JPH0780062A - Endotoxin removing device and production of purified blood - Google Patents

Abstract

PURPOSE:To provide the removing device for indirectly removing endotoxin and/or cytokine occurring from the endotoxin from blood by removing white blood cell components, such as granulocytes and monocytes and the process for production of the purified blood by using the removing device. CONSTITUTION:This removing device contains a granulocyte and/or monocyte capturing material having 30 to 90% pore volumetric rate of pores of pore sizes of 1 to 100mum, removes the endotoxin and/or the cytokine occurring from the endotoxin from the endotoxin-contg. blood by sampling the granulocytes and/or monocytes.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a technique for removing endotoxin in the blood and / or cytokine derived from endotoxin.

[0002]

BACKGROUND OF THE INVENTION Endotoxin exhibits various biological activities in blood, such as fever, blood pressure lowering, intravascular coagulation, and activation of Hageman factor. Particularly in the clinical setting, serious malignant symptoms are induced by endotoxin mixed in the blood of the patient after surgery, for example. For the purpose of treating or preventing this symptom, an endotoxin adsorbent in which polymyxin B having a binding property with endotoxin is covalently bonded to a polypropylene non-woven fabric is known. This adsorbent is intended to adsorb and remove endotoxin in plasma. However, although polymyxin B has a binding property with endotoxin in a state of being dissolved in a solution, according to the study of the present inventors, the insolubilization of polymyxin B significantly reduces the binding property with endotoxin. I understood it. This is probably because the non-woven fabric itself used as a removing material by insolubilization or the chemical bond with the non-woven fabric three-dimensionally inhibits the bond with endotoxin to lower the binding force, and the polymyxin B is in a dissolved state. It was considered that the contact between the polymyxin B and the endotoxin is extremely unlikely to occur in the insolubilized state. Moreover, since the removing material is a non-woven fabric, the surface area that effectively works for adsorbing endotoxin is small, and again the adsorbing capacity for endotoxin was not sufficient. In addition, endotoxin is also attached to blood cell components, and these endotoxins could hardly be removed by the polymyxin B, and therefore could not be practically used sufficiently.

[0003]

DISCLOSURE OF THE INVENTION An object of the present invention is to remove endotoxin and / or endotoxin-derived cytokines from blood, and in particular endotoxin and / or endotoxin having adhesion to leukocyte components such as granulocytes and monocytes. Another object of the present invention is to provide an excellent remover for removing a cytokine caused by the endotoxin and a method for producing blood from which an endotoxin and / or a cytokine caused by the endotoxin are removed by using the remover.

[0004]

[Means for Solving the Problems] Endotoxin-containing blood induces cancer necrosis factor more than leukocytes by endotoxin,
Various cytokines such as interleukin 1, interleucine 6 and interferon, and acid peroxide are released, and it is known that these excess cytokines have adverse physiological effects. Therefore, according to the study by the present inventors, the endotoxin causing the release of cytokines, particularly endotoxin attached to the surface of granulocytes and monocytes, is removed, or excessive cytokine production by endotoxin is prevented, and endotoxin-containing substances are contained. It has been found that improving blood conditions is important. The present invention intends to indirectly remove endotoxin, which is attached to leukocyte components such as granulocytes or monocytes, from blood by attaching granulocytes or monocytes to the capturing material. Furthermore, by capturing granulocytes and / or monocytes, it is intended to suppress the production of cytokines due to the endotoxin and prevent the increase of cytokine concentration in blood. Cytokines are originally metabolized rapidly in blood, and suppressing the excessive production of cytokines that exceeds the metabolic rate lowers the concentration in blood. At the same time, an increase in the concentration of acid peroxide released by leukocytes can be suppressed at the same time. Further, the granulocytes or monocytes attached to the capturing material can be washed with an aqueous solution containing a salt having a buffering action or cultured to recover the endotoxin-free granulocytes or monocytes. The granulocytes and monocytes can also be used for remixing with blood. Here, culturing refers to placing granulocytes and monocytes under physiological conditions for a certain period of time. More specifically, a remover containing a granulocyte and / or monocyte-capturing material for removing endotoxin and / or a cytokine resulting from the endotoxin from blood by collecting granulocytes and / or monocytes. The present invention relates to a method for producing blood from which endotoxin and / or cytokines derived from endotoxin have been removed from blood using the remover.

Description of endotoxin Endotoxin is lipopolysaccharide or a complex of lipopolysaccharide and a protein, which is known as a main component constituting the outer wall of Gram-negative bacterium cell wall. Lipopolysaccharides are known as various compounds of lipid A and polysaccharides.

Capture Material The granulocyte and / or monocyte capture material (hereinafter referred to as a capture material) according to the present invention has any shape such as a thread shape, a sponge shape, a bead shape, a chip shape, a hollow fiber shape or a flat membrane shape. It may be. Of these, thread-like, sponge-like, and bead-like ones are preferably used from the viewpoint of handleability, and particularly, the thread-like and sponge-like ones are the most excellent in the capturing performance. The thread-like material can be used as it is, for example, in the shape of cotton, or can be used as a woven cloth or a non-woven cloth, or can be cut into short pieces, but it is also a non-woven cloth from the viewpoint of handleability and capturing performance. Is preferred.

Capture rate The capture rate of the granulocytes or monocytes of the capture material can be expressed by the capture rate. If the capture rate is too low, it is not possible to sufficiently achieve the object of the present invention to remove endotoxin and / or cytokine derived from the endotoxin from blood by collecting granulocytes and / or monocytes. The upper limit is not required because the purpose is removal.
The capture rate here is the rate of decrease in the number of granulocytes and / or monocytes before and after the treatment with the remover when blood is perfused in an amount 5 times the internal volume of the remover (priming volume). At this time, the anticoagulant may be any of the commonly used citric acid-based agents, those having an antithrompine action such as heparin, or those having a protease inhibitory action such as nafamostat mesylate, but the material of the capturing material And may vary depending on the surface condition. Therefore, the following method shall be used for measurement. ACD-A liquid as an anticoagulant 11.
Blood of a cow containing 1% is put in a blood reservoir and kept at 37 ° C. Cattle blood shall be used within 5 hours after blood collection. At this time, gently stir so that blood cell components in the blood do not settle. This blood is 1 / of the internal volume of the remover
Perfuse at a flow rate of 3 volumes / min. Blood is perfused in an amount 5 times the internal volume of the remover, and blood at the outlet of the remover is collected. The blood in the blood reservoir at the inlet of the remover is stained with a conventional method, and the number of granulocytes and / or monocytes is measured under a microscope. The capture rate can be obtained from the measurement result. Capture rate (%) = [(1- (concentration at the outlet of the remover) / (concentration at the inlet of the remover) × 100]) The capture rate thus obtained is preferably 60% or more, and more preferably 70% or more. The higher the capture rate, the better for the purpose of removing endotoxin, but when it is used for extracorporeal circulation, for example, complete capture of granulocytes or monocytes is not always preferable because it may lead to a decrease in immune function. Therefore, the capture rate is 98
% Or less is preferable. More preferably, it is 96% or less.

Materials As the capturing material, polyesters such as polyethylene terephthalate, polybutylene terephthalate and polyoxyethylene terephthalate, polyacrylonitrile, nylon 6, polyamides such as nylon 6,6, aromatic polyamide, polystyrene and derivatives thereof, Polyolefins such as polyethylene, polypropylene, and polybutene; polymer compounds obtained by polymerizing methacrylic acid ester derivatives such as methyl methacrylate and ethyl methacrylate; and acrylic acid ester derivatives such as methyl acrylate and ethyl acrylate. Synthetic polymer compounds such as polymer compounds, polytrifluorochloroethylene, polyvinylmale, polysulfone, polyurethane, polyvinyl acetal, polycarbonate, etc. A polymer including a homopolymer, a copolymer, a block polymer of the monomer of the polymer compound and a blended and alloyed product of the above polymer compound, a regenerated fiber such as cellulose and / or a derivative thereof, and the above. Examples thereof include blends with the synthetic polymer compounds shown in (1) and those including alloyed ones. Among the above, particularly polyester terephthalate, polybutylene terephthalate, polyester-based polyoxyethylene terephthalate, and polyolefin-based synthetic polymer materials are formed by the moldability of the nonwoven fabric, the fiber diameter of the resulting nonwoven fabric, and the fibers. It is particularly preferable because the state of pores and the like are easy to control, and this greatly affects the capturing ability of granulocytes, monocytes and the like. The polyester-based synthetic polymer material is also preferable from the viewpoint of blood wettability described later.

Method for producing trapping material As a specific example of the method for producing the trapping material of the present invention, a foaming decomposition method such as an atmospheric pressure foaming method, a pressure foaming method, an extrusion foaming method, an injection foaming method, or a solvent vaporization method. Known methods such as a gas mixing method, a chemical reaction method, an elution method, a sintering method and the like can be mentioned. Further, it is more preferable that secondary processing such as hot press compression and swelling with an appropriate liquid is performed to control the pore size distribution defined in the present invention. Examples of the production of fibrous materials include methods such as melt blow method and flash spinning. Further, the produced fibers are subjected to secondary processing such as press compression, heat shrinkage, treatment with an appropriate liquid, and are specified in the present invention. It is more preferable that the pore size distribution is controlled so that

Definition of contact angle, optimum range of contact angle When the liquid is brought into contact with the surface of the capturing material and the angle formed by the liquid and the surface of the capturing material is defined as the contact angle θ, the hydrophilicity of the surface of the capturing material is expressed by the contact angle. Can be represented. The contact angle was measured by using a liquid having a surface tension of 100 dy / cm and measuring the angle formed between the tangent line at the contact point between the droplet and the capturing material and the capturing material plane when the droplet was dropped on the surface of the capturing material. Desired. When the contact angle is θ, when θ is 120 degrees or less, the moistening performed prior to the removal of endotoxin is easy. Further, since it is possible to prevent the adsorption of useful plasma proteins, it is preferable that the contact angle is 90 degrees or less.
Most preferred is 0 degrees or less.

Definition of pores The pores referred to in the present invention include, for example, a space formed by fibers in the case of a fibrous material such as a non-woven fabric, and a space formed in the material by a sponge-like porous material. For example, it means a three-dimensional mesh-like space and is used for the purpose of collecting white blood cells. For this reason, it is preferable that the hole in the present invention forms a flow path through which blood cells pass and penetrates. For the purpose of capturing granulocytes or monocytes, which is the object of the present invention, the pore diameter is preferably 1 μm or more, more preferably 3 μm or more, in order to secure a channel for blood cells and prevent clogging during blood processing. Is. Further, it is preferable to design so that the capturing material and the granulocytes or monocytes come into contact with each other more during blood treatment. If the pore size is too large, the leakage of granulocytes or monocytes increases, which is not preferable. The pore size is preferably 100 μm or less, more preferably 50 μm or less, and most preferably 30 μm or less.

Distribution of pores In order to improve the efficiency of capturing granulocytes or monocytes and to secure a surface area capable of adhering a large number of granulocytes or monocytes in a certain volume, a large number of pores having an optimum pore diameter may exist. preferable. Therefore, it is preferable that more pores having a pore diameter of 1 μm or more and 100 μm or less exist. When the amount of pores is expressed as the ratio of the volume occupied by pores of 1 μm or more and 100 μm or less to the volume of the capturing material in a practical state, that is, the pore volume ratio, it is preferably 30% or more. It is more preferably 40% or more. By the way, it has been found that when the pore volume ratio becomes too large, the strength of the trapping material tends to decrease, clogging occurs, and the pressure tends to rise. Therefore, the pore volume ratio is preferably 90% or less, more preferably 80% or less. When the pore diameter in a more preferable range is within the above range, even better results can be obtained. The most preferable example is when the pore volume ratio of the pores of 3 μm or more and 30 μm or less is 40% or more and 80% or less.

Determination of Volume and Area of Hole The volume and area of the hole can be obtained from the mercury intrusion curve obtained by the mercury intrusion method. The measurement by the mercury injection method is 1-265.
The value is measured in the pressure range of 0 psia. Here, it is preferable that the pores have a value in a state as close to practical use as possible, but in the study by the present inventors, in the fibrous trapping material, a part of the fiber is cut out and a change in the fiber length due to swelling or the like is detected as The measurement was made with a microscope, and the surface area was corrected by multiplying the rate of change of the fiber length by the square of the rate of change of the fiber length by the cube of the rate of change of the fiber length.
In addition, the volume of the trapping material is reduced to 1 by compression when the container is filled.
When it becomes / X ³ , it is assumed that the hole diameter becomes 1 / X, and the difference between the measurement of the volume and area of the hole and the time of practical use is corrected. On the other hand, in the case of a sponge, the length, width and thickness of the capturing material are measured to obtain the rate of change in volume, and it is assumed that the volume of the hole changes by this rate of change. The rate of change in area is assumed to be the 2/3 power of the rate of change in volume. In the case of beads or the like, the change in the diameter of the trapping material is measured by an optical microscope, and the surface area is corrected by multiplying the change rate of the trapping material diameter by the square of the change rate of the trapping material diameter. In addition to the mercury porosimetry, the average pore diameter can be obtained by calculating the area of a large number of pores from an image obtained by a scanning electron microscope and converting the diameter into a circle. For example, the surface of the capturing material is photographed with a scanning electron microscope, the area of 200 or more pores dispersed on the photographed surface is measured by visual observation, and the average pore diameter can be obtained from the diameter when converted to a circle. it can.

Definition of average pore size and total surface area The average pore size is defined as the number of pores dispersed on any cut surface of the trapping material in various forms and the diameters thereof are different. It is the average diameter. If the average pore size is less than 1 μm, the flow path of blood cells is too narrow, resulting in an increase in pressure loss. On the other hand, if the average pore size exceeds 100 μm, the capturing performance of granulocytes or monocytes is deteriorated. Therefore, the average pore diameter is preferably in the range of 1 to 100 μm.
A more preferable range is 1 to 30 μm. The thickness is more preferably 2 to 20 μm, most preferably 3 to 15 μm.

Optimum value of surface area The total surface area of the capturing material, that is, the surface area of the capturing surface of granulocytes or monocytes can be used. The surface area differs greatly depending on whether the trapping material has a large number of pores of 0.1 μm or less and when it does not have pores such as a nonwoven fabric. In the removal of endotoxin and / or cytokines caused by endotoxin via granulocytes or monocytes of the present invention, pores do not effectively act on removal, and therefore it is preferable to express by surface area excluding pores. . At this time, the surface area is 0.2
It is preferably 0 m ² / ml or more. It is more preferably 0.50 m ² / ml or more, and most preferably 0.5.
It is 70 m ² / ml or more. The surface area is 0.20 m ² /
If it is less than ml, the surface area that can capture the granulocytes or monocytes is small, so that the granulocytes or monocytes leak out, which is not preferable. On the other hand, a trapping material having a large number of pores is preferable because endotoxin liberated in plasma can be directly adsorbed and removed at the same time. At this time, the total surface area including pores is 10 m ² / ml
The above is preferable. The upper limit of the total surface area is not particularly limited, but if it is too large, nonspecific adsorption of proteins increases, which is not preferable. Therefore, it is preferably 100 m ² / ml or less. More preferably 50 m ² / ml
Or less, and most preferably 20 m ² / ml or less. The surface area excluding pores of 0.1 μm or less can be measured by the mercury intrusion method, and the total surface area including the pores can be measured by the nitrogen adsorption method (BET method).
Of these, the surface area of the pores of 1 to 30 μm is 5 of the total surface area.
It is desirable that it is 0% or more, more preferably 55% or more, and most preferably 60% or more. The surface area of the pores of 1 to 30 μm is 60% or more of the total surface area, and more preferably 6
It is desirable that it is 5% or more, and most preferably 70% or more. The surface area of the pores of less than 1 μm is 38% or less, more preferably 30% or less, most preferably 28% or less. Surface area of 1 to 30 μm pores is 50% of the total surface area
When it is less than the above range, lymphocyte leakage or clogging is induced, which is not preferable. Further, since pores of less than 1 μm are pores through which blood cells cannot pass easily, when the surface area of pores of less than 1 μm exceeds 38%, the recovery amount of red blood cells decreases,
After all it is not preferable.

Total pore volume of trapping material Pore volume refers to the ratio of the volume of pores per volume of trapping material. The total pore volume is preferably 0.95 ml / ml or less in order to have some mechanical strength. Further, from the viewpoint of the capturing performance of granulocytes and monocytes, there are many pores having a preferable pore size, and it is effective that the contact area is larger, so that the total pore volume is 0.4 ml / ml.
It is preferably not less than 0.45 ml / ml, more preferably not less than 0.50 ml
/ Ml or more.

Average fiber diameter of capture material, coefficient of variation of fiber diameter When a fibrous capture material such as a non-woven fabric is used, the fiber diameter contributes to the pore diameter and the pore distribution, so that the effective average fiber diameter may be shown. is important. The average fiber diameter of the present invention is determined by photographing the surface of the fibrous trapping material with a scanning electron microscope and visually measuring 100 or more randomly distributed yarn diameters on the photographed surface. The fiber diameter of the capturing material which is effective in the mechanical strength and the capturing performance of granulocytes or monocytes is 0.3 μm or more and 10 μm or less, and the smaller the yarn diameter is, the better the capturing performance of granulocytes or monocytes is. 0.3 μm or more and 5 μm or less, more preferably 0.3 μm
It is 3 μm or less. Further, the distribution of the variation of the fiber diameter from the average fiber diameter is preferably narrow, and the vertical variation is 2
It should be within 0 to 60%. Vertical fluctuation is preferably within 20 to 50%, more preferably 20 to 45%
When within, it gives a uniform capture material. When the fibrous trapping material swells when wet, the rate of change in fiber length between dry and wet is measured with an optical microscope, and the fiber diameter is changed by the rate of change and corrected.

Definition of hydrophilic coating layer In order to improve the affinity with an aqueous solution or blood, the capturing material is
It may be substantially covered with a hydrophilic polymer. The hydrophilic coating layer preferably has a contact angle of water droplets of 80 degrees or less when formed into a flat plate, which is obtained by a contact angle measuring method, and the polymer is exemplified by the name as a monomer of a polymer unit. Hydroxystyrene, hydroxymethylstyrene, vinyl alcohol, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, vinylamine, diethylaminoethylstyrene, diethylaminoethyl methacrylate, methoxytriethylene glycol methacrylate, dimethylaminoethyl (meth) acrylate segmentation Block copolymers such as polyurethane and segmented polyester, graft copolymers such as monomers having polyethylene oxide chains and other polymerized monomers, ethylene-vinyl alcohol, polyesters, polyesters. Glycol, etc. may be exemplified. Among these, it is preferable to have a hydroxyl group in the polymer because it is possible to prevent excessive capture of platelets. Since endotoxin is considered to be attached also to platelets, it is preferable that the platelet trapping property is also excellent for the purpose of endotoxin removal, but on the other hand, trapping excessive platelets is preferable because it significantly reduces the blood coagulation ability. Absent. Therefore, it is practically preferable that the platelet capture rate is, for example, 50% or less. There is no particular limitation on the bonding mode of the hydroxyl group in the polymer. Of these polymers, 2-hydroxyethyl methacrylate, ethylene-vinyl alcohol, polyethylene glycol, and methoxytriethylene glycol methacrylate are preferable in terms of hydrophilicity, and ethylene-vinyl alcohol is particularly active when introducing a basic functional group. It is more preferable because the group is easily introduced, and the hydrophilicity is less likely to be reduced by peeling when the basic functional group is introduced. The hydrophilic coating layer may be a homopolymer of the above polymer units, or may be a copolymer of two or more polymers, and may be a polymer such as a linear polymer, a graft polymer or a crosslinked polymer. Has nothing to do with.

Method for Obtaining Coating Layer The method for obtaining the hydrophilic coating layer on the trapping material is as follows: dipping the trapping material in a liquid in which the compound forming the coating layer is dissolved or spraying the liquid for coating. There are a method of covalently bonding to the surface of the capturing material by a grafting method using radiation or an electron beam, or a method of chemically bonding via a functional group on the surface of the capturing material by a chemical method. Among them, the coating method is industrially easy and advantageous. The coating method referred to here may be a method in which a polymerizable compound also coexists in the compound forming the coating layer and crosslinking is performed after coating. The amount of thus obtained coating layer, i.e. coating weight, when expressed by weight of the coating layer per covering capturing material surface area, 10 ^-5 g / m ² or more and 1 g / m ²
It was below. When the coating amount is less than 10 ^-5 g / m ² , the hydrophilicity is insufficient and the effect as the hydrophilic coating layer cannot be obtained.
If the coating amount exceeds 1 g / m ² , there is a risk of peeling or elution during practical use, which is not preferable. A particularly preferred amount of this coating layer is 10 ^-4 g / m ² or more and 10 ^-1 g / m 2.
It was less than ² .

Remover The trapping material of the present invention can be used as a remover by filling a container having an inlet and an outlet for blood. The shape of the container is not particularly limited as long as it is a container having an inlet and an outlet for blood, but, by way of example, a container that can be filled with a known trapping material in a laminated form, a columnar shape, a triangular columnar shape, or a rectangular columnar shape. , Hexagonal columnar, octagonal columnar, etc., and a container in which a capturing material can be rolled into a cylindrical shape and filled, or a blood flow enters from the outer circumference of the cylinder and flows inward, and collects at the innermost side A container or the like characterized by coming out of the outlet has a good shape. Further, a container or the like having a shape such that its cross-sectional area such as a cone shape decreases from the inlet to the outlet is used. If the ratio (cross-sectional area / thickness, S / L) of the blood filtration cross-sectional area (filtration area) of the capturing material to the blood filtration length (thickness of the capturing material) at this time is too small, the cross-sectional area becomes insufficient and the blood flow. It is not preferable because the resistance becomes too large, and when it is too large, nonuniformity of the flow such as one-way flow of blood is likely to occur and granulocytes and / or
Alternatively, the capture rate due to adhesion of monocytes is reduced, which is also not preferable. Preferred S / L is 10 cm or more 5
It is less than 00 cm.

Here, in order to prevent clogging due to larger solids such as agglomerates of blood components and to suppress an increase in pressure during use, a filter material having an average pore diameter larger than that of the present trapping material is used. It may be provided on the upstream side of blood. The average pore size of the filtration material here is preferably 10 to 300 μm, more preferably 10 to 150 μm, and most preferably 10 to 100 μm. Average pore size is 10μ
If it is less than m, it may cause clogging by blood cells on the surface of the filtration material and increase pressure loss, which is not suitable. On the other hand, when the average pore diameter is more than 300 μm, the effect as a filtering material is to remove components larger than blood cells such as aggregates of blood cells in advance before the capturing material to prevent clogging of the capturing material due to the component. It is not preferable because it cannot be obtained sufficiently.

Packing Density of Remover The packing density means the weight per fixed volume when the container is filled with the trapping material, and the packing density of the container is 0.
05G / cm ³ was filled above 0.5 g / cm ³ or less, it is preferable that the remover. Further, in order to prevent clogging, increase in pressure loss, and smooth flow, a preferable packing density is 0.1 g / cm ³ or more and 0.4 g / cm ³ or less, and a further preferable packing density is 0.1 g / cm ^3. cm ³ or more 0.
It is 3 g / cm ³ or less.

The cross-sectional area and thickness of the remover The preferred effective cross-sectional area of the remover may be any size, but it is 0.5 cm because of its ease of manufacture and blood throughput.
^It is preferably ² or more and 300 cm ² or less, more preferably 250 cm ² or less, and further preferably 200 cm ² or less from the viewpoint of miniaturization and operability. Although the total thickness of the capturing material depends on its shape, the thickness of the entire capturing material is preferably 0.1 mm or more and 500 mm or less as a practical range. Considering the priming property, preferably 0.1 m
m or more and 450 mm or less, more preferably 0.
It is practically 1 mm or more and 400 mm or less.

The priming volume of the remover The priming amount of the remover of the present invention may be any amount, but the amount is preferably small in terms of operability and shortening of processing time. An example of a preferable priming amount is 5 to 1000 ml, more preferably 10 to 3 ml.
00 ml, most preferably 50-300 ml.

Sterilization method of the remover Although any known method may be used, for example, autoclave heat sterilization, ethylene oxide gas (EOG) sterilization, γ-ray sterilization, electronic Radiation sterilization such as line sterilization and sterilization methods such as UV irradiation can be used.

Shape of use of remover As a specific example of the method of using the remover of the present invention, a blood bag, a blood circuit, a chamber, a clamp,
A roller clamp, a drip chamber, a needle, a drip chamber with a mesh, a tube for a blood pump, etc. or a plurality of them, for example, an extracorporeal circulation circuit or a blood transfusion circuit can be used. Further, the equipment incorporated in the circuit is not limited to the above. Further, for blood treatment, a pump such as a blood pump, a liquid feeding pump, or a suction pump can be incorporated in the circuit and used. Further, it is possible to favorably use even a drop due to the own weight of blood.

Capture Conditions The slower the flow rate of blood perfusion, the higher the capture rate, but the longer the processing time, which is not preferable. On the contrary, if it is faster, the processing time can be shortened, but the capture rate will be reduced. Therefore, the flow rate is preferably 1 ml / min or more and 1000 ml / min or less. More preferable flow rate is 5 ml / min or more, 500 ml
/ Min or less. Actually, it is useful to express the flow velocity as a value per effective cross-sectional area of filtration, that is, a linear velocity, regardless of the size and design of the remover. The preferable range of the linear velocity is 0.
It is not less than 05 cm / min and not more than 100 cm / min.
Most preferably, the linear velocity is 0.1 cm / min or more, and 5
It is when it is 0 cm / min or less. In addition, as a method for passing blood, it may be continuously passed or intermittently passed depending on the necessity of use or the device status of the facility. At this time, to obtain high capture performance,
The residence time is preferably long, and the residence time of blood in the remover is preferably 0.5 minutes or more. In addition, when blood remains in the remover for a long time, there is a risk of causing nonspecific adsorption of blood components and blood coagulation. Therefore, the residence time is 30
It is preferably not more than minutes.

Utilization of Captured Granulocytes / Monocytes Granulocytes and / or monocytes attached to the remover are oscillated by the remover, and physiological saline such as phosphate buffered saline or lactated Ringer's solution, or a very small amount of interface. Recovered by perfusing a physiological saline solution containing an active agent, and then washed with a physiological saline solution containing a slight amount of a surfactant or a physiological saline solution after recovery,
It can be reused.

[0029]

INDUSTRIAL APPLICABILITY The remover of the present invention enables efficient and easy removal of endotoxin and / or cytokines derived from endotoxin from blood containing endotoxin.

[0030]

【Example】

Example 1 A nonwoven fabric was obtained by melt extrusion of polyethylene terephthalate. This non-woven fabric has a basis weight of 40 g
/ M ² , thickness 0.32 mm, density 0.126, average fiber diameter 1.6 μm, CWST value 58 dyne / cm, average pore diameter 5.3 μm, and pore volume ratio of pores having a pore diameter of 1 μm or more and 100 μm or less was 76%. Using the non-woven fabric as an endotoxin trapping material, it was cut into a 7.5 cm width and a length of 240 cm, wound into a cylindrical shape, and both ends thereof were bonded and solidified with urethane. The amount of non-woven fabric was 12 g. This cylindrical nonwoven fabric was filled in a cylindrical container having an inner diameter of 4 cm and a length of 8 cm. The container was provided with an inlet and an outlet so that blood could flow from the outer peripheral side of the cylindrical non-woven fabric toward the central portion to form a remover. The remover was filled with phosphate buffered saline and autoclaved at 121 ° C. for 1 hour. The capture rate of this remover was 98%. An endotoxin-containing blood was prepared by administering 0.5 mg / kg body weight of lipopolysaccharide of Escherichia coli as endotoxin to a healthy dog having a shunt in the carotid part and having a weight of 22 kg. After the administration, a catheter was inserted into the shunt and blood was collected by an extracorporeal circulation circuit. The blood obtained through the catheter was mixed with 25 mg / hour of nafamostat mesylate as an anticoagulant and then perfused into the remover with a pump at a blood perfusion rate of 30 ml / min to obtain endotoxin-free blood. The blood from which endotoxin was removed by the remover was returned to a healthy adult dog over time. By observing the symptoms caused by endotoxin, the adsorption removal effect of endotoxin and cytokine was determined. Although a decrease in blood pressure due to endotoxin was observed, healthy dogs did not show the fatal symptoms due to endotoxin, and this remover was able to effectively remove endotoxin and cytokines due to endotoxin.

[0031]

Example 2 Inner Diameter 2 cm with Solution Inlet and Outlet
A 2 cm square container was filled with the endotoxin trapping material of Example 1 to a thickness of 5 mm to obtain a remover. White blood cells were collected from human blood by specific gravity centrifugation, and the white blood cell suspension was adjusted with Hanks 'solution (Hanks' solution) to a concentration of 5 × 10 ⁶ cells / ml. 0.05E in this leukocyte suspension
Dissolve U / ml endotoxin and incubate at 37 ° C for 2 hours,
The mixture was shaken to give an endotoxin-containing solution. The remover was perfused with 10 ml of the endotoxin-containing liquid at 0.5 ml / min,
The remover effluent was collected. No white blood cells were found in the remover effluent. When the endotoxin concentration in the effluent of this remover was measured by the diazo coupling method, it was found to be 0.
It was 03 EU / ml. With this remover, 40% of endotoxin could be removed by adsorption.

[0032]

Example 3 Endotoxin 0.5 mg / ml was added to 10 ml of BALB / c mouse blood and reacted at 37 ° C. for 2 hours with shaking. Next, this endotoxin-containing blood was perfused into the remover of Example 2 at 0.5 ml / min, and the remover outflow blood was collected. Remover 1 ml of outflowing blood BALB
When it was administered to the tail vein of 5 / c mice, only one death due to endotoxin was observed, and most of them survived.

[0033]

[Comparative Example 1] In the same manner as in Example 1, endotoxin was administered to a healthy adult dog to prepare endotoxin-containing blood, and the symptoms due to endotoxin were observed. He showed various symptoms.

[0034]

[Comparative Example 2] 1 ml of endotoxin-containing blood obtained in the same manner as in Example 3 was BAL without perfusion into a remover.
When administered to the tail vein of 5 B / c mice, the death due to endotoxin was 4 and most of them died.

Claims (2)

[Claims] 1. Collecting granulocytes and / or monocytes containing a granulocyte and / or monocyte-capturing material having a pore volume ratio of 30% or more and 90% or less of pores having a pore diameter of 1 μm or more and 100 μm or less. A remover for removing endotoxin and / or cytokines caused by endotoxin from blood containing endotoxin. 2. Granulocytes and / or monocytes are obtained by using a remover containing granulocytes and / or monocyte-capturing material having a pore volume ratio of pores having a pore diameter of 1 μm or more and 100 μm or less of 30% or more and 90% or less. A method for producing purified blood in which endotoxin and / or cytokines derived from endotoxin are removed from endotoxin-containing blood by collecting spheres.