Leukocyte Capture Device

Abstract

The present invention addresses the problem of providing a white blood cell capturing device that captures white blood cells with high efficiency while being unsusceptible to capturing other material. The problem is solved by this white blood cell capturing device, which captures white blood cells included in a blood-containing liquid, and includes a sample entrance, a buffer entrance, a white blood cell separation unit, a main part, an auxiliary part, and an exit, wherein: the main part has a flat portion and a large number of protrusions provided thereon, the main part being configured such that a buffer which has entered from the entrance passes above the main part and is discharged from the exit; the protrusions are provided in layered form on the surface of the flat portion, each layer includes a plurality of the protrusions, and the protrusions are configured such that a buffer that has passed through a layer on the entrance side passes through a layer on the exit side adjacent thereto; and the capture part faces the exit side of all or some of the bypass parts in a specific layer, and is disposed as a part of a separate layer adjacent thereto.

Description

[Technical Field]

[0001] The present invention relates to a leukocyte capturing device. [Background technology]

[0002] DNA is damaged by radiation exposure and the influence of the living environment. Damaged DNA is thought to be closely related to diseases such as cancer. Traditionally, the method used to analyze this DNA damage involves centrifuging blood to extract white blood cells, staining them, and then observing them on a glass slide.

[0003] Microchannels for capturing particles such as white blood cells have also been proposed (see Patent Document 1). Patent Document 1 discloses a microchannel device with a filter function that uses a microchannel equipped with a concave capture element to capture and separate only solids of a certain size or larger from a solid-liquid mixture. In Patent Document 1, one capture element accommodates one or more solids of a certain size or larger, and the objective is to completely capture all solids of a certain size or larger by the end of a separation element in which multiple capture elements are arranged. However, the capture of only one solid in one capture element to facilitate observation of the captured solid, such as a white blood cell, is not taken into consideration. Furthermore, the solid-liquid mixture must always flow from the inlet to the outlet of the microchannel to prevent solids once captured in the capture element from resuspending and flowing out of the capture element. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2009-109232 Summary of the Invention [Problem to be solved by the invention]

[0005] However, in conventional methods, the liquid in which white blood cells are captured contains red blood cells, proteins, etc., and these coagulated substances are captured along with the white blood cells, which can sometimes become noise when observing the white blood cells.

[0006] The present invention has an object to solve the above-mentioned problems, that is, to provide a leukocyte capturing device that captures leukocytes with high efficiency and is less likely to capture other cells. [Means for solving the problem]

[0007] The present invention includes the following (1) to (6). (1) A leukocyte capturing device that captures leukocytes contained in a blood-containing liquid by passing the blood-containing liquid through the device, a sample inlet into which the blood-containing liquid X1 is introduced; a buffer inlet into which buffer Y1 is introduced; the blood-containing liquid (X1) and the buffer (Y1) are configured to flow in the same specific direction (Z) on the surface of the flat surface and between the pillars and other pillars adjacent thereto so that they are adjacent to each other; when the surface of the planar portion is viewed in a direction parallel to the perpendicular line, the pillars form a plurality of rows, and each row is arranged so as to form an angle θ with respect to the same direction Z in which the blood-containing liquid X1 and the buffer Y1 flow; When the surface of the planar portion is viewed from a direction parallel to the perpendicular line, the distance between the pillar and the adjacent pillar is set to g in a direction perpendicular to the same direction Z. In this case, Dc=1.4g×(1 / tanθ) -0.48 The boundary diameter Dc defined by is smaller than the diameter of the leukocyte, a leukocyte separation unit which separates the leukocytes from the blood-containing liquid X1 during the process in which the blood-containing liquid X1 and the buffer Y1 flow in the same specific direction Z, and causes the leukocytes to be contained in the buffer Y1, thereby generating a buffer Y2 containing the leukocytes and a blood-containing liquid X2 as a remainder from which the leukocytes have been separated, and discharging these; a main part that passes the buffer Y2 discharged from the leukocyte separation unit and captures the leukocytes contained therein; an auxiliary section having the same flow path resistance as the main section, through which the blood-containing liquid X2 discharged from the leukocyte separation unit passes; an outlet through which the liquid flows after passing through the main portion and the auxiliary portion; and The main portion is the main portion has a flat portion and a number of convex portions provided thereon, and the buffer Y2 entering from the inlet passes over the surface of the flat portion of the main portion and between the convex portion and another convex portion adjacent thereto, and is discharged from the outlet; the convex portions are provided in layers on the surface of the flat portion, each layer including a plurality of the convex portions, and the buffer Y2 having passed through an inlet-side layer passes through an outlet-side layer adjacent thereto; In each layer, a capture section is formed in which the width between the convex section and another convex section adjacent thereto is set to 2 to 7.5 μm, and a bypass section is formed in which the width is set to 8 to 20 μm, The width of the inlet side portions of the two convex portions constituting the capture portion is chamfered so as to gradually narrow toward the back of the capture portion, A leukocyte capture device, wherein the capture section is disposed as part of another layer adjacent to a particular layer, facing the outlet side of all or part of the bypass section in that particular layer. (2) The leukocyte capturing device according to (1) above, wherein the auxiliary part has the same structure as the main part. (3) The leukocyte capturing device according to (1) or (2) above, wherein the width between a specific layer and another layer adjacent thereto in the main portion is 8 to 30 μm. (4) The leukocyte capturing device according to any one of (1) to (3) above, wherein in the main portion, the ratio of the width of the bypass portion to the width of the capturing portion is greater than 1 and is 3 or less. (5) A leukocyte capture device according to any one of (1) to (4) above, wherein in the main portion, the portion other than the capture portion on the inlet-side end face of the convex portion extends parallel to the layer direction, and the end face constituting the bypass portion of the convex portion extends perpendicular to the layer direction. (6) The leukocyte capturing device according to any one of (1) to (5) above, wherein the boundary diameter Dc is 5 μm or more and less than 10 μm. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a leukocyte capturing device that captures leukocytes with high efficiency and is less likely to capture other cells. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic plan view showing a leukocyte capturing device of the present invention. [Figure 2] FIG. 2 is a cross-sectional view (schematic cross-sectional view) taken along line AA in FIG. [Figure 3] FIG. 3 is an enlarged view of the junction between the flow channels 5 and 6 and the inlet 10 in of the leukocyte separation unit 10 . [Figure 4] FIG. 4 is an enlarged (schematic) view of the surface of FIG. [Figure 5] FIG. 5 is a schematic diagram of the surface of the main portion 50 as viewed in a direction parallel to this perpendicular line. [Figure 6] FIG. 6 is an enlarged view (schematic diagram) of B in FIG. [Figure 7] FIG. 7 is a cross-sectional view taken along line CC in FIG. [Figure 8] 1 is a microscopic photograph (eyepiece 10x x objective 10x) of a leukocyte capturing device according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0010] The leukocyte capturing device of the present invention will now be described. In the leukocyte capture device of the present invention, a blood-containing liquid that has entered through an inlet is forced to flow toward an outlet by a pump (such as a syringe pump), hydrostatic pressure, electroosmotic flow, etc. During this process, leukocytes are transferred from the blood-containing liquid into a buffer, and then captured by a capture section located between a convex portion on the main section and another convex portion adjacent to it.

[0011] The leukocyte capturing device of the present invention can be produced, for example, by pouring rubber into a mold in which a flow channel pattern has been formed to obtain a flow channel chip, and then bonding this to a glass substrate.

[0012] The leukocyte capturing device of the present invention will be described with reference to the drawings. FIG. 1 is a schematic plan view showing a leukocyte capturing device 1 of the present invention. The leukocyte capturing device 1 of the present invention shown in FIG. 1 is obtained by laminating a channel chip, which is obtained by pouring rubber into a mold in which a channel pattern has been formed, onto a glass substrate. However, a lid (described later) that covers the top surface of the leukocyte capturing device 1 of the present invention is not shown in FIG.

[0013] The leukocyte capturing device of the present invention will be described below with reference to the drawings as a preferred embodiment and is an example, but the leukocyte capturing device of the present invention is not limited to the embodiment shown in the drawings.

[0014] The leukocyte capturing device 1 of the present invention has a sample inlet 3 , a buffer inlet 4 , a leukocyte separation unit 10 , a main section 50 , an auxiliary section 70 , and an outlet 9 .

[0015] <Sample entrance> A blood-containing liquid X1 is introduced into the sample inlet 3. The sample inlet 3 may be a hole in which the introduced blood-containing liquid X1 can temporarily remain. For example, in the embodiment shown in FIG. 1, a through-hole is formed in a channel chip obtained by pouring rubber into a mold on which a channel pattern has been formed, and this is then attached to a glass substrate to form a hole in which the blood-containing liquid X1 can temporarily remain, which can be used as the sample inlet 3.

[0016] The sample inlet 3 and the inlet 10in of the leukocyte separation unit 10 are connected by a flow path 5, and the blood-containing liquid X1 introduced into the sample inlet 3 passes through the flow path 5 and is introduced into the leukocyte separation unit 10 from the inlet 10in.

[0017] The channel 5 may be formed, for example, by pillars. The pillars prevent the channel 5 from being blocked when through-holes are formed in a channel chip obtained by pouring rubber into a mold on which a channel pattern has been formed, and the chip is then attached to a glass substrate.

[0018] Here, the blood-containing liquid X1 is not particularly limited as long as it contains the blood of an animal, including a human, and may be, for example, a mixed liquid in which human blood is added to a phosphate buffer solution (PBS), an anticoagulant, a staining solution, etc. Furthermore, the blood-containing liquid may be the blood itself of an animal, including a human.

[0019] <Buffer inlet> The buffer inlet 4 is used to introduce the buffer Y1. The buffer inlet 4 may be a hole where the introduced buffer Y1 can temporarily stay. For example, in the embodiment shown in FIG. 1, a through-hole is formed in a channel chip obtained by pouring rubber into a mold on which a channel pattern has been formed, and this is then attached to a glass substrate to form a hole in which buffer Y1 can temporarily remain, which can be used as the buffer inlet 4.

[0020] The buffer inlet 4 and the inlet 10in of the leukocyte separation unit 10 are connected by a flow path 6, and the buffer Y1 introduced into the buffer inlet 4 passes through the flow path 6 and is introduced into the leukocyte separation unit 10 from the inlet 10in. Like the flow channel 5, the flow channel 6 may be formed of, for example, a pillar.

[0021] Here, buffer Y1 is not particularly limited as long as it is a reagent used in the analysis of DNA damage, and may be, for example, phosphate buffered saline (PBS), paraformaldehyde (PFA), DAPI, or the like.

[0022] <Leukocyte Separation Unit> The leukocyte separation unit 10 will be described with reference to FIGS. 2, 3 and 4 in addition to FIG. Figure 2 is a cross-sectional view (schematic cross-sectional view) along line AA in Figure 1, Figure 3 is an enlarged view of the connection between flow path 5 and flow path 6 and inlet 10in of white blood cell separation unit 10, and Figure 4 is an enlarged view (schematic view) of the surface of white blood cell separation unit 10 in Figure 1.

[0023] As shown in FIG. 2, the leukocyte separation unit 10 has a flat surface 11 and a plurality of pillars 13 extending in the direction perpendicular to the flat surface 11. As described above, in this example, a channel chip is obtained by pouring rubber into a mold in which a channel pattern is formed, and then the channel chip is bonded to a glass substrate, and the glass substrate forms the flat portion 11. Furthermore, the leukocyte separation unit 10 shown in Fig. 2 has a plate-shaped lid 15 that covers the top of the pillars 13 (illustration of the lid 15 is omitted in Figs. 1 and 4). In Fig. 2, the boundary between the pillars 13 and the lid 15 is indicated by a dotted line, but the pillars 13 and the lid 15 may be integral with each other or may be configured to be separable. By forming the shape of the mold used to obtain the channel chip by pouring rubber into a channel pattern that integrates the pillars 13 and the lid 15, a channel chip in which the pillars 13 and the lid 15 are formed integrally can be obtained.

[0024] Surface 11 of flat portion 11 S Above and between the pillar 13 and the adjacent pillar 13, the blood-containing liquid X1 and the buffer Y1 flow.

[0025] 3, the blood-containing liquid X1 that arrives at the inlet 10in of the leukocyte separation unit 10 from the flow path 5 and flows into the inside of the leukocyte separation unit 10 from the inlet 10in, and the buffer Y1 that arrives at the inlet 10in of the leukocyte separation unit 10 from the flow path 6 and flows into the inside of the leukocyte separation unit 10 from the inlet 10in, both flow in a specific direction (the same direction Z) inside the leukocyte separation unit 10. That is, the direction F in which the blood-containing liquid X1 flows in the leukocyte capturing device 10 is the same as the direction F in which the blood-containing liquid X1 flows inside the leukocyte capturing device 10. X and the direction F in which buffer Y1 flows. Y are substantially parallel to each other, and their directions are the same direction Z.

[0026] Furthermore, the blood-containing liquid X1 and the buffer Y1 each form a roughly laminar flow. X Arrows and direction F Y The arrows pointing in the same direction Z and the dotted line between them are parallel to the arrows pointing in the same direction Z, and they flow next to each other. This is mainly due to the extremely low Reynolds numbers of the blood-containing solution X1 and the buffer Y1.

[0027] FIG. 4 shows the surface 11 of the flat portion 11. S This is an enlarged view of the surface 11 S 4 corresponds to a view of the planar portion 11 viewed from a direction parallel to the perpendicular line of the plate 1. As shown in FIG. 4, the pillars 13 are arranged in multiple rows, and the white blood cells contained in the blood-containing fluid X1 are separated from other components such as red blood cells and platelets by a deterministic lateral displacement (DLD) method. The diameter of white blood cells in blood is about 10 to 20 μm, the diameter of red blood cells is about 7 to 8 μm, and the diameter of platelets is 2 to 3 μm. Since white blood cells are the largest, they can be separated by this DLD method. The DLD method is disclosed in "Development of Deterministic Lateral Displacement Device for Separation of Particles" by Naotomo Tottori and three others, Proceedings of the 2015 Japan Society for Precision Engineering Spring Meeting Academic Lecture Series, Japan Society for Precision Engineering, 2015, pp. 743-744, and in International Publication No. 2016 / 136273. The DLD method is a technology that uses a large number of pillars regularly arranged in a microfluidic device to separate large and small particles from a liquid flow in which particles are dispersed.

[0028] As shown in FIG. 4, the pillars 13 are arranged in a plurality of rows, each row forming an angle θ with respect to the same direction Z in which the blood-containing liquid X1 and the buffer Y1 flow. The white blood cells 20 move diagonally along the inclination of the row of pillars 13. On the other hand, small particles 22, which have a smaller diameter than the white blood cells 20, change their flow direction due to the pillars 13 and move in the direction of the flow (F X and the same direction Z), but generally moves linearly along the laminar flow. Therefore, when the blood-containing liquid X1 and the buffer Y1 flow adjacent to each other on the flat surface portion 11 of the leukocyte capturing device 10 in the same specific direction Z, the leukocytes 20 in the blood-containing liquid X1 move toward the buffer Y1, and from the interface between the laminar flow of the blood-containing liquid X1 and the laminar flow of the buffer Y1, the leukocytes 20 move into the buffer Y1.

[0029] The flow direction of the blood-containing fluid X1 (F X According to "Development of a Particle Separation Device Using a Deterministic Lateral Displacement Method" and WO 2016 / 136273, the boundary diameter Dc between small particles moving in the same direction (and the same direction Z) and large particles moving obliquely can be calculated using the following equation 1: Equation 1...Dc=1.4gN -0.48 Here, g is the surface 11 of the flat portion 11 as shown in FIG. S In the drawing when the plane portion 11 is viewed from a direction parallel to the perpendicular line of Xis the spacing between the pillars 13 in the direction perpendicular to the same direction Z), and N is the number of flow streams, which is expressed by the following formula: N=1 / ε Here, ε is the row shift fraction, i.e., the shift rate of the pillar 13, and ε=tan θ.

[0030] According to WO 2016 / 136273, the boundary diameter Dc can be calculated using the following formula, which is equivalent to Formula 1: Dc=1.4g·ε 0.48 Particles with diameters less than the boundary diameter Dc move generally along the direction of the flow, while particles with diameters greater than the boundary diameter Dc move diagonally. As mentioned above, since the white blood cells 20 have a larger diameter than the others, Dc = 1.4g × (1 / tan θ) -0.48 By setting the boundary diameter Dc defined by the above to be smaller than the diameter of the white blood cells and larger than the diameter of other particles to be separated (such as platelets), the white blood cells 20 can be moved diagonally along the slope of the row of pillars 13 and into buffer Y1 as described above.

[0031] For example, if the boundary diameter Dc is set to 5 μm or more and less than 10 μm, the boundary diameter Dc will be smaller than the diameter of white blood cells (approximately 10 to 20 μm) and larger than other particles to be separated (such as platelets), allowing for efficient separation of white blood cells.

[0032] It should be noted that the number and positions of the pillars 13 are not necessarily accurate in Fig. 4. Although the cross-sectional shape of the pillars 13 is circular in Fig. 4, the cross-sectional shape may be other shapes as long as the above-mentioned separating action is exerted.

[0033] The buffer Y1 containing the leukocytes separated from the blood-containing liquid X1 as described above will be referred to as buffer Y2 below. The remainder of the blood-containing fluid X1 after the white blood cells have been separated will be referred to as blood-containing fluid X2 below. Then, each of these is discharged from the leukocyte separation unit 10. The buffer Y2 discharged from the leukocyte separation unit 10 flows toward the main section, which will be described later. Furthermore, the blood-containing fluid X2 discharged from the leukocyte separation unit 10 flows toward the auxiliary section, which will be described later.

[0034] <Main section> The main part 50 is a tip that passes the buffer Y2 and captures the white blood cells contained in the buffer Y2. The main portion 50 will be defined using Figures 5 to 7. Figure 5 is a schematic diagram of the surface of the main portion 50 as viewed from a direction parallel to the perpendicular line, Figure 6 is an enlarged view (schematic diagram) of B in Figure 5, and Figure 7 is a cross-sectional view taken along line CC in Figure 5. However, a lid (described later) that covers the top surface of the main portion 50 is not shown in FIGS.

[0035] The main portion 50 illustrated in FIG. 5 has an inlet 50in through which the buffer Y2 enters the interior thereof, and an outlet 50out through which the buffer Y2 that has passed through the main portion 50 is discharged. The configuration of the main portion 50 is not limited to the examples shown in FIGS.

[0036] As shown in FIGS. 5 to 7, the main portion 50 includes a flat portion 52 and a number of protrusions 54 provided thereon. As described above, in this example, a channel chip is obtained by pouring rubber into a mold in which a channel pattern is formed, and then the channel chip is bonded to a glass substrate, and the glass substrate forms the flat portion 52. 5 to 7 has a plate-shaped lid 56 that covers the top of the protrusion 54 (illustration of the lid 56 is omitted in FIGS. 5 and 6). In FIG. 7, the boundary between the protrusion 54 and the lid 56 is indicated by a dotted line, but the protrusion 54 and the lid 56 may be integral with each other or may be configured to be separable. By forming the mold used to obtain the channel chip by pouring rubber into a channel pattern that integrates the protrusion 54 and the lid 56, a channel chip in which the protrusion 54 and the lid 56 are formed integrally can be obtained.

[0037] As shown in FIG. 5, the protrusions 54 are provided in layers on the flat surface portion 52. In Figure 5, the layer closest to the inlet 50 in is designated as layer 1, and the layer adjacent to layer 1 on the outlet side (downstream side) is designated as layer 2. Also, a certain layer is designated as layer P, the layer adjacent to layer P on the outlet side (downstream side) is designated as layer P+1, and the layer adjacent to layer P on the outlet side (downstream side) is designated as layer P+2. Each layer includes a plurality of protrusions 54. Although Fig. 5 shows an example in which each layer includes seven protrusions 54, the number of protrusions 54 included in each layer is not particularly limited. Furthermore, the number of layers is not particularly limited.

[0038] Buffer Y2 entering the main portion 50 from the inlet 50in flows over the surface of the flat portion 52, first passing through the flow paths between the convex portions 54 in the first layer, and then passing through the flow paths between the convex portions 54 in the second layer. Thereafter, the buffer Y2 is configured to pass through the flow paths between the convex portions 54 in the Pth layer, and then passing through the flow paths between the convex portions 54 in the P+1th layer in the same manner.

[0039] As shown in FIG. 6, each layer has a capture section 61 in which the width (flow path width) L1 between adjacent convex sections 54 is set to 2 to 7.5 μm, and a bypass section 63 in which the width L2 is set to 8 to 20 μm.

[0040] In the example of Fig. 6, in each of the Pth, P+1th, and P+2th layers, capture sections 61 and bypass sections 63 are alternately formed as flow paths between the multiple convex sections 54. However, in the main section 50, the capture sections and bypass sections formed in each layer do not have to be alternately formed as in Fig. 6. For example, multiple capture sections may be continuously formed within a layer.

[0041] Furthermore, a capture section 61 is arranged on the outlet side of a bypass section 63 in a specific layer as part of another adjacent layer. That is, in the example of Fig. 6, a capture section 61 in the (P+1)th layer is arranged on the outlet side (downstream side) of the bypass section 63 in the Pth layer. 6, it is preferable that the bypass section 63 in the Pth layer and the capture section 61 in the P+1th layer are arranged side by side in a direction perpendicular to the layer direction. More specifically, it is preferable that the bypass section 63 in the Pth layer and the capture section 61 in the P+1th layer are arranged so that when a straight line perpendicular to the layer direction is drawn, the straight line passes through the bypass section 63 in the Pth layer and the capture section 61 in the P+1th layer (i.e., the straight line does not contact the convex section 54).

[0042] In the examples shown in Figures 5 and 6, white blood cells contained in buffer Y2 that has flowed from the inlet side (upstream side) and reached layer P cannot, in principle, pass through capture section 61, so at least some of the white blood cells are captured by capture section 61 in layer P. When white blood cells are captured, capture section 61 is blocked. On the other hand, even if components other than white blood cells (red blood cells, platelets, etc.) are contained in buffer Y2, they are not captured by capture section 61 in layer P, but pass through capture section 61 or bypass section 63 and reach layer P+1. Furthermore, all components contained in buffer Y2 that has reached layer P can pass through bypass section 63. Therefore, white blood cells that are not captured by capture section 61 in layer P pass through bypass section 63 in layer P and reach layer P+1, and at least some of them are captured by capture section 61 in layer P+1. Here, since the capture section 61 in the P+1th layer is arranged on the outlet side (downstream side) of the bypass section 63 in the Pth layer, white blood cells that pass through the bypass section 63 in the Pth layer are easily captured by the capture section 61 in the P+1th layer.

[0043] 5 and 6, the convex portion 54 is rectangular in shape, with the four corners partially chamfered (e.g., by straight or curved lines). The area of ​​the chamfered portion (e.g., by straight or curved lines) is preferably smaller than the area (projected area) of the white blood cells to be captured. 6, the inlet portions of the two protrusions 54 that make up the capture part 61 are chamfered so that they become gradually narrower toward the back of the capture part 61. This is because white blood cells are more easily captured by the capture part 61. Also, once captured by the capture part 61, white blood cells are deformed and become trapped in the chamfered portions, making them less likely to flow out of the capture part.

[0044] When the chamfer is formed by cutting off a straight line, the angle of the chamfered line with respect to the direction perpendicular to the layer direction (the direction from the inlet to the outlet) is preferably 30 to 60 degrees. Here, when the chamfer is not linear but, for example, spoon-shaped or rounded, the average angle of the tangents is preferably 30 to 60 degrees. If this angle is less than 30 degrees, the flow rate of white blood cells into the bypass section 63 tends to increase, resulting in lower capture efficiency. Furthermore, if this angle is greater than 60 degrees, the probability of multiple white blood cells being captured by one capture section 61 tends to increase.

[0045] As long as the chamfering gradually narrows toward the back of the capturing portion 61, both entry-side portions of the two convex portions that make up the capturing portion 61 may be chamfered, or only one entry-side portion may be chamfered. Furthermore, if both entry-side portions are chamfered, the angles of the chamfering may be the same or different.

[0046] When the convex portions 54 are rectangular in shape with some of the four corners cut off (for example, by straight lines or curves) to be chamfer them, leukocytes that reach the capture portion 61 where another leukocyte has already been captured tend to move in the layer direction along the end faces of the convex portions 54, move from the bypass portion 63 to the adjacent downstream layer, and are more likely to be captured by the capture portion 61 in the downstream layer. As a result, the inventors have found that the leukocyte capture efficiency is improved.

[0047] As shown in Figure 6, in the main part 50, it is preferable that the part other than the capture part 61 on the inlet side end face of the convex part 54 extends parallel to the layer direction, and that the end face constituting the bypass part 63 in the convex part 54 extends perpendicular to the layer direction. In particular, as shown in Figure 6, if the inlet portions of the two convex portions 54 that make up the capture section 61 are chamfered (preferably linearly) so as to gradually narrow continuously toward the back of the capture section 61, and the inlet end faces of the convex portions 54, other than the capture section 61, extend parallel to the layer direction, and the bypass section 63 extends perpendicular to the layer direction, this effect becomes more pronounced, and the efficiency of capturing leukocytes is further improved, which is preferable. If the convex portion 54 is not based on a rectangle (for example, if it is circular or elliptical), its outer shape includes a radius, and the white blood cells may move along the radius and not move to the capture portion 61 in the adjacent layer downstream.

[0048] The width L1 of the capture part 61 is 2 to 7.5 μm, preferably 3 to 6 μm, and more preferably 4 to 5 μm. The width L2 of the bypass portion 63 is 8 to 20 μm, preferably 8.5 to 15 μm, and more preferably 9 to 10 μm. It should be noted that the width L1 and the width L2 refer to the shortest distance between the convex portion 54 and the convex portion 54 adjacent thereto in each layer.

[0049] Furthermore, the ratio (L2 / L1) of the width L2 of the bypass section 63 to the width L1 of the capture section 61 is preferably greater than 1 and not greater than 3, and more preferably 1.5 to 2.5. In this case, the flow to the bypass section 63 is appropriately suppressed, making it easier for white blood cells to be captured by the capture section.

[0050] Furthermore, the width L3 between the Pth layer and the P+1th layer is preferably 8 to 30 μm, and more preferably 9 to 10 μm. It should be noted that the width L3 means the shortest distance between the Pth layer and the P+1th layer. Furthermore, the maximum width L4 of the chamfered portion on the inlet side of the capture part 61 is preferably 10 to 35 μm, and more preferably 15 to 25 μm.

[0051] The height h of the protrusions 54 shown in FIG. 7 is preferably 8 to 30 μm, and more preferably 9 to 15 μm.

[0052] <Auxiliary part> The auxiliary section 70 allows the blood-containing fluid X2 discharged from the leukocyte separation unit 10 to pass through. The structure of the auxiliary section 70 has the same flow path resistance as the main section 50 described above. Here, the same flow path resistance means that the difference between the pressure loss from the inlet to the outlet of the main section and the pressure loss from the inlet to the outlet of the auxiliary section is within 20% (preferably within 10%) of the higher pressure loss. The auxiliary section 70 preferably has the same structure as the aforementioned main section 50. When the auxiliary section 70 has the same structure as the aforementioned main section 50, the flow path resistance to the buffer Y2 and the blood-containing liquid X2 becomes the same, and as a result, the flow rates of the blood-containing liquid X1 and the buffer Y1 flowing within the leukocyte separation unit 10 can be controlled to be the same, and the separation efficiency of leukocytes in the leukocyte separation unit 10 can be improved.

[0053] The leukocyte capturing device 1 of the present invention preferably has a partition wall 80 between the main section 50 and the auxiliary section 70 to separate them. The partition wall 80 is not particularly limited in shape, etc., as long as it has the function of separating the main portion 50 and the auxiliary portion 70.

[0054] <Exit> After passing through the main section 50 and the auxiliary section 70 , the liquid passes through the flow path 7 and flows out from the outlet 9 . The outlet 9 may be a hole through which the liquid can exit after passing through the main section 50 and the auxiliary section 70 . For example, in the embodiment shown in Figure 1, a through-hole is formed in a channel chip obtained by pouring rubber into a mold on which a channel pattern has been formed, and this is then attached to a glass substrate, thereby forming a hole through which liquid can flow out after passing through main section 50 and auxiliary section 70, and this hole can be used as outlet 9.

[0055] The outlet 50out of the main section 50 and the outlet of the auxiliary section 70 are connected to the outlet 9 by a flow path 7, and the liquid discharged from the main section 50 and the auxiliary section 70 passes through the flow path 7 and reaches the outlet 9.

[0056] The flow channel 7 may be configured with a material having the same functions as the above-described flow channels 5 and 6. The flow channel 7 may be configured with, for example, a pillar.

[0057] As described above, the leukocyte capturing device 1 of the present invention may be obtained by laminating a flow channel chip, which is obtained by pouring rubber into a mold on which a flow channel pattern has been formed, to a glass substrate. The size and material of the flow channel chip are not particularly limited. For example, the chip may be formed from a resin such as silicone rubber, acrylic resin, polycarbonate, cyclic olefin polymer, cyclic olefin copolymer, polystyrene, polyethylene, or polyethylene terephthalate. Furthermore, the substrate to which the rubber is affixed is preferably glass, but may be made of a material other than glass.

[0058] The leukocyte capture device of the present invention separates only leukocytes from a blood-containing liquid into a buffer, and then captures the leukocytes. In other words, when capturing the leukocytes, the buffer containing them does not contain red blood cells, proteins, etc. This prevents these coagulated substances from being captured along with the leukocytes. Therefore, there is no need to remove unnecessary blood or clean the flow path. [Example]

[0059] <Creating the main part> First, silicone rubber (SILPOT184, Dow Corning) was poured into a mold in which a flow path pattern had been formed. Next, the silicone rubber was vulcanized at 120°C for 30 minutes. Next, the silicone rubber was peeled off from the silicon wafer to form a channel-forming chip. Next, holes were made using a punch to serve as the sample inlet 3, buffer inlet 4 and outlet 9.

[0060] <Joining> Both the channel-forming chip with holes drilled as described above and the glass substrate were irradiated with vacuum ultraviolet light (L12530-01, Hamamatsu Photonics) for 15 seconds. The irradiated surfaces were then bonded together to obtain the leukocyte-trapping device 1 of the present invention shown in FIG.

[0061] <Experiment> Peripheral blood obtained from an adult male was diluted 10-fold with PBS to obtain blood-containing solution X1, and PBS was used as buffer Y1. Next, blood-containing liquid X1 was added dropwise to sample inlet 3, and buffer Y1 was added dropwise to buffer inlet 4. Next, the liquid was delivered from outlet 9 by negative pressure of a syringe pump.

[0062] The state of the liquid transfer was then observed using a microscope. A micrograph (eyepiece 10x x objective 10x) is shown in Figure 8. As shown in Figure 8, it was confirmed that the blood-containing liquid X1 and buffer Y1 were flowing in a laminar flow. It was also confirmed that the blood-containing liquid X2 was not flowing into the main part 50. It was also confirmed that white blood cells were separated from the blood-containing liquid X1 and transferred to the buffer Y1. Furthermore, when the liquid was continuously sent for 30 minutes, no clogging of the flow path in the main portion 50 was observed. It was also confirmed that leukocytes were captured in the main portion 50.

[0063] This application claims priority based on Japanese Patent Application No. 2022-51142, filed on March 28, 2022, the disclosure of which is incorporated herein in its entirety. [Explanation of symbols]

[0064] 1. Leukocyte capturing device of the present invention 3. Sample inlet 4 Buffer Exit 5, 6, 7 Flow paths 9 exit 10 Leukocyte Separation Unit 10in Leukocyte Separation Unit Inlet 11 Plane part 11 S Flat surface 13 Pillars 15 Lid 20 white blood cells 22 Small particles 50 Main Section 50in main entrance 50out Main section exit 52 Plane part 54 Convex part 56 Lid 61 Capture unit 63 Bypass section 70 Auxiliary part 80 Bulkhead

Claims

1. A leukocyte capturing device that captures leukocytes contained in a blood-containing liquid by passing the blood-containing liquid through the device, a sample inlet into which the blood-containing liquid X1 is introduced; a buffer inlet into which buffer Y1 is introduced; the blood-containing liquid (X1) and the buffer (Y1) are configured to flow in the same specific direction (Z) on the surface of the flat surface and between the pillars and other pillars adjacent thereto so that they are adjacent to each other; when the surface of the planar portion is viewed in a direction parallel to the perpendicular line, the columns are arranged in a plurality of rows, and each row is arranged so as to form an angle θ with respect to the same direction Z in which the blood-containing liquid X1 and the buffer Y1 flow, When the deterministic lateral displacement method (DLD method) is used, the distance between the pillar and the adjacent pillar in the direction perpendicular to the same direction Z when the surface of the planar portion is viewed from a direction parallel to the perpendicular line is set to g, and Dc = 1.4g × (1 / tan θ) -0.48 The boundary diameter Dc defined by is smaller than the diameter of the leukocyte, a leukocyte separation unit that separates the leukocytes from the blood-containing liquid X1 during the process in which the blood-containing liquid X1 and the buffer Y1 flow in the same specific direction Z and causes the leukocytes to be contained in the buffer Y1, thereby generating a buffer Y2 containing the leukocytes and a blood-containing liquid X2 as a remainder from which the leukocytes have been separated, and discharging these; a main part that passes the buffer Y2 discharged from the leukocyte separation unit and captures the leukocytes contained therein; an auxiliary section having the same flow path resistance as the main section, through which the blood-containing liquid X2 discharged from the leukocyte separation unit passes; an outlet through which the liquid flows after passing through the main portion and the auxiliary portion; and The main portion is a flat portion and a number of convex portions provided thereon, and the buffer Y2 entering from an inlet passes over the surface of the flat portion of the main portion and between the convex portion and another convex portion adjacent thereto, and is discharged from an outlet; the convex portions are provided in layers on the surface of the flat portion, each layer including a plurality of the convex portions, and the buffer Y2 having passed through an inlet-side layer passes through an adjacent outlet-side layer; In each layer, a capture section is formed in which the width between the convex section and another convex section adjacent thereto is set to 2 to 7.5 μm, and a bypass section is formed in which the width is set to 8 to 20 μm, The width of the inlet side portions of the two convex portions constituting the capture portion is chamfered so as to gradually narrow toward the back of the capture portion, A leukocyte capture device, wherein the capture section is disposed as part of another layer adjacent to a particular layer, facing the outlet side of all or part of the bypass section in that particular layer.

2. The leukocyte capturing device according to claim 1 , wherein the auxiliary section has the same structure as the main section.

3. 3. The leukocyte capturing device according to claim 1, wherein the width between a specific layer and another layer adjacent thereto in the main portion is 8 to 30 μm.

4. 3. The leukocyte capturing device according to claim 1, wherein in the main portion, the ratio of the width of the bypass portion to the width of the capture portion is greater than 1 and is not greater than 3.

5. 3. The leukocyte capturing device according to claim 1, wherein in the main portion, the portion other than the capture portion on the inlet-side end face of the convex portion extends parallel to the layer direction, and the end face constituting the bypass portion of the convex portion extends perpendicular to the layer direction.

6. The leukocyte capturing device according to claim 1 or 2, wherein the boundary diameter Dc is 5 μm or more and less than 10 μm.

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