White blood cell capturing 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

Leukocyte Capture Device

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

[0002] DNA is damaged by radiation exposure and the influence of the living environment. Damaged DNA is believed to be closely related to diseases such as cancer. Traditionally, to analyze this DNA damage, blood has been centrifuged to extract white blood cells, which are then stained and observed 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 captures and separates only solids of a certain size or larger from a solid-liquid mixture using a microchannel with a concave capture element. In Patent Document 1, each 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 section in which multiple capture elements are arranged. However, the capture of only one solid in each capture element, which would facilitate the observation of captured solids such as white blood cells, is not considered. 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.

[0004] JP 2009-109232 A

[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.

[0007] The present invention provides the following (1) to (6): (1) A leukocyte capturing device for passing a blood-containing liquid and capturing leukocytes contained in the blood-containing liquid, the device comprising: a sample inlet into which the blood-containing liquid X1 is introduced; a buffer inlet into which a buffer Y1 is introduced; a flat surface; and a plurality of pillars extending in a direction perpendicular to the flat surface, the blood-containing liquid X1 and the buffer Y1 flow adjacent to each other in the same specific direction Z on the surface of the flat surface and between a pillar and another pillar adjacent thereto; when the surface of the flat surface is viewed in a direction parallel to the perpendicular, the pillars form a plurality of rows, and each row is arranged 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) -0.48is smaller than the diameter of the white blood cells, and the device has a white blood cell separation unit that separates the white blood cells from the blood-containing liquid X1 and contains them in the buffer Y1 during the process in which the blood-containing liquid X1 and the buffer Y1 flow in the same specific direction Z, thereby generating a buffer Y2 containing the white blood cells and a blood-containing liquid X2 as a remainder from which the white blood cells have been separated, and discharging these; a main section that passes the buffer Y2 discharged from the white blood cell separation unit and captures the white blood cells contained therein; an auxiliary section that passes the blood-containing liquid X2 discharged from the white blood cell separation unit, and has the same flow path resistance as the main section; and an outlet from which the liquid flows out after passing through the main section and the auxiliary section, wherein the main section is (2) A leukocyte capturing device according to (1), comprising a flat portion and a number of protrusions provided thereon, wherein the buffer Y2 entering through an inlet passes over the surface of the flat portion of the main portion and between a protrusion and another protrusion adjacent thereto, and is discharged through an outlet, the protrusions are provided in layers on the surface of the flat portion, each layer including a plurality of the protrusions, and the buffer Y2 passing through an inlet layer passes through an outlet layer adjacent thereto, each layer having a capture portion in which the width between a protrusion and an adjacent convex portion is set to 2 to 7.5 μm and a bypass portion in which the width is set to 8 to 20 μm, the widths of the inlet-side portions of the two protrusions constituting the capture portion are chamfered so that they gradually narrow toward the back of the capture portion, and the capture portion is disposed opposite the outlet side of all or part of the bypass portion in a specific layer, as part of another layer adjacent thereto. (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 the ratio of the width of the bypass portion to the width of the capture portion in the main portion is greater than 1 and not greater than 3.(5) The leukocyte capturing 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.

[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.

[0009] FIG. 1 is a schematic plan view showing a leukocyte capturing device of the present invention. FIG. 2 is a cross-sectional view (schematic cross-sectional view) taken along line A-A in FIG. 1. FIG. 3 is an enlarged view of the junction between flow paths 5 and 6 and the inlet 10in of the leukocyte separation unit 10. FIG. 4 is an enlarged view (schematic view) of the surface of FIG. 1. FIG. 5 is a schematic view of the surface of the main portion 50 as viewed from a direction parallel to the perpendicular line. FIG. 6 is an enlarged view (schematic view) of B in FIG. 5. FIG. 7 is a cross-sectional view (schematic view) taken along line C-C in FIG. 5. These are microscopic photographs (eyepiece 10x x objective 10x) of a leukocyte capturing device of the present invention according to an embodiment.

[0010] The leukocyte capture 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, or the like. During this process, leukocytes are transported from the blood-containing liquid into a buffer, and then the leukocytes are 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 bonding a channel chip obtained by pouring rubber into a mold on which a channel pattern has been formed to a glass substrate. However, a lid (described below) that covers the top surface of the leukocyte capturing device 1 of the present invention is not shown in Fig. 1.

[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 inlet> 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, thereby forming a hole in which the blood-containing liquid X1 can temporarily remain, and this can serve 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 flow channel 5 may be formed, for example, by pillars. The pillars prevent the flow channel 5 from being blocked when through-holes are formed in a flow channel chip obtained by pouring rubber into a mold on which a flow channel pattern is formed, and the resulting chip is then bonded to a glass substrate.

[0018] Here, the blood-containing liquid X1 is not particularly limited as long as it is a liquid containing the blood of an animal, including a human, and may be, for example, a mixed liquid obtained by adding human blood 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> Buffer Y1 is introduced through the buffer inlet 4. The buffer inlet 4 may be a hole in which the introduced buffer Y1 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 the channel chip is then attached to a glass substrate, thereby forming a hole in which buffer Y1 can temporarily remain, and this hole can serve 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. The flow path 6 may be formed, for example, by a pillar, similar to the flow path 5.

[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 using Figures 2, 3, and 4 in addition to Figure 1. Figure 2 is a cross-sectional view (schematic cross-sectional view) taken along line A-A in Figure 1, Figure 3 is an enlarged view of the junction between the flow paths 5 and 6 and the inlet 10in of the leukocyte separation unit 10, and Figure 4 is an enlarged view (schematic view) of the surface of the leukocyte separation unit 10 in Figure 1.

[0023] As shown in FIG. 2 , the leukocyte separation unit 10 has a planar portion 11 and a plurality of pillars 13 extending perpendicular to the planar portion 11. As described above, in this example, a flow path chip obtained by pouring rubber into a mold with a flow path pattern is bonded to a glass substrate, and the glass substrate constitutes the planar portion 11. The leukocyte separation unit 10 shown in FIG. 2 also has a plate-shaped lid 15 that covers the top of the pillars 13 (the lid 15 is not shown in FIGS. 1 and 4 ). While the boundary between the pillars 13 and the lid 15 is indicated by a dotted line in FIG. 2 , the pillars 13 and the lid 15 may be integral or separable. By forming the mold used to obtain the flow path chip by pouring rubber into a flow path pattern that integrates the pillars 13 and the lid 15, a flow path chip in which the pillars 13 and the lid 15 are integrally formed can be obtained.

[0024] Surface 11 of the 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. In other words, 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 the 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 have a generally laminar flow. X Arrows indicating direction F Y The blood-containing liquid X1 and the buffer Y1 flow side by side, separated by a dotted line parallel to the arrow indicating the same direction Z, which exists between the arrow indicating the same direction Z and the blood-containing liquid X1. This is mainly due to the extremely low Reynolds number of the blood-containing liquid X1 and the buffer Y1.

[0027] FIG. 4 shows the surface 11 of the flat portion 11. S , but 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 column 13. As shown in this Figure, the columns 13 are arranged in multiple rows, and the white blood cells contained in the blood-containing solution 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 Figure 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 obliquely along the inclination of the rows of pillars 13. In contrast, 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 liquid 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 (Z) and large particles moving obliquely can be calculated by the following formula 1: Formula 1...Dc=1.4 g N -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 X and the same direction Z), N is the number of flow streams, and is expressed by the following formula: N=1 / ε where ε is the row shift fraction, i.e., the shift rate of the pillars 13, and ε=tan θ.

[0030] According to WO 2016 / 136273, the boundary diameter Dc can be calculated by the following formula, which is equivalent to Formula 1: Dc = 1.4 g ε 0.48 Particles with a diameter smaller than the boundary diameter Dc move generally along the direction of the flow, while particles with a diameter larger than the boundary diameter Dc move obliquely. 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 (platelets, etc.) to be separated, the white blood cells 20 can be moved diagonally along the inclination of the row of pillars 13 and into the buffer Y1 as described above.

[0031] For example, if the boundary diameter Dc is set to be 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 (platelets, etc.), 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] Buffer Y1 containing white blood cells separated from blood-containing liquid X1 as described above will be referred to as buffer Y2 below. Furthermore, the remainder of blood-containing liquid X1 after white blood cells have been separated will be referred to as blood-containing liquid X2 below. These are then each discharged from the white blood cell separation unit 10. Buffer Y2 discharged from the white blood cell separation unit 10 flows toward the main section, which will be described later. Furthermore, blood-containing liquid X2 discharged from the white blood cell separation unit 10 flows toward the auxiliary section, which will be described later.

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

[0035] 5 includes an inlet 50in through which the buffer Y2 enters the main part 50, and an outlet 50out through which the buffer Y2 is discharged after passing through the main part 50. The configuration of the main part 50 is not limited to those illustrated in FIGS. 5 to 7.

[0036] As shown in FIGS. 5 to 7 , the main section 50 includes a flat section 52 and a number of convex sections 54 provided thereon. As described above, in this example, a channel chip is formed by pouring rubber into a mold in which a channel pattern is formed, and then bonding the resulting channel chip to a glass substrate, with the glass substrate constituting the flat section 52. The main section 50 shown in FIGS. 5 to 7 also includes a plate-shaped lid 56 that covers the top of the convex sections 54 (the lid 56 is not shown in FIGS. 5 and 6 ). In FIG. 7 , the boundary between the convex sections 54 and the lid 56 is indicated by a dotted line, but the convex sections 54 and the lid 56 may be integral or may be separable. By forming the mold used to obtain the channel chip by pouring rubber into a channel pattern in which the convex sections 54 and the lid 56 are integrated, a channel chip in which the convex sections 54 and the lid 56 are integrally formed can be obtained.

[0037] As shown in FIG. 5 , the convex portions 54 are arranged in layers on the flat portion 52. In FIG. 5 , the layer closest to the inlet 50 in is designated the first layer, and the layer adjacent to the first layer on the outlet side (downstream side) is designated the second layer. A certain layer is designated the Pth layer, the layer adjacent to the Pth layer on the outlet side (downstream side) is designated the P+1th layer, and the layer adjacent to the Pth layer on the outlet side (downstream side) is designated the P+2th layer. Each layer includes multiple convex portions 54. While FIG. 5 shows an example in which each layer includes seven convex portions 54, the number of convex portions 54 included in each layer is not particularly limited. Furthermore, the number of layers is also not particularly limited.

[0038] Buffer Y2 entering the main portion 50 from the inlet 50in flows on 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 similarly pass through the flow paths between the convex portions 54 in the Pth layer, and then pass through the flow paths between the convex portions 54 in the P+1th layer.

[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 plurality of 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, a plurality of capture sections may be continuously formed within a layer.

[0041] Furthermore, a capture section 61 is disposed on the outlet side of the bypass section 63 in a specific layer as part of another adjacent layer. That is, in the example of FIG. 6 , the capture section 61 in the P+1th layer is disposed on the outlet side (downstream side) of the bypass section 63 in the Pth layer. As illustrated in FIG. 6 , the bypass section 63 in the Pth layer and the capture section 61 in the P+1th layer are preferably disposed side by side in a direction perpendicular to the layer direction. More specifically, the bypass section 63 in the Pth layer and the capture section 61 in the P+1th layer are preferably disposed so that, when a line perpendicular to the layer direction is drawn, the line passes through the bypass section 63 in the Pth layer and the capture section 61 in the P+1th layer (i.e., the line does not contact the convex portion 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 have 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] In the plan views shown in Figures 5 and 6, the convex portion 54 is rectangular, with some of its four corners cut off (e.g., by straight or curved lines) to form a chamfered shape. The area of ​​the chamfered portion cut off (e.g., by straight or curved lines) is preferably smaller than the area (projected area) of the white blood cells to be captured. As shown in Figure 6, the inlet portions of the two convex portions 54 that make up the capture portion 61 are chamfered so that they become gradually narrower toward the back of the capture portion 61. This is because white blood cells are more easily captured by the capture portion 61. Furthermore, once captured by the capture portion 61, the white blood cells are deformed and become trapped in the chamfered portions, making them less likely to escape from the capture portion.

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

[0045] As long as the chamfering gradually narrows toward the back of the capture portion 61, both entry-side portions of the two convex portions constituting the capture 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 (e.g., by straight or curved lines) to be chamfered, 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 Fig. 6, in the main section 50, it is preferable that the inlet-side end faces of the convex sections 54, other than the capture section 61, extend parallel to the layer direction, and the end faces of the convex sections 54 that form the bypass section 63 extend perpendicular to the layer direction. In particular, as shown in Fig. 6, the inlet-side portions of the two convex sections 54 that form the capture section 61 are chamfered (preferably linearly) so as to gradually narrow toward the back of the capture section 61, and the inlet-side end faces of the convex sections 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 is particularly pronounced, and the leukocyte capture efficiency is further improved, which is preferable. If the convex sections 54 are not based on a rectangle (e.g., if they are circular or elliptical), their outer shape includes a curve, which may cause leukocytes to migrate along the curve and not reach the capture section 61 in the adjacent downstream layer.

[0048] The width L1 of the capture portion 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. Note that the widths L1 and L2 refer to the shortest distance between two adjacent convex portions 54 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 into the bypass section 63 is appropriately suppressed, making it easier for white blood cells to be captured by the capture section.

[0050] 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. Note that the width L3 refers to the shortest distance between the Pth layer and the P+1th layer. The maximum width L4 of the chamfered portion on the inlet side of the capture section 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 Section> The auxiliary section 70 allows the blood-containing liquid X2 discharged from the leukocyte separation unit 10 to pass through. The auxiliary section 70 has the same structure as the main section 50 described above in terms of flow path resistance. Here, "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 main section 50 described above. Having the same structure as the main section 50 described above makes the flow path resistance to the buffer Y2 and the blood-containing liquid X2 the same, thereby controlling the flow rates of the blood-containing liquid X1 and the buffer Y1 flowing within the leukocyte separation unit 10 to be the same and improving the leukocyte separation efficiency in the leukocyte separation unit 10.

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

[0054] <Outlet> The liquid that has passed through the main section 50 and the auxiliary section 70 passes through the flow path 7 and flows out from the outlet 9. The outlet 9 may be a hole through which the liquid that has passed through the main section 50 and the auxiliary section 70 can flow out. For example, in the embodiment shown in Fig. 1 , a through-hole is formed in a flow path chip obtained by pouring rubber into a mold on which a flow path pattern has been formed, and this is then bonded to a glass substrate, thereby forming a hole through which the liquid that has passed through the main section 50 and the auxiliary section 70 can flow out, and this hole can serve as the 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 in which a flow channel pattern is formed, to a glass substrate. The size and material of the flow channel chip are not particularly limited. For example, the flow channel 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.

[0059] <Creating the Main Part> First, silicone rubber (SILPOT184, Dow Corning) was poured into the mold with the flow path pattern 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 flow path-forming chip. Next, holes were formed using a punch to serve as the sample inlet 3, buffer inlet 4, and outlet 9.

[0060] <Bonding> 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 liquid X1. PBS was also used as buffer Y1. Next, blood-containing liquid X1 was dripped into sample inlet 3, and buffer Y1 was dripped into buffer inlet 4. Next, the liquids were delivered from outlet 9 by negative pressure of a syringe pump.

[0062] The state of liquid transfer was then observed under a microscope. A micrograph (eyepiece 10x magnification x objective lens 10x magnification) 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 being separated from the blood-containing liquid X1 and moving to the buffer Y1. After continuing the liquid transfer for 30 minutes, no clogging of the flow path in the main part 50 was confirmed. It was also confirmed that white blood cells were being captured in the main part 50.

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

[0064] REFERENCE SIGNS LIST 1 Leukocyte capture device of the present invention 3 Sample inlet 4 Buffer outlet 5, 6, 7 Flow path 9 Outlet 10 Leukocyte separation unit 10in Inlet of leukocyte separation unit 11 Flat surface 11 S Surface of flat portion 13 Pillar 15 Lid 20 White blood cell 22 Small particle 50 Main portion 50in Inlet of main portion 50out Outlet of main portion 52 Flat portion 54 Convex portion 56 Lid 61 Capture portion 63 Bypass portion 70 Auxiliary portion 80 Partition

Claims

1. A leukocyte capturing device that captures leukocytes contained in a blood-containing liquid by passing the blood-containing liquid through the blood-containing liquid, 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 a specific same direction (Z) on the surface of the planar portion and between the adjacent pillars 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, In a configuration using a deterministic lateral displacement method (DLD method) in which the distance between the pillar and another adjacent pillar in a 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 g, Dc = 1.4g × (1 / tan θ) -0.48 The boundary diameter Dc defined as is smaller than the diameter of the white blood cell, 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 discharges 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 resistance as the main section, through which the blood-containing liquid X2 discharged from the leukocyte separation unit passes; an outlet through which liquid flows after passing through the main portion and the auxiliary portion; having 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 includes 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 portion of each 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, in which the capture section is disposed as part of another layer adjacent thereto, facing the outlet side of all or a part of the bypass section in a 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, a ratio of a width of the bypass portion to a width of the capture portion is greater than 1 and is equal to or smaller than 3.

5. 3. The leukocyte capturing device according to claim 1 or 2, wherein in the main portion, a 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 in 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.