JP7184247B2 - Ultraviolet light irradiation method for photopheresis, microdevice for photopheresis, and ultraviolet light irradiation apparatus for photopheresis - Google Patents

Description

The present invention relates to a microdevice for photopheresis, an ultraviolet light irradiation apparatus including the microdevice, and an ultraviolet light irradiation method for photopheresis using the ultraviolet light irradiation apparatus.

Immunity of the human body plays a role in protecting the human body from foreign enemies (parasites) such as bacteria, viruses, cancer, and parasites, and it can be said that immunity is an indispensable mechanism for human survival. On the other hand, however, immune reactions also cause skin diseases such as atopic dermatitis and psoriasis, and autoimmune diseases such as rheumatoid arthritis and collagen diseases such as systemic lupus erythematosus. An autoimmune disease is a condition in which the immune system, which is originally intended to recognize and eliminate foreign substances other than itself, such as bacteria, viruses, and tumors, overreacts and attacks its own normal cells and tissues. It is a general term for symptoms that develop due to

In recent years, one of the most important issues in modern medicine is to appropriately control such immune reactions and suppress excessive immune reactions with few side effects. It is well known that control of immune response is the most important issue in the field of organ transplantation as well.
Conventionally, a method of administering an immunosuppressant has been generally used to suppress excessive immunity, but side effects due to the administration of the immunosuppressant have been regarded as a problem.

Against this background, in recent years, photopheresis (extracorporeal circulation photochemotherapy) has attracted attention in the field of immunosuppression. Photopheresis generally involves adding a photosensitizing drug such as a psoralen derivative to the blood taken out of the body, allowing it to be absorbed by activated T cells, and then irradiating the blood with ultraviolet light. This is a method of activating a photosensitizer to induce immune tolerance by causing apoptosis or damage to the T cells, thereby suppressing an excessive immune response.
In photopheresis, the addition of a photosensitive medicine is not always necessary. is included, the activated state of the photosensitizing drug will not continue once the irradiation of ultraviolet light is finished, so side effects may occur in the patient compared to the immunosuppressive method by administering an immunosuppressive drug. is extremely low. For this reason, photopheresis is attracting attention as an immunosuppressive method with fewer side effects than the administration of immunosuppressants. In addition, in Western countries, the clinical application of photopheresis for treatment of autoimmune diseases to which immunosuppressants such as steroids are ineffective and for immunosuppression during transplantation treatment such as bone marrow transplantation is rapidly increasing.

When performing photopheresis, it is necessary to irradiate T cells, which are a type of white blood cell in blood, with ultraviolet light as described above. However, blood contains erythrocytes in addition to T cells, and hemoglobin contained in erythrocytes has a property of exhibiting high absorbance to ultraviolet light. Moreover, among the blood cells contained in blood, white blood cells account for about 3%, while red blood cells account for about 96%. Therefore, when blood containing red blood cells is irradiated with ultraviolet light, the irradiated ultraviolet light is absorbed by the hemoglobin in the red blood cells. However, there is a problem that the ultraviolet light cannot be effectively irradiated. In addition, in order to irradiate the T cells with the necessary amount of ultraviolet light to induce immune tolerance, it is necessary to irradiate the T cells with high-intensity ultraviolet light from a light source. There is also the problem that exposure of blood components to ultraviolet light may cause adverse effects such as hemolysis of red blood cells and denaturation of plasma proteins.

In order to solve such problems, when conventional photopheresis is performed, after adding a photosensitive drug to blood collected from a patient, the blood is centrifuged to take out only leukocytes, and the leukocytes thus taken out are It is placed in a flat container with a thickness of about several mm and has ultraviolet light permeability, and is irradiated with ultraviolet light through the container in the thickness direction of the container. I was in the process of getting it back.
However, such conventional photopheresis has problems such as requiring complicated procedures, increasing the physical burden on the patient, and rising medical costs.

In order to solve such problems, Patent Document 1 discloses a photopheresis treatment method that can be performed without requiring a complicated and expensive treatment such as centrifugation. Specifically, a method has been proposed in which blood is introduced into fine through-holes formed in a porous body and, in this state, the porous body is irradiated with ultraviolet light from the outside.

However, the photopheresis treatment method disclosed in Patent Document 1 has the following problems. For example, since the through-holes formed in the porous body are not uniform in the penetration direction, the irradiation amount of ultraviolet light to each T cell is not uniform, and therefore the effect of apoptosis of T cells is sufficient. There is a problem that it is not possible to obtain In addition, the diameter of the through-holes formed in the porous body is set to 15 to 50 μm for the purpose of making it slightly larger than the diameter of white blood cells (15 μm). Since it is difficult to make the diameter of the pores uniform with high accuracy, the blood clogs the porous body when the blood is introduced, or even if the blood does not clog, it takes a long time to introduce the blood. There is a problem.

Japanese Patent No. 4468976

The present invention has been made based on the circumstances as described above, and an object of the present invention is to prepare a liquid sample for photopheresis with high accuracy without the need for complicated processing such as centrifugation. An ultraviolet light irradiation method for photopheresis capable of inducing immune tolerance by causing apoptosis or damage to the treated cells contained therein, a microdevice used in the ultraviolet light irradiation method, and the microdevice An object of the present invention is to provide an ultraviolet light irradiation device.

The microdevice for photopheresis of the present invention is provided with a flow path in the wall portion, into which the liquid sample for photopheresis is introduced, and the liquid sample for photopheresis introduced into the flow path On the other hand, ultraviolet light is irradiated through the wall,
The length of the distribution path in the irradiation direction of the ultraviolet light is 70 μm or more and 500 μm or less,
The microdevice for photopheresis comprises a cylindrical rod having ultraviolet light transmissivity, and a plurality of flow passages formed of through holes extending in the longitudinal direction of the rod are provided independently of each other. ,
The flow path is formed of a wide slit, and two mutually independent flow paths are provided so that the plane directions in which the slits extend are parallel to each other .

In the microdevice for photopheresis of the present invention, the liquid sample for photopheresis is blood,
It is preferable that the wall transmit ultraviolet light including light in a wavelength range of 280 nm or more and 320 nm or less.

The ultraviolet light irradiation device for photopheresis of the present invention comprises the above microdevice for photopheresis,
a light source for irradiating the flow path with ultraviolet light from the outside of the microdevice for photopheresis through the wall portion of the microdevice for photopheresis.

In the ultraviolet light irradiation device for photopheresis of the present invention, the light source may emit ultraviolet light including light in a wavelength range of 280 nm or more and 320 nm or less.

In the ultraviolet light irradiation device for photopheresis of the present invention, the light source may be arranged so that the ultraviolet light is irradiated in a direction different from the direction in which the liquid sample for photopheresis flows in the flow path. preferable.

In the ultraviolet light irradiation method for photopheresis of the present invention, a liquid sample for photopheresis containing cells to be treated is introduced into a flow passage whose length in the irradiation direction of ultraviolet light is regulated to 70 μm or more and 500 μm or less. be done. As a result, clogging of the liquid sample for photopheresis in the flow path can be suppressed, so that the liquid sample for photopheresis can be rapidly irradiated with ultraviolet light while the liquid for photopheresis can be dispensed. The cells to be treated contained in the sample can be irradiated with ultraviolet light with high uniformity. Therefore, immune tolerance can be induced by causing apoptosis or damage to the treated cells contained in the liquid sample for photopheresis with high accuracy without the need for centrifugation. As a result, in photopheresis, the time required for complicated processing such as centrifugation of the liquid sample for photopheresis can be greatly reduced, and the burden on the patient can be greatly reduced.

1 is a cross-sectional view along the flow direction of a liquid sample for photopheresis, schematically showing a main part in an example of the configuration of the ultraviolet light irradiation device for photopheresis of the present invention; FIG. 1 is a cross-sectional view along a direction perpendicular to the flow direction of a liquid sample for photopheresis, showing an example of the configuration of the microdevice for photopheresis of the present invention. FIG. FIG. 3 is a cross-sectional view along a direction perpendicular to the flow direction of the liquid sample for photopheresis, showing another configuration example of the microdevice for photopheresis of the present invention. FIG. 4 is a cross-sectional view along a direction perpendicular to the flow direction of the liquid sample for photopheresis, showing still another configuration example of the microdevice for photopheresis of the present invention. FIG. 4 is a cross-sectional view along a direction perpendicular to the flow direction of the liquid sample for photopheresis, showing still another configuration example of the microdevice for photopheresis of the present invention. FIG. 4 is a cross-sectional view along a direction perpendicular to the flow direction of the liquid sample for photopheresis, showing still another configuration example of the microdevice for photopheresis of the present invention. 4 is a graph showing the results of Experimental Example 1. FIG. 1 is a schematic diagram illustrating an outline of an experimental photopheresis apparatus used in Experimental Example 2. FIG. 7 is a graph showing the results of Experimental Example 2. FIG.

Embodiments of a microdevice for photopheresis, an ultraviolet light irradiation apparatus, and an ultraviolet light irradiation method for photopheresis according to the present invention will be described below.

[Ultraviolet light irradiation device]
FIG. 1 is a cross-sectional view along the flow direction of a liquid sample for photopheresis, schematically showing the main part of an example of the configuration of the ultraviolet light irradiation apparatus for photopheresis of the present invention.
The ultraviolet light irradiation device for photopheresis of the present invention (hereinafter simply referred to as "ultraviolet light irradiation device") irradiates cells to be treated in a series of photopheresis treatments performed in photopheresis (extracorporeal circulation photochemotherapy). It is an apparatus for irradiating a liquid sample for photopheresis contained therein with ultraviolet light. Specifically, the ultraviolet light irradiation device is a microdevice for photopheresis (hereinafter simply referred to as a "microdevice") that includes a wall portion 11 that transmits ultraviolet light and a flow path 12 that penetrates the wall portion 11. ) 10, and a light source 20 for irradiating the photopheresis liquid sample S introduced into the flow passage 12 from the outside of the microdevice 10 through the wall portion 11 of the microdevice 10 with ultraviolet light. is provided.
The microdevice 10 is disposable and replaceable for each photopheresis process.

[Liquid sample for photopheresis]
The liquid sample S for photopheresis is a cell-containing liquid containing target cells (hereinafter also referred to as "treated cells") to undergo apoptosis by irradiation with ultraviolet light. Examples include cerebrospinal fluid, lymph, and liquids obtained by diluting these with physiological saline or the like, and particularly include blood (whole blood) from which blood components have not been separated.
When the liquid sample for photopheresis is blood, the cells to be treated are, for example, white blood cells, especially T cells.

[Micro device]
A microdevice 10 of the present invention includes a wall portion 11 having ultraviolet light transparency and a flow path 12 extending through the wall portion 11 . In the microdevice 10 of the present invention, the liquid sample S for photopheresis containing cells to be treated is introduced into the flow path 12, and the liquid sample S for photopheresis introduced into the flow path 12 is wall-mounted. Ultraviolet light from a light source 20 is irradiated through the portion 11 .
Specifically, as shown in FIG. 2, the microdevice 10 can consist of a columnar tubule. The inside of the narrow tube is used as the flow passage 12 and the tube wall is used as the wall portion 11 of the microdevice 10 . In this microdevice 10, the flow path 12 has an inlet port and an outlet port for the photopheresis liquid sample S at both ends, and the inlet port and the outlet port are communicated by a single path. Say.

In the microdevice 10 of the present invention, the length of the flow path 12 in the irradiation direction of the ultraviolet light from the light source 20 (hereinafter, also referred to as the "irradiation direction length") is set to 70 μm or more and 500 μm or less, and 100 μm. It is preferably 300 μm or more, and particularly preferably 200 μm or more and 300 μm or less.
Since the length of the flow passage 12 in the irradiation direction is 500 μm or less, the ultraviolet light from the light source 20 can reach the bottom of the flow passage 12 in the irradiation direction of the ultraviolet light (lower part in FIGS. 1 and 2), Therefore, an effect capable of causing apoptosis or damaging the treated cells to induce immune tolerance is obtained with a high degree of certainty. Further, since the length of the flow path 12 in the ultraviolet light irradiation direction is 70 μm or more, regardless of the components contained in the liquid sample S for photopheresis, the components of the liquid sample S for photopheresis can be detected in the flow path 12 . In particular, when the photopheresis liquid sample S is blood, blood cell components of the blood clog the flow path 12. can be deterred.

The length of the distribution path 12 in the irradiation direction of the ultraviolet light (irradiation direction length) refers to the length along the traveling direction C (vertical direction in FIG. 2) of the ultraviolet light irradiated to the distribution path 12 . For example, when using a light source that irradiates diffused light including an oblique light component, such as a discharge lamp, the irradiation direction is the traveling direction C of the ultraviolet light shown in FIG.
2, the irradiation direction length d of the microdevice 10 is, for example, the flow direction of the liquid sample S for photopheresis with respect to the microdevice 10 (the flow direction of the thin tube forming the flow path 12). When the ultraviolet light is irradiated in the direction perpendicular to the extending direction), it corresponds to the inner diameter of the narrow tube that constitutes the microdevice 10 .

Further, the width of the flow path 12, that is, the irradiation direction of the ultraviolet light (the Y direction in FIG. 1)) and the direction perpendicular to the flow direction of the liquid sample S for photopheresis (the X direction in FIG. 1) (the direction shown in FIG. 1). The width (hereinafter also referred to as “flow path width”) in the direction perpendicular to the paper surface (Z direction) is, for example, 70 to 500 μm.
In addition, the length of the flow direction of the photopheresis liquid sample S (hereinafter also referred to as the “irradiation length”) of the region R in the flow path 12 irradiated with ultraviolet light (hereinafter also referred to as the “light irradiation region”) ) can be appropriately determined based on the type and intensity of the ultraviolet light, the flow velocity of the liquid sample S for photopheresis in the flow path 12, and the like.

The thickness of the wall portion 11, that is, the thickness t of the wall portion 11 in the irradiation direction of the ultraviolet light varies depending on the material constituting the wall portion 11, particularly the transmittance of the ultraviolet light, but is, for example, about 1 to 5 mm. is preferred.
If the thickness of the wall portion 11 is too small, the microdevice 10 may be inferior in durability. In addition, when the thickness of the wall portion 11 is excessively large, ultraviolet light having an energy amount sufficient to induce immunological tolerance by causing apoptosis or damaging the cells to be treated is circulated with the desired accuracy. It may not be possible to reach the bottom of the channel 12 .

The fine tubes constituting the microdevice 10 are made of glass such as quartz glass, alkali glass, and borosilicate glass; silicone resin, cycloolefin resin (cycloolefin polymer (COP), cycloolefin copolymer (COC), etc.), acrylic resin, etc. can be made of a synthetic resin or the like.

〔light source〕
The light source 20 provided in the ultraviolet light irradiation apparatus of the present invention emits ultraviolet light, and preferably includes ultraviolet light including light in a wavelength range of 280 nm or more and 320 nm or less.
Light in the wavelength region of 280 nm or more and 320 nm or less is light in the middle wavelength ultraviolet region, so-called UVB. By using such UVB for photopheresis, apoptosis or damage to the treated cells can be caused only by irradiating the liquid sample S for photopheresis with UVB without using a photosensitive drug such as a psoralen derivative. can induce immune tolerance.
In the case of photopheresis using long-wavelength ultraviolet light (320 to 400 nm) called UVA, UVA has a longer wavelength than UVB and has less energy. In order to cause apoptosis, it is necessary to irradiate the liquid sample S for photopheresis with UVA while adding a photosensitive medicine such as a psoralen derivative.

As the light source 20 emitting such UVB, for example, an LED element, a XeCl excimer discharge lamp, a metal halide lamp, a fluorescent lamp, a mercury lamp, or the like can be used.
The light source 20 may be provided with an optical filter that cuts off light in a wavelength range other than UVB in the wavelength range of 280 nm or more and 320 nm or less among the light emitted from the LED element or the lamp.

The light source 20 emits light having a shape that can irradiate ultraviolet light over the entire width of the flow path 12 in the microdevice 10 and over a desired length (irradiation length of the light irradiation region R). You can use what you can. Specifically, it is preferable to use a rod-shaped light source as the light source 20 for the microdevice 10 that is a thin tube having one flow passage 12, for example. Further, as will be described later, in a microdevice in which a plurality of flow paths are arranged in a planar manner so as to extend parallel to each other, it is preferable to use a planar light source as the light source.

The light source 20 is preferably arranged so that the ultraviolet light emitted from the light source 20 is irradiated in a direction different from the direction in which the liquid sample S for photopheresis flows in the flow path 12 of the microdevice 10 . Specifically, it is preferable to irradiate in a direction substantially perpendicular to the flow direction of the liquid sample S for photopheresis.

[Ultraviolet light irradiation method]
The ultraviolet light irradiation method for photopheresis of the present invention is a method of irradiating a treated cell with ultraviolet light in a series of photopheresis treatments performed in photopheresis (extracorporeal circulation photochemotherapy). a step of introducing the photopheresis liquid sample S into the flow path 12 of the device 10; and irradiating with light.
Specifically, the ultraviolet light irradiation method according to the present invention is performed as follows using the ultraviolet light irradiation apparatus as described above. In the following description, the photopheresis liquid sample S is assumed to be blood.

First, blood collected from a patient is introduced as a photopheresis liquid sample S into the flow path 12 of the microdevice 10 through an inlet at one end of the flow path 12 by, for example, an infusion pump. The blood introduced into the flow path 12 flows through the flow path 12 in one direction (rightward in FIG. 1) at a constant flow rate. Ultraviolet light from the light source 20 is transmitted through the wall portion 11 of the microdevice 10 and applied to the blood flowing through the light irradiation region R in the flow path 12 .
In the flow path 12, the cells to be treated (white blood cells S1) contained in the blood are mixed with the red blood cells S2 as they flow. In the flow path 12, the length of the direction in which the ultraviolet light is irradiated is in the range of 70 to 500 μm, which is slightly larger than that of the white blood cells. A comfortable environment is created. Therefore, part of the ultraviolet light that has passed through the wall portion 11 of the microdevice 10 is irradiated to the cells to be processed (white blood cells S1) without being absorbed by the red blood cells S2 in the blood flowing through the flow path 12. FIG. The cells to be treated (white blood cells S1) receive ultraviolet light from the light source 20 from the beginning to the end of the light irradiation region in the flow path 12 . When the leukocyte S1 is irradiated with ultraviolet light, the leukocyte S1 undergoes apoptosis or is damaged to induce immune tolerance. The blood that has been irradiated with the ultraviolet light then flows through the flow path 12, is discharged from the outlet, and is returned to the patient's body.

The flow velocity of the liquid sample S for photopheresis in the flow path 12 depends on the type of the liquid sample S for photopheresis, the type of the light source 20, the wavelength and irradiation amount of the emitted ultraviolet light, the width of the flow path in the flow path 12, and the light intensity. Although it depends on the length of the irradiation area, if the speed is enough to obtain the effect of apoptosis or damage to the treated cells and induce immune tolerance good.

In the ultraviolet light irradiation method as described above, the liquid sample S for photopheresis containing cells to be treated is introduced into the flow path 12 whose length in the ultraviolet light irradiation direction is regulated to 70 μm or more and 500 μm or less. As a result, clogging of the liquid sample S for photopheresis in the flow path 12 can be suppressed, so that the liquid sample S for photopheresis can be rapidly irradiated with ultraviolet light, and moreover, the photopheresis liquid sample S can be rapidly irradiated with ultraviolet light. Even if the liquid sample S for pheresis contains components other than the cells to be processed together with the cells to be processed and is circulated in the flow path, the cells to be processed contained in the liquid sample S for photopheresis are irradiated with ultraviolet light with high uniformity. Light can be applied. Therefore, although no photosensitive medicine is used and centrifugation is not required, the cells to be treated contained in the liquid sample S for photopheresis can be apoptotic or damaged with high accuracy. Tolerance can be induced. As a result, in photopheresis, the time required for complicated processing such as centrifugation of the photopheresis liquid sample S can be greatly reduced, and the burden on the patient can be greatly reduced.

Although the embodiments of the ultraviolet light irradiation method, the microdevice, and the ultraviolet light irradiation apparatus for photopheresis of the present invention have been specifically described above, the present invention is not limited to the above examples, and can be used in various ways. changes can be made.
For example, the microdevice may have a plurality of flow paths independently of each other.
Specifically, as shown in FIG. 3, the microdevice 30 includes a cylindrical ultraviolet light-transmitting rod 31 and a plurality of flow passages 32 formed of through holes extending in the longitudinal direction of the rod 31. , may be provided in parallel independently of each other. In this rod body 31, a plurality of flow passages 32 are arranged in parallel and at regular intervals on a virtual plane A separated substantially parallel to the plane containing the central axis of the rod body 31, and the virtual plane A and a plurality of flow passages 32 are arranged in parallel and at regular intervals on a virtual plane B which is plane-symmetrical with respect to the plane including the central axis of the rod body 31 .
In the ultraviolet light irradiation apparatus provided with this microdevice 30 , the light sources are arranged so that the ultraviolet light from the light source is applied to all the flow paths 32 without passing through other flow paths 32 . Specifically, for this microdevice 30, ultraviolet light is transmitted through the rod body 31 from the virtual plane A side (upper side in FIG. 3) and the virtual plane B side (lower side in FIG. 3). is preferably irradiated. Furthermore, the light source for irradiating ultraviolet light is preferably a planar light source having a width corresponding to the arrangement width of the flow path 32 .
In this microdevice 30, it is preferable that the irradiation direction length of all flow paths 32 is set to 70 μm or more and 500 μm or less.

Further, as shown in FIG. 4, the microdevice 40 has two flow passages 42 formed of wide slits extending in the longitudinal direction through a cylindrical ultraviolet light transmissive rod 41 . Alternatively, the slits may be provided so that the plane directions in which the slits extend independently of each other are parallel to each other. In this rod body 41 , two flow passages 42 are arranged symmetrically so as to sandwich a plane including the central axis of the rod body 41 .
In the ultraviolet light irradiation apparatus having this microdevice 40 , the light sources are arranged so that the two flow paths 42 are irradiated with ultraviolet light from the light sources without passing through the other flow path 42 . Specifically, with respect to this microdevice 40, the rod body 41 is attached from the upper side (upper side in FIG. 4) and the lower side (lower side in FIG. 4) of the plane including the central axis of the rod body 41. It is preferable to irradiate ultraviolet light through it. Further, the light source for irradiating the ultraviolet light is preferably a planar light source having a width corresponding to the width of the slit forming the flow path 42 (the width in the left-right direction in FIG. 4).
In this microdevice 40, it is preferable that each of the two flow paths 42 has a length of 70 μm or more and 500 μm or less in the irradiation direction.

Further, as shown in FIG. 5, the microdevice 50 includes a cylindrical rod having ultraviolet light transmissivity, and a plurality of flow passages formed of through holes extending in the longitudinal direction of the rod. can be provided. In the rod body 51 of the microdevice 50, the flow passages 52 made of a plurality of holes extending in the longitudinal direction of the rod body 51 through the rod body 51 are formed in a regular manner surrounding the central axis P of the rod body 51. are arranged independently of each other. The flow passages 52 in the rod body 51 are equidistant from the central axis P and arranged side by side so that the adjacent flow passages 52 are equidistant from each other.
In the ultraviolet light irradiation apparatus having this microdevice 50 , the light sources are arranged so that the ultraviolet light from the light source is applied to all the flow paths 52 without passing through other flow paths 52 . It is preferable to irradiate the microdevice 50 with ultraviolet light passing through the rod body 51 from three different directions. Specifically, three light sources (not shown) have their optical axes aligned in the direction of flow of the liquid sample S for photopheresis in the flow path 52 of the microdevice 50 (the direction perpendicular to the paper surface in FIG. 5). They are arranged so as to be substantially perpendicular and intersect each other on the central axis P at an intersecting angle of 120 degrees.
In this microdevice 50, it is preferable that the irradiation direction length of all the flow paths 52 is 70 μm or more and 500 μm or less.

Furthermore, the microdevice may be configured by combining a plurality of capillaries each having one flow path.

According to the microdevice having a plurality of channels as described above, the total width of the channels can be increased, so that the speed of the photopheresis process can be improved.

Also, for example, microdevices are not limited to cylindrical ones such as thin tubes and rod bodies, and as shown in FIG. It may consist of a flat plate-like microchip 60 in which microchannels are formed by microfabrication technology.
Specifically, the microchip 60 is composed of a pair of microchip substrates 61A and 61B, and the pair of microchips is formed in a state in which a microchannel (channel forming portion) is formed on the surface of one of the microchip substrates 61A. By joining the substrates 61A and 61B so as to face each other to close the microchannel, a flow path 62 through which the liquid sample S for photopheresis flows is formed. In this microchip 60, one microchip substrate 61A on which microchannels are formed is made of silicone rubber such as polydimethylsiloxane (PDMS), and the other microchip substrate 61B is made of quartz glass.
One or a plurality of flow paths 62 are formed in the microchip 60 . In FIG. 6, six flow passages 62 are formed.
In the ultraviolet light irradiation device having this microchip 60 , the light sources are arranged so that the ultraviolet light from the light source is applied to all the flow passages 62 without passing through other flow passages 62 . Specifically, for this microchip 60, ultraviolet light is transmitted through the other microchip substrate 61B from the substrate side made of a material having good ultraviolet light transmittance, that is, from the other microchip substrate 61B side. Irradiation is preferred. Furthermore, the light source for irradiating ultraviolet light is preferably a planar light source having a width corresponding to the arrangement width of the flow path 62 .
In this microchip 60, it is preferable that the irradiation direction length of all flow paths 62 is 70 μm or more and 500 μm or less.

In addition, the ultraviolet light irradiation method for photopheresis of the present invention is carried out by flow irradiation, that is, continuously while circulating the liquid sample for photopheresis in the flow path of the microdevice from the viewpoint of improving the processing speed. is preferred, but batch irradiation can also be carried out.

Experimental examples of the present invention will be described below.

[Experimental example 1]
The main mechanism of photopheresis is that ultraviolet light irradiation induces apoptosis of activated T cells, resulting in immunosuppression. In the following, it was confirmed whether the method according to the present invention could induce apoptosis of T cells.
Untreated Jurkat cells: A cell proliferation assay reagent "CellTrace Far Red Cell Proliferation Kit" (manufactured by Thermo Fisher Scientific) was added to a human acute T-cell leukemia cell line (manufactured by Riken Cell Bank), and 37 After culturing for 10 minutes under conditions of 5% CO ₂ at 37° C., culture medium was added and culture was continued for 5 minutes under conditions of 37° C. and 5% CO ₂ . After that, centrifugation was performed and the supernatant was removed to obtain labeled Jurkat cells. The resulting labeled Jurkat cells were diluted with PBS (phosphate-buffered saline) to 1×10 ⁷ cells/mL to prepare a Jurkat cell suspension. Jurkat cell suspension: blood by volume 0% (100:0), 5% (95:5), 10% (90:10), 20% (80:20), 50% (50:50) , and 70% (30:70) mixtures [1] to [6] were prepared.
Each of the mixtures [1] to [6] was filled in a quartz tube having a length of 50 mm and irradiated with ultraviolet light using a spectral irradiator. The irradiated ultraviolet light has an irradiation wavelength of 290 nm and an irradiation amount of 10 mJ/cm ² (irradiance: 0.16 mW/cm ² , irradiation time: 63 seconds). Quartz narrow tubes with inner diameters of 200 μm, 300 μm, 600 μm and 900 μm were used. All quartz tubules have an outer diameter of 3 mm.
After irradiation with ultraviolet light, the mixtures [1] to [6] were collected, the culture medium was added, and the mixture was cultured for 24 hours under conditions of 37° C. and 5% CO ₂ , followed by FACS (fluorescence activated cell sorting) analysis. , examined the rate of AnnexinV positivity or apoptosis induction of labeled Jurkat cells. The results are shown in FIG.

From the results of Experimental Example 1, it was confirmed that the smaller the diameter of the quartz tubule, the higher the apoptosis induction ratio, that is, the effect of causing apoptosis of the labeled Jurkat cells. The values in the graph of FIG. 7 are the average values of N=3, and the error bars indicate the standard deviation.

[Experimental example 2]
Untreated Jurkat cells: A cell proliferation assay reagent "CellTrace Far Red Cell Proliferation Kit" (manufactured by Thermo Fisher Scientific) was added to a human acute T-cell leukemia cell line (manufactured by Riken Cell Bank), and 37 After culturing for 10 minutes at 5% CO ₂ at 37° C., the culture solution was added and cultured for 5 minutes at 37° C. and 5% CO ₂ . After that, centrifugation was performed and the supernatant was removed to obtain labeled Jurkat cells. The resulting labeled Jurkat cells were diluted with PBS (phosphate-buffered saline) to 1×10 ⁷ cells/mL to prepare a Jurkat cell suspension. A mixed solution [A] was prepared so that the concentration of Jurkat cell suspension:blood was 20% (80:20) by volume.
On the other hand, an experimental photopheresis device shown in FIG. 8 was produced. Specifically, infusion tubes 73 and 74 made of silicon tubes are attached to both ends of a quartz tube 70 having a length of 50 mm, an outer diameter of 3 mm, and an inner diameter of 300 μm. 77 intervened. An LED illuminator 80 was provided above the quartz tube 70 so that the quartz tube 70 was irradiated with ultraviolet light.
Using this experimental photopheresis apparatus, an ultraviolet light irradiation treatment was performed by irradiating ultraviolet light with an irradiation wavelength of 290 nm from the LED irradiator 80 while the liquid mixture [A] was being circulated in the quartz narrow tube. Specifically, the liquid mixture [A] (indicated by symbol SA in FIG. 8) contained in the supply sample tube 78 is transported by the transport pump 77 to the quartz tubule 70 via the infusion tube 73, and the quartz tubule is 70 and the other infusion tube 74 were collected in a collection sample tube 79 while flowing. The flow rate of the liquid mixture [A] was set so that the passage time of the quartz narrow tube, that is, the ultraviolet light irradiation time was 5.6 seconds.
This ultraviolet light irradiation treatment was performed under the conditions that the irradiation amount of ultraviolet light was 0 mJ/cm ² , 6 mJ/cm ² , 11 mJ/cm ² , 20 mJ/cm ² , 35 mJ/cm ² and 71 mJ/cm ² . The dose of UV light was varied by changing the irradiance by current control according to Table 1.
After irradiation with ultraviolet light, the mixed solution [A] was collected, the culture solution was added, and cultured under conditions of 37°C and 5% CO ₂ for 24 hours. The rate of AnnexinV positivity of cells, ie induction of apoptosis, was examined. The results are shown in FIG.

From the results of Experimental Example 2, the effect of apoptosis of the labeled Jurkat cells can be obtained even in the flow irradiation method, that is, the method in which the mixed solution [A] is continuously circulated in the quartz tubule while being irradiated with ultraviolet light. It was confirmed that the apoptosis induction rate increased dose-dependently. The values in the graph of FIG. 9 are the average values of N=3, and the error bars indicate the standard deviation.

10 microdevice 11 wall 12 flow path 20 light source 30 microdevice 31 rod body 32 flow path 40 microdevice 41 rod body 42 flow path 50 microdevice 51 rod body 52 flow path 60 microchips 61A, 61B microchip substrate 62 flow path 70 Quartz tubules 73, 74 Infusion tube 77 Transport pump 78 Supply sample tube 79 Recovery sample tube 80 LED illuminator C Traveling direction P Central axis R Light irradiation area S Liquid sample for photopheresis S1 White blood cell S2 Red blood cell

Claims (6)

A flow passage is provided in the wall portion, a photopheresis liquid sample is introduced into the flow passage, and ultraviolet light is transmitted through the wall portion to the photopheresis liquid sample introduced into the flow passage. A photopheresis microdevice irradiated with
The length of the distribution path in the irradiation direction of the ultraviolet light is 70 μm or more and 500 μm or less,
The microdevice for photopheresis comprises a cylindrical rod having ultraviolet light transmissivity, and a plurality of flow passages formed of through holes extending in the longitudinal direction of the rod are provided independently of each other. ,
1. A microdevice for photopheresis, wherein said flow path is formed of a wide slit, and said two flow paths independent of each other are provided so that plane directions in which said slits extend are parallel to each other. The liquid sample for photopheresis is blood,
2. The microdevice for photopheresis according to claim 1, wherein said wall transmits ultraviolet light including light in a wavelength range of 280 nm or more and 320 nm or less. 3. A microdevice for photopheresis according to claim 1 or claim 2, characterized in that the channel has a length of at least 50 mm. a microdevice for photopheresis according to any one of claims 1 to 3;
a light source that irradiates the flow path with ultraviolet light from the outside of the microdevice for photopheresis through the wall portion of the microdevice for photopheresis. Ultraviolet light irradiation device for 5. The ultraviolet light irradiation device for photopheresis according to claim 4, wherein the light source emits ultraviolet light including light in a wavelength range of 280 nm or more and 320 nm or less. 6. The photovoltaic device according to claim 4, wherein the light source is arranged so that the ultraviolet light is emitted in a direction different from a direction in which the liquid sample for photopheresis flows in the flow path. Ultraviolet light irradiation device for pheresis.

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