for selectively removing leukocytes obtained by using the coating solution of the present invention is usually used by filling it into a suitable filter container for blood filtration having a well-known blood inlet and outlet.

The filling density of the filter at the time of filling varies depending on the type of the filter base material before coating,
It is preferably ^{g / cm 3 ~0.7g / cm 3} , 0.15g / cm 3 ~
More preferably, it is 0.38 g / cm ³ . If it is less than 0.02 g / cm ³ , the mechanical density is insufficient, and the blood may be deformed at the time of blood filtration. If it exceeds 0.7 g / cm ³ , the resilience of the filter is too high and it is difficult to fill the inside of the container, which is not preferable. Here, the packing density of the leukocyte selective removal filter is a value obtained by dividing the weight of the filter with respect to the filtration cross-sectional area in the state of being contained in the container by (effective filtration area × thickness) of the filter.

When the filter substrate is a woven or non-woven fabric, it may be used alone depending on the thickness thereof,
A plurality of sheets may be stacked and used. The number of sheets to be overlapped depends on the conditions of blood filtration and is not critical, but usually several to several tens of sheets are used.

Examples of the present invention will be described below, but the present invention is not limited to these examples.

Example 1 2-hydroxyethyl methacrylate 97
5.96% by weight of a copolymer consisting of 3% by mole of dimethylaminoethyl methacrylate and 3% by mole of dimethylaminoethyl methacrylate is dissolved at 40 ° C. in a mixed solvent of 4.69% by weight of iso-propyl alcohol and 89.35% by weight of ethanol at 40 ° C. and homogeneously. Solution. pH was 7.6.

A non-woven fabric made of polyethylene terephthalate fiber having an average fiber diameter of 1.2 μm (40 g / m ² basis weight, porosity 79
%) Is cut into a circle having a diameter of 25 mm, immersed in the above-mentioned solution at 40 ° C for 1 minute, and then set in a filter holder four times, and vacuum-suction the excess solution absorbed by the nonwoven fabric for 4.5 minutes. To remove. After taking it out of the holder, it was vacuum dried at 25 ° C. for 16 hours. The remaining amount of iso-propanol and ethanol in the obtained filter was 1 ppm or less.

The four sheets thus coated were set in a filter holder (manufactured by Shibata Scientific Instruments Co., Ltd.) (filling density of filter: 0.16 g / cm ³ ), and ACD (acid- citrate-dextrose) was added to a human 1-day-preserved blood (6 ml) using a syringe pump (2.7 ml).
It flowed at room temperature at a constant speed of / min.

Before and after passing through the nonwoven fabric, a fixed amount of blood was collected, diluted with a Turck's solution, and the leukocyte concentration was measured using a hemocytometer. The concentration of platelets in blood was measured with a multi-item automatic blood cell counter (K-4500, manufactured by SYSMEX) using Stomalizer 3 WP (manufactured by Toa Medical Electronics Co., Ltd.) as a diluent. The leukocyte removal rate and platelet passage rate were calculated by the following equations.

[0049]

(Equation 1)

[0050]

(Equation 2)

Table 1 shows the leukocyte removal rate and platelet passage rate at this time. High results were obtained for both leukocyte removal ability and platelet passage rate.

[0052]

Example 2 The same procedure as in Example 1 was carried out except that sodium hydroxide powder (concentration of sodium hydroxide in the solution was 0.02% by weight) was added to the homogeneous solution to adjust the pH to 13. The same operation as in Example 1 was performed. Table 1 shows the results. High results were obtained for both leukocyte removal ability and platelet passage rate.

[0053]

Example 3 5.96% by weight of the copolymer used in Example 1 was n-
4.69% by weight of butyl alcohol and 89.35% by weight of ethanol
The same operation as in Example 1 was performed except that a homogeneous solution (pH 7.6) dissolved at 40 ° C. in a mixed solvent consisting of was used. Table 1 shows the results. High results were obtained for both leukocyte removal ability and platelet passage rate.

[0054]

Comparative Example 1 The same operation as in Example 1 was carried out except that iso-propyl alcohol was used alone as a solvent. Table 1 shows the results.

[0055]

Comparative Example 2 The same operation as in Example 1 was performed except that N, N-dimethylformamide was used alone as a solvent. Table 1 shows the results.

[0056]

Comparative Example 3 The same operation as in Example 1 was performed except that ethanol was used alone as a solvent. Table 1 shows the results.

[0057]

[Table 1]

[0058]

The selective leukocyte removal filter obtained from the coating solution of the present invention removes leukocytes from a cell suspension typified by blood containing both platelets and leukocytes while suppressing the loss of platelets, Since it has excellent platelet passage rate and excellent leukocyte removal performance, it can be used for medical uses, medical uses, and general industrial uses.

Continued on the front page F term (reference) 4D006 GA02 KE02P MA03 MA07 MA22 MA24 MA40 MB06 MC11 MC18 MC22 MC23 MC24 MC28 MC29 MC32 MC37X MC39 MC48X MC53 MC54 MC58 MC59 MC62 MC71 MC77 MC78 MC85 MC88 NA46 NA64 PB09 PB45 PB46 PC41

Claims (4)

[Claims] 1. A coating solution for a leukocyte selective removal filter, wherein a hydrophilic polymer is dissolved in a mixed solvent comprising two or more organic solvents having different solubilities of the hydrophilic polymer. 2. The coating solution for a leukocyte selective removal filter according to claim 1, wherein the hydrophilic polymer is a polymer having a nonionic hydrophilic group and a basic nitrogen-containing functional group. 3. The coating solution for a leukocyte selective removal filter according to claim 1, further comprising an additive for controlling a solution viscosity and a dissolution state. 4. A filter for selectively removing leukocytes produced using the coating solution according to claim 1.