JP2018017720A - Method of evaluating biocompatibility of hollow fiber - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method that can evaluate the biocompatibility of a hollow fiber device by a convenient method with improved accuracy, using a small amount of blood, and giving a low mechanical stimulus to blood.SOLUTION: Blood is introduced into a hollow fiber device and the device is rotated. Then, a blood component derived from inside the hollow fiber and/or a blood component attached to the internal surface of the hollow fiber is measured, to allow a convenient evaluation of the biocompatibility of the hollow fiber device.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a biological (blood) compatibility evaluation method for the inner surface of a hollow fiber that is preferably used in a hollow fiber device or the like 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 there have been fewer troubles such as blood coagulation during the extracorporeal circulation and the inability to continue extracorporeal circulation. The main trouble at the time of extracorporeal circulation treatment represented by hemodialysis starts when a biological reaction is caused by the contact between the surface of a foreign material and blood. Major clinical symptoms include blood clotting, anaphylactic shock, transient decrease in leukocytes (leucopenia), fever, decrease or increase in blood pressure. The mechanism of the occurrence of problems during extracorporeal circulation with these acute clinical symptoms has been studied for a long time. The surface of the material activates platelets when it comes into contact with blood, and the surface has many hydroxyl groups such as cellulose. This is thought to be caused by activation of complement components in blood by contact with blood, production of bradykinin by contact between negatively charged surfaces and blood, and endotoxin influx from the dialysate to the blood side.
Conventional 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 materials more accurately, it is necessary to collect and evaluate blood from patients during actual dialysis. To that end, the safety and performance of the developed product are satisfied. Only then can you evaluate. Therefore, in order to make it as close to clinical conditions as possible outside the body, connect the hollow fiber module and a circuit for circulating blood to circulate or circulate blood with a pump, etc., and allow blood to flow through the hollow fiber module. Evaluation is generally performed. (For example, see Patent Document 1).
However, in that case, an extra blood volume is required to fill the circuit part connected to the module, the blood is activated by mechanical stimulation by the pump, or the connection between the circuit and the module If there is a level difference, blood stagnation occurs, and the evaluation results such as blood coagulation are affected by factors unrelated to the inner surface of the hollow fiber to be evaluated.

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

On the other hand, Patent Document 3 discloses a method for evaluating the biocompatibility of only the inner surface of a 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 the five hollow fiber membranes are arranged in the length direction. Paste it all together. A gap gauge having a thickness of about half the diameter of the hollow fiber membrane is placed on both sides of the hollow fiber membrane and cut into two pieces with a microtome blade to expose the inner surface of the hollow fiber membrane. This film is punched into a circular shape having a diameter of 2 cm, and a sample sample is attached to the bottom of a polystyrene tube of 18 mmφ 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 a heparin concentration of 50 U / ml is placed in a tube containing the sample and immersed at a shaking speed of 120 times / min at 37 ° C. for 60 min.
However, in this method, the hollow fiber membranes are aligned in the length direction and attached to the film, and then, the operation is extremely complicated, such as cutting the hollow fiber so as to cut about half the diameter of the hollow fiber. There remains a defect that the biocompatibility of the inner surface of the hollow fiber cannot be accurately evaluated due to contact of blood with a tube other than the surface or contact with air.

  Accordingly, it has been desired to develop a method capable of accurately and simply evaluating the biocompatibility and blood compatibility of the inner surface of the hollow fiber material while reducing the amount of blood necessary for the evaluation.

JP 2006-305333 A JP 2010-82068 A JP-A-2005-69966

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

In order to solve the above problems, the present inventors have conducted intensive research, introduced blood into the hollow fiber, and rotated the hollow fiber as it is, without using a blood pump or a blood circuit. Thus, it was found that the flow of blood is sufficiently generated and the interaction between the inner surface of the hollow fiber and the blood can be increased to the same level as that actually used in hemodialysis or the like.
And based on such knowledge, if the blood derived from the inside of the hollow fiber that has been introduced into the interior of the hollow fiber and stirred and rotated and / or the blood adhering to the inner surface of the hollow fiber is evaluated, the hollow fiber is easily and accurately evaluated. The inventors have conceived that biocompatibility can be evaluated and have completed the present invention.

That is, the present invention is as follows.
[1] The step of introducing blood into the hollow fiber, the step of rotating the hollow fiber, and the blood derived from the hollow fiber and / or the blood adhering to the inner surface of the hollow fiber A method for evaluating the biocompatibility of a hollow fiber, comprising the steps of measuring the concentration in this order.
[2] The hollow fiber biocompatibility evaluation method according to [1], wherein in the rotation step, the hollow fiber is rotated in a state of being accommodated in a hollow fiber device.
[3] The hollow fiber biocompatibility evaluation method according to [1] or [2], wherein the rotation is performed using a rotator.
[4] The components are red blood cells, platelets, white blood cells, plasma proteins, white blood cell-derived products, platelet-derived products, blood coagulation factors, blood coagulation factor activators, blood fibrinolytic activators, and complements. The hollow fiber biocompatibility evaluation method 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 hollow fiber biocompatibility evaluation method of the present invention, the blood volume required for the evaluation can be reduced because no blood pump or blood circuit is used, and the biocompatibility of the hollow fiber can be accurately evaluated by a simple method. it can.

It is the schematic which shows the state which set the comparatively big hollow fiber device to the rotator. It is the schematic which shows the state which set the comparatively small hollow fiber device to 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, this invention is not limited to this, A various deformation | transformation is possible in the range which does not deviate from the summary.

In the present embodiment, a hollow fiber that is a target for biocompatibility evaluation is housed in a hollow fiber device or the like and can be used to separate a specific substance in blood.
Here, the hollow fiber device is a device using a hollow fiber as a part for separating a specific substance from a fluid, and usually a flexible or rigid container having a fluid inlet and a fluid outlet, and the container It is comprised from the hollow fiber bundle accommodated in the inside.
Examples of the hollow fiber device include hemodialyzers, hemofilters, hemodialyzers, plasma separators, plasma component filters, etc., and these are particularly used in blood purification therapy, mainly artificial dialysis ( Widely used in the field of filtration) and apheresis therapy.

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

  Although there is no special restriction | limiting in the length of the hollow fiber used in the case of evaluation, Usually 1-100 cm in length, More preferably, the thing of the range of 5 cm to 50 cm is used well. The thickness of the membrane of the hollow fiber is not limited, and generally 5 μm to 100 μm, more preferably 10 μm to 50 μm is more suitably used. Although there is no limitation also in the internal diameter of a hollow fiber, 100-300 micrometers is preferable from a viewpoint of flowing blood inside a hollow fiber and mass exchange efficiency.

In this embodiment, blood refers to a solution containing blood components or a solution containing cultured cells derived from blood cells. Blood components include erythrocytes, platelets, leukocytes, plasma proteins, leukocyte-derived products, platelet-derived products, blood clotting factors, blood clotting factor activators, blood fibrinolytic activators, and complements. This refers to activated substances.
In the present embodiment, any of the above can be used as blood. However, considering that blood cell measurement can be performed with a Coulter counter and the operation is simple, particularly, a relatively large amount of white blood cells and platelets are contained. A solution containing whole blood or cultured cells, which is a contained solution, is preferably used.

The origin of blood is not particularly limited as long as it is animal blood. 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 human use, and human blood is preferably used.
Moreover, there are no particular limitations on the type of cells derived from blood cells, but for example, human monocytic cell lines THP-1 cells and mouse macrophage-derived cell lines RAW264.7 cells. Stocks can be used.

  In the present embodiment, the biocompatibility evaluation of the hollow fiber is a change (specifically) of blood after contact with the inner surface of the hollow fiber or blood attached to the inner surface of the hollow fiber from before contact (adhesion) (specifically, Means quantitative or qualitative evaluation of changes in the amount and quality of various components.

  Specifically, for example, quantitative changes in blood components after contact with the inner surface of the hollow fiber include blood cells such as platelets and leukocytes; and complements such as C3a, C5a, and SC5b-9. Complement activating components that increase with activation; TAT and PIC that increase with activation of blood coagulation system and blood fibrinolysis system; β-TG and PF-4 that are produced with platelet activation ; Cytokines such as granulocyte elastase, myeloperoxidase, IL-1, TNF-α, IL-6, and chemokines such as MIP-1, MCP-1, and RANTES, which are produced upon activation of leukocyte cells The amount (concentration) can be changed. Furthermore, in recent years, PAO (Potential Anti-Oxidant), BAP (Biological Anti-oxidant Potential), which is an index of blood antioxidant capacity, MDA (Malonaldehyde), which is one of lipid peroxidation degradation products, and superoxide anion have been added. SOD (Super Oxide Dismutase), which is an enzyme that degrades, and the amount (change in concentration) of hydroperoxide, which is a marker of the degree of oxidative stress, are also representative measurement items for evaluating biocompatibility.

  In addition, as a qualitative change in blood components after contact with the inner surface of the hollow fiber, integrin CD11b / CD18 (also known as Mac-1), which is a surface marker of granulocyte cells that are leukocyte cells Examples also include expression or increase of CD62P that accompanies platelet activation.

  Furthermore, the measurement of changes in blood adhering to the inner surface of the hollow fiber can be performed by SEM observation of the number and form of platelets and white blood cells adhering to the inner surface of the hollow fiber, or fibrinogen interacting with platelets on the inner surface of the hollow fiber For example, the amount of adsorption and the degree of denaturation thereof can be measured.

By the way, as a hollow fiber excellent in 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 dialysis treatment using a polysulfone hollow fiber with vitamin E immobilized, TAT is lower than when using polysulfone hollow fiber with no vitamin E immobilized. There is a report that the increase in the blood coagulation system was suppressed (Terashima et al., Sendai Red Cross Medical Miscellaneous Vol. 12, N0.1, 95-103, 2003). In addition, after the dialysis patient used polysulfone hollow fiber for 6 months and changed to a dialyzer containing vitamin E-immobilized polysulfone hollow fiber, the concentration of complement activation product C3a 15 minutes after the start of dialysis was significantly increased. There is a report that it was low (Motohashi et al., 2007 Vitamembrane Study Group, Clinical Evaluation of Vitamin E Immobilized Polysulfone Membrane (VPS)). In addition, when using a vitamin E-immobilized hollow fiber dialyzer, the values of CRP and IL-6, which are markers of inflammation, are significantly higher than when using a hollow fiber dialyzer that does not immobilize vitamin E. (Panichi et. Al. Blood Purification, 2011; 32; 7-14.). Furthermore, the value of d-ROM during dialysis of 66 maintenance dialysis patients did not change in the vitamin E-immobilized polysulfone hollow fiber dialyzer, but in the polysulfone hollow fiber dialyzer without vitamin E immobilized, The value increased significantly in the second half of dialysis. That is, it is speculated that the vitamin E-immobilized polysulfone hollow fiber dialyzer is not easily affected by oxidative stress during dialysis (Takahashi et al., 2014 Vitamembrane Study Group, Oxidative Stress Level of Maintenance Dialysis Patients (d-ROM) and Examination of antioxidant power).
As described above, it has been reported that the vitamin E-immobilized polysulfone hollow fiber is superior in biocompatibility compared to the polysulfone hollow fiber not immobilized with vitamin E.

  In this embodiment, first, blood is introduced into the hollow fiber. The amount of introduction is not limited, but it is preferable to fill the hollow fiber with blood. When the hollow fiber is accommodated in the container as described later, for example, the container is preferably filled with blood.

Next, the evaluation method of this 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 can be very low, so accurate data reflecting clinical practice can be obtained. Can not.
However, in the present embodiment, accurate data reflecting clinical practice is achieved by performing a simple operation of rotating the hollow fiber after blood introduction.

In the present embodiment, when the hollow fiber is rotated, it is preferable that blood introduced into the hollow fiber is not spilled. For example, the end of the hollow fiber is closed, or the hollow fiber is stored in a container that can be sealed. And rotating it.
There is no limitation on the container that can seal the hollow fiber, and any shape can be used. For example, it is also preferable to use the above-described 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 hindrance when performing rotation, the length is 1 cm to 100 cm, respectively. Is preferably 1 mm to 50 cm, mass is 0.01 g to 10 kg, and 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. Furthermore, in that case, it is more preferable that the hollow fiber or its extension line passes near the center of rotation. When the hollow fiber is rotated in such a direction, a blood flow is more efficiently generated in the hollow fiber, and the interaction between the inner surface of the hollow fiber and the blood can be further enhanced.
Moreover, in this embodiment, it is preferable to perform rotation using a rotator. Here, the rotator refers to a device that rotates an object to be rotated on a rotating body that is a flat plate-like rotating body that rotates within the plane.
There is no limitation on the method of setting the rotator of the hollow fiber or hollow fiber device to the rotating body. However, 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 so that does not touch the ground plane. Specifically, as shown in FIG. 1, the hollow fiber device may be set along the diameter of the rotator so that the hollow fiber (device) is approximately at the center of the rotator rotator. Alternatively, when the hollow fiber (device) is relatively small, as shown in FIG. 2, it is perpendicular to the circumference (tangent) along the circumference of the rotating body of the rotator (the center of the rotating body is on the extension line of the hollow fiber). The hollow fibers (devices) may be set side by side so as to ride. Moreover, you may install a hollow fiber (device) so that it may protrude from a rotary body.

  The angle of the rotator with respect to the ground contact surface of the rotating body is not particularly limited. However, when the rotator is parallel to the ground contact surface on which the rotator is installed, the blood cell components are biased toward both ends of the hollow fiber due to centrifugal force. This is not preferable because it becomes impossible to uniformly contact the inner surface of the yarn. Therefore, the angle formed between the rotating body of the rotator and the ground contact surface is preferably in the range of 5 degrees to 175 degrees, more preferably in the range of 45 degrees to 135 degrees, and still more preferably in the range of 60 degrees to 120 degrees. It is very preferable that the rotating body is perpendicular to the ground plane.

  In this embodiment, the specific method of rotation is not particularly limited, but it takes into consideration that the hollow fiber is not damaged, that extreme blood cell destruction does not occur, the simplicity of the process, etc. The hollow fiber is accommodated in a hollow fiber device, and the hollow fiber device is placed on a rotating body of a rotator and rotated to bring the hollow fiber inner surface and blood into relatively gentle contact with each other.

  Further, 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. For example, the diameter is 1 cm to 200 cm, more preferably 5 cm to 100 cm. A 10 cm to 50 cm disk is preferable. As a rotator having such a rotating body, for example, “ROTATORRT50” (manufactured by Taitec Corporation, Japan) and the like can be mentioned. Note that the rotating body does not necessarily have a width or diameter larger than the length of the hollow fiber (device).

  The rotational speed is not particularly limited, but the high speed at which the blood component moves by centrifugal force cannot sufficiently bring the blood component into contact with the inner surface of the hollow fiber, and in terms of operational safety. It is not preferable. Therefore, it is preferable to stir at a relatively slow rotational speed, and 1 to 10 rpm is practical, and 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 that data correlating with the biocompatibility evaluation result in actual clinical practice can be obtained as much as possible. From such a viewpoint, the rotation 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 time of actual clinical practice, 30 minutes to 24 hours are preferable, and 60 minutes to 4 hours are 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, immediately transferred to a blood bag containing heparin as an anticoagulant so that the heparin concentration was 1000 U / L, and mixed well.

Polysulfone hollow fiber 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 have higher biocompatibility than dialyzer 1, is immobilized Hollow fiber dialyzer VPS-08HA (manufactured by Asahi Kasei Medical Co., Ltd.) (hereinafter abbreviated as dialyzer 2) was prepared, and each of the inner surface of the hollow fiber on the blood contact surface side in advance with physiological saline (2 L), and The outer surface side of the hollow fiber, which is the dialysate side, was washed. Thereafter, the physiological saline remaining on the outer surface side (dialysis solution side) of the hollow fiber was discarded, and the dialysate introduction outlet was sealed with a cap.
Next, 60 ml of whole blood with a heparin concentration of 1000 U / L is slowly introduced into the dialyzer 1 and dialyzer 2 from the blood inlet side using a 100 ml syringe, and the liquid discharged from the blood outlet port is discharged. Then, a cap was fitted on the blood outlet and the blood inlet and sealed.
Thereafter, each dialyzer is passed through a rotator “ROTATOR RT-50” (made by Taitec Co., Ltd., Japan) with a disk-shaped rotating body having a diameter of 20 cm so that the dialyzer passes near the center of the rotating body as shown in FIG. The mixture was set and rotated and stirred at a speed of 5 rpm for 240 minutes.

  The blood taken out from the blood outlet of each dialyzer after completion of rotary stirring 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 Corporation, 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 introduction into the dialyzer were expressed 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, and is close to the original blood value. became. This indicates that the dialyzer 2 does not adhere white blood cells and platelets as compared to the dialyzer 1, that is, the dialyzer 2 has better biocompatibility than the dialyzer 1, and is evaluated by the evaluation method of the present invention. It was confirmed that the result was consistent with the actual biocompatibility.

In addition, the blood taken out from the dialyzer 1 and the dialyzer 2 was centrifuged at 3500 rpm for 10 minutes in a table top centrifuge 4000 (manufactured by KUBOTA, Japan), respectively, and six types of cytokines in the obtained plasma Of IL-1β, IL-5, IL-6, IL-8, IFN-γ, TNF-α and chemokines (MIP1-α, RANTES) were measured using the Bio-Plex system (Bio-Rad Bio -Plex Pro human cytokine GI27-plex panel).
The results are shown in Table 2. In addition, the result measured about the original blood was represented as pre. As a result, the blood in contact with the inner surface of the hollow fiber of the dialyzer 2 shows a lower value for both cytokines and chemokines than the blood in contact with the inner surface of the hollow fiber of the dialyzer 1. It was close to the value. This indicates that the dialyzer 2 has better biocompatibility than the dialyzer 1, and it has been 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, immediately transferred to a blood bag containing heparin as an anticoagulant so that the heparin concentration was 1000 U / L, and mixed well.

The same dialyzer 1 and dialyzer 2 used in Example 1 were prepared, respectively, and previously with physiological saline (2 L), the inner surface of the hollow fiber that is the blood contact surface side, and the dialysate side. The outer surface side of the hollow fiber was washed. Thereafter, the physiological saline remaining on the outer surface side (dialysis solution side) of the hollow fiber was discarded, and the dialysate introduction outlet was sealed with a cap.
Next, 60 ml of whole blood with a heparin concentration of 1000 U / L is slowly introduced into the dialyzer 1 and dialyzer 2 from the blood inlet side using a 100 ml syringe, and the liquid discharged from the blood outlet port is discharged. Then, a cap was fitted on the blood outlet and the blood inlet and sealed.
Thereafter, each dialyzer is passed through a rotator “ROTATOR RT-50” (made by Taitec Co., Ltd., Japan) with a disk-shaped rotating body having a diameter of 20 cm so that the dialyzer passes near the center of the rotating body as shown in FIG. The mixture 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. With respect to the extracted blood, a) TAT (thrombin / antithrombin III complex) which is an indicator of blood coagulation factor activation, b) β-TG (β-thromboglobulin) which is an indicator of platelet activation, c) anti PAO (Potential Anti-Oxidant), which is an index of oxidative ability, and d) BAP (Biological Anti-oxidant Potential), which shows antioxidant ability, were measured by the following procedure.
a) TAT
Centrifuge the “Hybrid high-speed cooling centrifuge 6200” (manufactured by KUBOTA, Japan) for 1.8 ml of blood at room temperature and 10 minutes at 3,500 rpm to obtain plasma, and TAT in the plasma 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. and 2000 G for 30 minutes 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 at 3,500 rpm for 10 minutes at room temperature in a centrifuge “Hybrid high-speed cooling centrifuge 6200” (manufactured by KUBOTA, Japan) to obtain plasma, and using the plasma, Antioxidant activity measurement kit “PAO” (Nihon Kensile Co., Ltd., Japan Aging Control Laboratory) was used for measurement.
d) BAP
0.5 ml of blood was centrifuged at 3,500 rpm for 10 minutes at room temperature in a centrifuge “Hybrid high-speed cooling centrifuge 6200” (manufactured by KUBOTA, Japan) to obtain plasma, and using the plasma, Measurement was performed using a FREE Carrio Duo free radical analyzer (made by Wismer, Inc., Japan).

Further, after the rotary stirring was completed, blood was taken out from the blood outlet of each dialyzer, and then 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 with a length of 10 cm and a number of 100 hollow erythrocytes and cutting it into 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 protein adsorbed on the inner surface of the hollow fiber membrane 4.0 ml of 1.0% SDS solution was added to the cut hollow fiber membrane and stirred for 4 hours. Thereafter, using the supernatant as a sample, the total protein amount was measured with Micro BCA Protein Assay Reagent (Thermo Fisher Scientific, USA) and calculated as the total protein adsorption amount per hollow fiber inner membrane area.
f) Amount of fibrinogen adsorbed on the inner surface of the hollow fiber membrane 2.0 ml of 0.5% Triton X-100 solution was added to the cut hollow fiber membrane and stirred for 1 hour. Thereafter, using the supernatant as a sample, the amount of fibrinogen was measured by an AssayMax ^™ 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) are shown in Table 3. As a result, it was confirmed that the dialyzer 2 suppresses fibrinogen adsorption compared to the dialyzer 1, is less prone to activation of blood coagulation factors and platelets, and further has an antioxidant ability. It 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 results obtained by the evaluation method of the present invention coincided with the actual biocompatibility.

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

Claims (5)

Introducing blood into the hollow fiber;
Rotating the hollow fiber; and
Measuring blood properties derived from the inside of the hollow fiber and / or blood adhering to the inner surface of the hollow fiber, and / or the concentration of the component,
A method for evaluating the biocompatibility of hollow fibers, including this order.   The hollow fiber biocompatibility evaluation method according to claim 1, wherein, in the rotation step, the hollow fiber is rotated in a state of being accommodated in a hollow fiber device.   The hollow fiber biocompatibility evaluation method according to claim 1 or 2, wherein the rotation is performed using a rotator.   The component is erythrocyte, platelet, leukocyte, plasma protein, leukocyte-derived product, platelet-derived product, blood coagulation factor, blood coagulation factor activator, blood fibrinolytic activator, and complement activator The method for evaluating the biocompatibility of a hollow fiber 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.