JP6864553B2 - Biocompatibility evaluation method for hollow fibers - Google Patents

Description

The present invention relates to a method for evaluating biocompatibility of the inner surface of a hollow fiber, which is suitably used for a hollow fiber device for separating a specific substance in blood such as a hemodialyzer or a plasma separator.

In recent years, medical devices with blood contact, especially extracorporeal circulation type medical devices, have made remarkable progress, and troubles such as blood coagulation during extracorporeal circulation and the inability to continue extracorporeal circulation have decreased. The main trouble during extracorporeal circulatory treatment represented by hemodialysis begins with the induction of a biological reaction by the contact of blood with the surface of a foreign material. The main clinical symptoms range from blood coagulation, anaphylactic shock, transient decrease in white blood cells (leucopenia), fever, and decrease or increase in blood pressure. The mechanism of problem occurrence during extracorporeal circulation accompanied by these acute clinical symptoms has been studied for a long time, and platelets are activated by contact between the material surface and blood, and a surface having many hydroxyl groups such as cellulose is used. It is thought that the cause is activation of complement components in blood by contact with blood, production of brazikinin by contact between negatively charged surface and blood, and regurgitation of endotoxin from dialysate to blood side.
Traditional medical devices have taken these mechanisms into account and have improved the problem by improving the biocompatibility or blood compatibility of the material.

In order to evaluate the biocompatibility and blood compatibility of more accurate materials, it is necessary to collect blood from actual dialysis patients and evaluate it, but for that purpose, the safety and performance of the developed product are satisfied. Evaluation cannot be done until after that. Therefore, in order to bring the clinical conditions as close as possible to the clinical conditions in vitro, the hollow fiber type module is connected to a circuit for circulating blood, and blood is flowed or circulated by a pump or the like, and the blood is passed through the hollow fiber type module. It is common practice to make an evaluation. (See, for example, Patent Document 1).
However, in that case, extra blood volume is required to fill the circuit part connected to the module, blood is activated by mechanical stimulation by the pump, and the connection between the circuit and the module. If there is a step, blood stagnate, and the evaluation result such as blood coagulation is affected by factors unrelated to the inner surface of the hollow fiber to be evaluated.

Patent Document 2 discloses a method for eliminating the problem of blood coagulation due to stagnation. However, in the evaluation method using in vitro blood provided with a circuit and a pump, in reality, the blood is activated by the stimulation by the pump or the stimulation by the contact with the circuit, so that the blood is activated in the module. There is a problem that the biocompatibility of only the inner surface of the hollow fiber cannot be evaluated accurately.

On the other hand, Patent Document 3 discloses a method for evaluating the biocompatibility of only the inner surface of the hollow fiber. As a specific method, for the purpose of evaluating the blood compatibility of the inner surface of the hollow fiber, a double-sided adhesive tape is attached to a polyethylene terephthalate film having a thickness of 0.1 mm, and five hollow fiber membranes are formed on the hollow fiber membrane in the length direction. Align and paste. Feeler gauges with a thickness of about half the diameter of the hollow fiber membrane are placed on both sides of the hollow fiber membrane and cut into two pieces using a microtome blade to expose the inner surface of the hollow fiber membrane. This film is punched into a circle with a diameter of 2 cm, and a sample sample is attached to the bottom of an 18 mmφ polystyrene tube as a sample sample, and processed so that blood can be held inside. A method is disclosed in which 1 ml of human blood adjusted to have a heparin concentration of 50 U / ml is placed in a tube containing the sample and immersed at 37 ° C. for 60 min at a shaking rate of 120 times / min.
However, in this method, the hollow fiber membranes are aligned in the length direction and attached to the film, and then the hollow fiber membrane is cut so as to cut off about half of the diameter of the hollow fiber. The drawback of not being able to accurately evaluate the biocompatibility of the inner surface of the hollow fiber remains, such as blood contact with tubes other than the surface and contact with air.

Therefore, it has been desired to develop a method capable of accurately and easily evaluating the biocompatibility and blood compatibility of the inner surface itself of the hollow fiber material while reducing the blood volume required for the evaluation.

Japanese Unexamined Patent Publication No. 2006-30533 Japanese Unexamined Patent Publication No. 2010-82068 Japanese Unexamined Patent Publication No. 2005-69966

An object of the present invention is to provide a method capable of more accurately evaluating the biocompatibility of a hollow fiber device with a small amount of blood without giving mechanical stimulation to the blood by a simple method.

The present inventors conducted diligent research to solve the above problems, introduced blood into the hollow fiber, and simply rotated the hollow fiber as it was, without using a blood pump or a blood circuit, and inside the hollow fiber. It was found that the blood flow is sufficiently generated in the above, and the interaction between the inner surface of the hollow fiber and the blood can be enhanced to the same extent as that actually used in hemodialysis or the like.
Then, based on such findings, if the blood derived from the inside of the hollow fiber and / or the blood adhering to the inner surface of the hollow fiber, which is rotated and stirred after introducing the blood into the inside, is evaluated, the hollow fiber can be easily and accurately obtained. The idea of being able to evaluate biocompatibility led to the completion of the present invention.

That is, the present invention is as shown below.
[1] The characteristics and / or components of the step of introducing blood into the hollow fiber, the step of rotating the hollow fiber, and the blood derived from the inside of the hollow fiber and / or the blood adhering to the inner surface of the hollow fiber. A method for evaluating the biocompatibility of hollow fibers, which comprises the steps of measuring the concentration in this order.
[2] The method for evaluating biocompatibility of a hollow fiber according to [1], wherein in the rotation step, the hollow fiber is rotated while being housed in the hollow fiber device.
[3] The method for evaluating the biocompatibility of a hollow fiber according to [1] or [2], wherein the rotation is performed using a rotator.
[4] The components are erythrocytes, platelets, leukocytes, plasma proteins, leukocyte-derived products, platelet-derived products, blood coagulation factors, blood coagulation factor activators, blood fibrinolytic activators, and complements. The method for evaluating biocompatibility of hollow filaments according to any one of [1] to [3], which is at least one selected from the group consisting of activating substances.
[5] The method according to [4], wherein the component is a cytokine or chemokine.

According to the method for evaluating the biocompatibility of hollow yarns of the present invention, since a blood pump or blood circuit is not used, the amount of blood required for evaluation can be reduced, and the biocompatibility of hollow yarns can be accurately evaluated by a simple method. it can.

It is the schematic which shows the state which set the relatively large hollow fiber device in the rotator. It is the schematic which shows the state which set the relatively small hollow fiber device in the rotator.

Hereinafter, a mode for carrying out the present invention (hereinafter, referred to as “the present embodiment”) will be described in more detail. In detail, the present invention is not limited to this, and various modifications can be made without departing from the gist thereof.

In the present embodiment, the hollow fiber to be evaluated for biocompatibility can be housed in a hollow fiber device or the like and used to separate a specific substance in blood.
Here, the hollow fiber device is a device that uses a hollow fiber as a component for separating a specific substance from a fluid, and is usually a flexible or rigid container having a fluid inlet and a fluid outlet, and the container. It is composed of a hollow fiber bundle housed inside.
Examples of the hollow thread device include a hemodialysis device, a blood filter, a hemodiadiafiltration device, a plasma separator, a plasma component filter, and the like, which are used especially in blood purification therapy and are mainly used for artificial dialysis (artificial dialysis). Widely used in the fields of (filtration) therapy and aferesis therapy.

In the present embodiment, the hollow fiber is a filamentous body having a hollow portion parallel to the longitudinal direction thereof inside and having a plurality of pores on the wall surface. Generally, the hollow fiber has a large number of pores on its wall surface, and various substances can be exchanged inside and outside the hollow fiber according to the particle size.
The material of the hollow yarn is not particularly limited, and is, for example, polysulfone, a polysulfone polymer such as a copolymer composed of polysulfone and polyvinylpyrrolidone, vitamin E-immobilized polysulfone, ethylene vinyl alcohol, polyacrylic nitrile, polyethylene, regenerated cellulose. , Cellulose diacetate, cellulose triacetate, polyethersulfone, polymethylmethacrylate and the like.

The length of the hollow fiber used in the evaluation is not particularly limited, but a hollow fiber having a length usually in the range of 1 to 100 cm, more preferably 5 cm to 50 cm is often used. The thickness of the hollow fiber membrane is not limited, and generally, 5 μm to 100 μm, more preferably 10 μm to 50 μm is more preferably used. The inner diameter of the hollow fiber is not limited, but 100 to 300 μm is preferable from the viewpoint of allowing blood to flow inside the hollow fiber and the efficiency of substance exchange.

In the present embodiment, the blood refers to a solution containing a blood component or a solution containing subcultured cells derived from blood line cells. The blood components include red blood cells, platelets, leukocytes, plasma proteins, leukocyte-derived products, platelet-derived products, blood coagulation factors, blood coagulation factor activators, blood fibrinolytic activators, and complements. It refers to an activating substance, etc.
In the present embodiment, any of the above can be used as the blood, but considering that the blood cell measurement can be measured by a Coulter counter or the like and the operation is simple, a relatively large amount of white blood cells and platelets are particularly used. A solution containing whole blood or cultured cells, which is a contained solution, is preferably used.

The origin of the blood is not particularly limited as long as it is animal blood, and for example, blood from humans, cows, pigs, rats, mice, rabbits, goats, sheep, monkeys and the like can be used. In particular, hollow fiber devices are often intended for use in humans, and human blood is preferably used.
Regarding cultured cells derived from blood line cells, the type of cells is not particularly limited. For example, THP-1 cells, which are human monocytic cell lines, and RAW264.7 cells, which are cell lines derived from mouse macrophages. Stocks and the like can be used.

In the present embodiment, the biocompatibility evaluation of the hollow fiber means a change (specifically) of blood after contact with the inner surface of the hollow fiber or blood adhering to the inner surface of the hollow fiber from before contact (adhesion). Means to quantitatively or qualitatively evaluate (changes in quantity and quality of various components, etc.).

Specifically, for example, as a quantitative change in the components of blood after contact with the inner surface of the hollow thread, blood cells such as platelets and white blood cells; complements such as C3a, C5a, SC5b-9 and the like Complement activating components that increase with activation; TAT and PIC that increase with activation of blood coagulation system and blood fibrinolytic system; β-TG and PF-4 produced with activation of platelets ; Cytokines such as granulocyte elastase, myeloperoxidase, IL-1, TNF-α, IL-6 produced by activation of leukocyte cells, and chemokines such as MIP-1, MCP-1, RANTES, etc. Changes in quantity (concentration) can be mentioned. Furthermore, in recent years, PAO (Potential Anti Oxidant) and BAP (Biological Anti-oxidant Potential), which are indicators of blood antioxidant capacity, MDA (Malondiardide), which is one of the lipid peroxidation products, and superoxide anion have been introduced. SOD (Superoxide Dismutase), which is an enzyme that decomposes, and the amount (change in concentration) of hydroperoxide, which is a marker of the degree of oxidative stress, are also listed as typical measurement items for evaluating biocompatibility.

In addition, as a qualitative change in blood components after contact with the inner surface of the hollow thread, integrin CD11b / CD18 (also known as Mac-1), which is a surface marker of granulocyte cells, which are leukocyte cells, is used. Expression or increase of CD62P associated with expression or increase of platelets and activation of platelets may also be mentioned.

Furthermore, as a measurement of changes in blood adhering to the inner surface of the hollow thread, the number and morphology of platelets and leukocytes attached to the inner surface of the hollow thread can be observed by SEM, or fibrinogen interacting with the platelets can be measured on the inner surface of the hollow thread. For example, measuring the amount of adsorption and the degree of modification thereof.

By the way, as a hollow fiber having excellent biocompatibility, a hollow fiber made of polysulfone in which vitamin E is immobilized on the inner surface is known.
The following has been reported as an example of the clinical evaluation. For example, as a result of performing dialysis treatment using a dialysis machine containing a polysulfone hollow fiber on which vitamin E is immobilized, the TAT is lower than when a polysulfone hollow fiber on which vitamin E is not immobilized is used. It has been reported that the increase in blood coagulation system was suppressed (Terashima et al., Sendai Red Cross Disease Medical Miscellaneous Vol.12, N0.1, 95-103, 2003). In addition, as a result of the dialysis patient changing to a dialyzer containing the vitamin E-immobilized polysulfone hollow fiber after using the polysulfone hollow fiber for 6 months, the concentration of C3a, which is a complement activation product, was significantly increased 15 minutes after the start of dialysis. It was reported to be low (Honbashi et al., 2007 Vitamembrane Study Group, clinical evaluation of vitamin E-immobilized polysulfone membrane (VPS)). In addition, when a vitamin E-immobilized hollow fiber dialyzer is used, the values of CRP and IL-6, which are markers of the degree of inflammation, are significantly higher than when a vitamin E-immobilized hollow fiber dialyzer is used. Decreases (Panichi et. Al. Blood Purification, 2011; 32; 7-14.). Furthermore, the d-ROM values during dialysis of 66 maintenance dialysis patients did not change with the vitamin E-immobilized polysulfone hollow fiber dialyzer, but with the non-vitamin E-immobilized polysulfone hollow fiber dialyzer. , The value increased significantly in the latter half of dialysis. That is, it is speculated that the vitamin E-immobilized polysulfone hollow fiber dialyzer is less susceptible to oxidative stress during dialysis (Takahashi et al., 2014 Vitamembrane Study Group, oxidative stress level (d-ROM) of maintenance dialysis patients and Examination of antioxidant power).
As described above, it has been reported that the vitamin E-immobilized polysulfone hollow fiber is superior in biocompatibility to the non-vitamin E-immobilized polysulfone hollow fiber.

In the present embodiment, first, blood is introduced into the hollow fiber. The amount to be introduced is not limited, but it is preferable to fill the inside of the hollow fiber with blood. When the hollow fiber is contained in the container as described later, it is preferable to fill the container with blood, for example.

Next, the evaluation method of the present embodiment includes a step of rotating the hollow fiber into which blood is introduced.
By simply introducing blood into the hollow fiber, the frequency of contact between blood components, especially blood cell components such as white blood cells and platelets, and the inner surface of the hollow fiber is extremely low, so accurate data that reflects clinical practice can be obtained. Can not.
However, in the present embodiment, accurate data reflecting clinical practice has been obtained by performing a simple operation of rotating the hollow fiber after introducing blood.

In the present embodiment, when rotating the hollow fiber, it is preferable to prevent the blood introduced into the hollow fiber from spilling. For example, the end of the hollow fiber is closed or the hollow fiber is housed in a container that can be sealed. It is preferable to rotate it.
The container that can contain the hollow fiber and can be sealed is not limited, and any shape can be used. For example, it is preferable to use the above-mentioned hollow fiber device as such a container. At that time, there are no particular restrictions on the length, thickness, mass, blood retention capacity, etc. of the hollow fiber device, but considering that there is no problem in performing rotation, the length is 1 cm to 100 cm and the thickness, respectively. Is preferably 1 mm to 50 cm, the mass is 0.01 g to 10 kg, and the blood retention capacity is preferably in the range of about 10 μl to 200 ml.

In the present embodiment, the rotation is preferably performed in a plane substantially parallel to the longitudinal direction of the hollow fiber. Further, at that time, it is more preferable that the hollow fiber or an extension line thereof passes near the center of rotation. When the hollow fiber is rotated in such a direction, blood flow is more efficiently generated inside the hollow fiber, and the interaction between the inner surface of the hollow fiber and blood can be further enhanced.
Further, in the present embodiment, the rotation is preferably performed by using a rotator. Here, the rotator refers to a device that rotates an object to be rotated by installing it on a rotating body that is a flat plate-shaped rotating body and that rotates in the plane.
There is no limitation on the method of setting the hollow fiber or the rotator of the hollow fiber device on the rotating body, but when the rotating body is perpendicular to the ground plane, the hollow fiber is set when the hollow fiber is set on the rotating body. It is preferable to set it so that it does not touch the ground surface. Specifically, as shown in FIG. 1, the hollow fiber device may be set along the diameter of the rotating body so that the hollow fiber (device) is substantially centered on the rotating body of the rotator. Alternatively, when the hollow fiber (device) is relatively small, as shown in FIG. 2, the center of the rotating body is on the extension line of the hollow fiber perpendicular to the circumference (tangent) along the circumference of the rotating body of the rotator. Hollow fibers (devices) may be set side by side (as if riding). Further, the hollow fiber (device) may be installed so as to protrude from the rotating body.

The angle of the rotating body of the rotator with respect to the ground plane is not particularly limited, but when the rotator is parallel to the ground plane on which the rotator is installed, the blood cell component is biased to both ends of the hollow fiber due to centrifugal force, and the hollow fiber is hollow. This is not preferable because it will not be possible to make uniform contact with the inner surface of the thread. Therefore, the angle formed by the rotating body of the rotator and the ground plane is preferably in the range of 5 degrees to 175 degrees, more preferably in the range of 45 degrees to 135 degrees, and even more preferably in the range of 60 degrees to 120 degrees. Therefore, it is very preferable that the rotating body is perpendicular to the ground plane.

In the present embodiment, the specific method of rotation is not particularly limited, but takes into consideration such as not causing damage to the hollow fiber membrane, preventing extreme destruction of blood cells, and simplicity of the process. A method in which the hollow fiber is housed in the hollow fiber device and the hollow fiber device is placed on the rotating body of the rotator and rotated to bring the inner surface of the hollow fiber into relatively gentle contact with blood is preferable.

The size of the rotating body of the rotator is not particularly limited, but a rotating body having a relatively small size and a small radius is preferable from the viewpoint of handleability, and for example, the diameter is 1 cm to 200 cm, more preferably 5 cm to 100 cm, and further. A disk of 10 cm to 50 cm is preferable. Examples of the rotator having such a rotating body include "ROTATORRT50" (manufactured by TIETECH Co., Ltd., Japan). The width and diameter of the rotating body do not necessarily have to be larger than the length of the hollow fiber (device).

The rotation speed is also not particularly limited, but a high speed at which the blood component moves due to centrifugal force does not allow the blood component to sufficiently contact the inner surface of the hollow thread, and in terms of operational safety. Not preferred. Therefore, it is preferable to stir at a relatively slow rotation speed, 1 to 10 rpm is practical, and even more preferably 1 to 5 rpm.

In the present embodiment, the rotation time is not particularly limited, but it is necessary to set the time so as to obtain data that correlates with the biocompatibility evaluation result in actual clinical practice as much as possible. From such a viewpoint, the rotary stirring time may be 10 seconds or more, but 20 minutes or more is preferable from the viewpoint of accuracy. Further, taking into consideration the execution time in actual clinical practice, 30 minutes to 24 hours is preferable, and 60 minutes to 4 hours is more preferable.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

[Example 1]
180 ml of blood was collected from a healthy person using a disposable syringe and an 18G winged needle, and immediately transferred to a blood bag containing heparin as an anticoagulant so that the heparin concentration became 1000 U / L, and mixed well.

Polysulfone hollow thread dialyzer APS-08SA (manufactured by Asahi Kasei Medical Co., Ltd.) (hereinafter abbreviated as dialyzer 1) and polysulfone on which vitamin E, which has already been confirmed to be more biocompatible than dialyzer 1, is immobilized. A hollow thread dialyzer VPS-08HA (manufactured by Asahi Kasei Medical Co., Ltd.) (hereinafter abbreviated as dialyzer 2) is prepared, and the inner surface of the hollow thread on the blood contact surface side and the inner surface of the hollow thread on the blood contact surface side are prepared in advance with physiological saline (2 L). , The outer surface side of the hollow thread, which is the dialysate side, was washed. After that, the physiological saline remaining on the outer surface side of the hollow fiber (dialysate side) was drained, and the dialysate introduction outlet was sealed with a cap.
Next, 60 ml of total blood having a heparin concentration of 1000 U / L is slowly introduced into each of the dialyzer 1 and the dialyzer 2 from the blood inlet side using a 100 ml syringe, and the liquid discharged from the blood outlet is discharged. Then, a cap was put on the blood outlet and the blood inlet to seal the blood.
After that, each dialyzer is placed on a disk-shaped rotating body with a diameter of 20 cm of the rotator "ROTATOR RT-50" (manufactured by Titec, Japan) so that the dialyzer passes near the center of the rotating body as shown in FIG. It was set and rotated and stirred at a speed of 5 rpm for 240 minutes.

After the rotation stirring is completed, the blood taken out from the blood outlet of each dialyzer is transferred to another 2 ml Eppendorf tube, and the white blood cell count and platelet count are measured with a blood cell measuring device "XT-1800i" (manufactured by Sysmex, Japan). It was measured.

The results are shown in Table 1. In Table 1, the white blood cell count and platelet count of the original blood before being introduced into the dialyzer are shown as pre. As a result, the blood in contact with the inner surface of the hollow fiber of the dialyzer 2 has a larger number of white blood cells and platelets than the blood in contact with the inner surface of the hollow fiber of the dialyzer 1, which is close to the value of the original blood. became. This indicates that the dialysis machine 2 does not adhere leukocytes and platelets as compared with the dialysis machine 1, that is, the dialysis machine 2 has better biocompatibility than the dialysis machine 1, and is evaluated by the evaluation method of the present invention. It was confirmed that the results were consistent with the actual biocompatibility.

In addition, blood taken out from dialyzer 1 and dialyzer 2 was centrifuged at 3500 rpm for 10 minutes with a tabletop centrifuge 4000 (manufactured by KUBOTA, Japan), and 6 types of cytokines in the obtained plasma were obtained. Bio-Plex system (Bio-Rad Bio) for the concentrations of chemokines (MIP1-α, RANTES) and chemokines (IL-1β, IL-5, IL-6, IL-8, IFN-γ, TNF-α) -Plex Pro human cytokine GI27-plex panel) was used for measurement.
The results are shown in Table 2. The result of measurement on the original blood was expressed as pre. As a result, the blood that came into contact with the inner surface of the hollow fiber of the dialyzer 2 showed lower values for both cytokines and chemokines than the blood that came into contact with the inner surface of the hollow fiber of the dialyzer 1, and the blood of the original blood. It was close to the value. This indicates that the dialyzer 2 has better biocompatibility than the dialyzer 1, and it was confirmed that the evaluation result by the evaluation method of the present invention matches the actual biocompatibility.

[Example 2]
180 ml of blood was collected from a healthy person using a disposable syringe and an 18G winged needle, and immediately transferred to a blood bag containing heparin as an anticoagulant so that the heparin concentration became 1000 U / L, and mixed well.

The same dialyzer 1 and dialyzer 2 used in Example 1 are prepared, and the inner surface of the hollow fiber on the blood contact surface side and the dialysate side are prepared in advance with physiological saline (2 L), respectively. The outer surface side of the hollow fiber was washed. After that, the physiological saline remaining on the outer surface side of the hollow fiber (dialysate side) was drained, and the dialysate introduction outlet was sealed with a cap.
Next, 60 ml of total blood having a heparin concentration of 1000 U / L is slowly introduced into each of the dialyzer 1 and the dialyzer 2 from the blood inlet side using a 100 ml syringe, and the liquid discharged from the blood outlet is discharged. Then, a cap was put on the blood outlet and the blood inlet to seal the blood.
After that, each dialyzer is placed on a disk-shaped rotating body with a diameter of 20 cm of the rotator "ROTATOR RT-50" (manufactured by Titec, Japan) so that the dialyzer passes near the center of the rotating body as shown in FIG. It was set and rotated and stirred at a speed of 5 rpm for 240 minutes.

After the rotary stirring was completed, blood was taken out from the blood outlet of each dialyzer. Regarding the extracted blood, a) TAT (thrombin / antithrombin III complex), which is an index of blood coagulation factor activation, b) β-TG (β-thromboglobulin), which is an index of platelet activation, and c) anti-antioxidant. PAO (Potential Anti Oxidant), which is an index of oxidative ability, and BAP (Biological Anti-oxidant Potential), which shows d) antioxidant power, were measured by the following procedure.
a) TAT
1.8 ml of blood was centrifuged at a centrifuge "Hybrid High Speed Cooling Centrifuge 6200" (manufactured by KUBOTA, Japan) at room temperature for 10 minutes at 3,500 rpm to obtain plasma, and TAT in the plasma was obtained. The concentration was measured by enzyme immunoassay.
b) β-TG
2.7 ml of blood was ice-cooled for 15 minutes and centrifuged at 4 ° C. for 30 minutes at 2000 G in a centrifuge "Hybrid High Speed Cooling Centrifuge 6200" (manufactured by KUBOTA, Japan) to obtain plasma. Β-TG in plasma was measured by enzyme immunoassay.
c) PAO
0.5 ml of blood was centrifuged in a centrifuge "Hybrid High Speed Cooling Centrifuge 6200" (manufactured by KUBOTA, Japan) at room temperature for 10 minutes at 3,500 rpm to obtain plasma, and the plasma was used. The measurement was performed using the antioxidant capacity measurement kit "PAO" (Japan, Nikken Zile Co., Ltd. Japan Aging Control Laboratory).
d) BAP
0.5 ml of blood was centrifuged in a centrifuge "Hybrid High Speed Cooling Centrifuge 6200" (manufactured by KUBOTA, Japan) at room temperature for 10 minutes at 3,500 rpm to obtain plasma, and the plasma was used. Measurement was performed using a FREE Carrio Duo free radical analyzer (manufactured by Wismer Co., Ltd., Japan).

After the blood was taken out from the blood outlet of each dialyzer after the rotation stirring was completed, physiological saline (500 mL) was flowed from the blood inlet at a flow rate of 100 ml / min to wash the inner surface of the hollow fiber. After disassembling the housing of each dialyzer, taking out a hollow fiber having a length of 10 cm and 100 pieces with no red blood cells remaining and cutting it to a few mm, e) the total amount of protein adsorbed on the inner surface of the hollow fiber membrane and f) The amount of fibrinogen adsorbed on the inner surface of the hollow fiber membrane was measured by the following procedure.
e) Total amount of protein adsorbed on the inner surface of the hollow fiber membrane 4.0 ml of a 1.0% SDS solution was added to the cut hollow fiber membrane, and the mixture was stirred for 4 hours. Then, using the supernatant as a sample, the total protein amount was measured by Micro BCA Protein Assay Reagent (Thermo Fisher Scientific, USA), and calculated as the total protein adsorption amount per hollow fiber membrane area.
f) Amount of fibrinogen adsorbed on the inner surface of the hollow fiber membrane 2.0 ml of a 0.5% Triton X-100 solution was added to the cut hollow fiber membrane, and the mixture was stirred for 1 hour. Then, using the supernatant as a sample, the amount of fibrinogen was measured with an AssayMax ^TM Human Fibrinogen ELISA Kit (Assaypro LLC, USA), and calculated as the amount of fibrinogen adsorbed per hollow fiber inner membrane area.

The measurement results of a) to f) above are shown in Table 3. As a result, it was confirmed that the dialyzer 2 suppresses fibrinogen adsorption, is less likely to activate blood coagulation factors and platelets, and has antioxidant ability as compared with the dialyzer 1. Was done. That is, it was shown that the dialyzer 2 has better biocompatibility than the dialyzer 1, and it was confirmed that the evaluation result by the evaluation method of the present invention matches the actual biocompatibility.

According to the method of the present invention, the biocompatibility of the hollow fiber can be easily and accurately evaluated with a small amount of blood, which is suitable for the evaluation of the hollow fiber for use in various devices such as a hemodialyzer and a plasma separator. Available.

Claims (5)

The process of introducing blood into the hollow fiber,
The process of rotating the hollow fiber and
A step of measuring the characteristics and / or the concentration of components of blood derived from the inside of the hollow fiber and / or blood adhering to the inner surface of the hollow fiber.
A method for evaluating the biocompatibility of hollow fibers, which includes this order. The biocompatibility evaluation method for a hollow fiber according to claim 1, wherein in the rotation step, the hollow fiber is rotated while being housed in the hollow fiber device. The biocompatibility evaluation method for hollow fibers according to claim 1 or 2, wherein the rotation is performed using a rotator. The components are erythrocytes, platelets, leukocytes, plasma proteins, leukocyte-derived products, platelet-derived products, blood coagulation factors, blood coagulation factor activators, blood fibrinolytic system activators, and complement activators. The method for evaluating biocompatibility of hollow yarn according to any one of claims 1 to 3, which is at least one selected from the group consisting of. The method of claim 4, wherein the component is a cytokine or chemokine.

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