Cell screening device and cell screening kit

Abstract

A cell screening device (1) has: a bottom plate part (2); a cell placement membrane (6) provided on the bottom plate part (2) and forming a cell placement surface (4); a pair of fluid injection parts (8) provided on the bottom plate part (2) and partitioned from the cell placement membrane (6); and a flow channel (10) provided between the bottom plate part (2) and the cell placement membrane (6) and having flow channel end portions (12) extending to the fluid injection parts (8). On the cell placement surface (4) of the cell placement membrane (6) are formed a plurality of wells having a size that enables the wells to individually accommodate cells (C) to be screened, and through holes extending from the inner bottom surfaces of the wells and communicating with the flow channel (10).

Description

[Technical field]

[0001] The present invention relates to a cell screening device and a cell screening kit for screening cells. This application claims priority based on Japanese Patent Application No. 2019-178989 filed on September 30, 2019, the contents of which are incorporated herein by reference. [Background technology]

[0002] In recent years, especially in the field of drug discovery, the target of cell analysis has been subdivided from the cell group level to the single cell level, and a method is used in which a cell screening device is used to capture cells one by one in a minute well, and a screening test is performed on a large number of cells at once to select cells with the desired characteristics. For example, a method of screening cells is used in which cells captured in a large number of wells are contacted with a liquid in which a reagent such as a catcher that binds to a specific antibody is dispersed, and cells that secrete a secretion that binds to the catcher are found and collected from the well.

[0003] Patent Document 1 shows a cell screening device previously proposed by the present applicants. This cell screening device has a first substrate and a second substrate arranged in a horizontally stacked manner, and a number of wells each capable of accommodating a single cell are formed on the surface of the first substrate, and each well has a through hole extending from the bottom of the well to the back surface of the first substrate. These through holes are so thin that cells cannot pass through them. The second substrate has a storage section that receives secretions that flow out of the wells through the through holes, and the secretions react with a reagent in the storage section to identify target cells. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. WO2017 / 057234A1 Brochure Summary of the Invention [Problem to be solved by the invention]

[0005] In a cell screening device such as that described in Patent Document 1, it is necessary to provide a reagent introduction section to supply a reagent to the accumulation section. For example, if a reagent introduction section communicating with the accumulation section is provided at the end of the device, when cells are captured in the wells and the cell screening device is carried or tilted from a horizontal position with the accumulation section filled with dispersion liquid and reagent, the dispersion liquid and reagent will flow in the longitudinal direction of the accumulation section (called the sloshing phenomenon), and some of the dispersion liquid and reagent will flow into the wells through the through-holes, lifting up the cells captured in the wells and causing the cells to detach from the wells. [Means for solving the problem]

[0006] The cell screening device according to aspect [1] of the present invention comprises a bottom plate portion, a cell mounting portion provided on the bottom plate portion and constituting a cell mounting surface, a fluid injection portion provided on the bottom plate portion and separated from the cell mounting portion, a plurality of wells formed on the cell mounting surface of the cell mounting portion and having a size capable of individually accommodating cells to be screened, a flow path provided between the bottom plate portion and the cell mounting portion and having an end portion of the flow path extending to the fluid injection portion, a through hole leading from the inner bottom surface of the well to the flow path and having an inner diameter that does not allow the cells to be screened to pass through, a lid portion provided in the fluid injection portion and closing the end portion of the flow path, and a fluid injection port provided in the lid portion and having an opening communicating with the flow path.

[0007] According to such a cell screening device, the end of the flow channel is closed with a lid, and the lid is provided with a fluid injection port having an opening communicating with the flow channel, so that the movement of the fluid in the flow channel is suppressed by the lid, and the fluid can be introduced into and removed from the flow channel through the fluid injection port. Therefore, even if the cell screening device is carried or tilted with cells trapped in the wells and a fluid such as a dispersion liquid in the flow channel, the fluid is unlikely to move excessively along the flow channel, and the sloshing phenomenon can be suppressed. Therefore, it is possible to suppress the problem that a part of the dispersion liquid flows into the well through the through-hole and causes the cells trapped in the well to detach.

[0008] The cell screening device of aspect [2] is the same as the aspect [1], except that a fluid storage section capable of storing fluid overflowing from the opening of the fluid injection port is formed in the lid section around the fluid injection port, and a partition section is provided between the cell placement surface and the lid section to prevent the fluid from flowing to the cell placement section when the fluid is stored in the fluid storage section. In this case, even if the fluid overflows from the opening of the fluid injection port, the fluid storage section can receive the fluid and prevent it from re-entering the flow path from the fluid injection port, thereby reducing the risk of contamination, for example. In addition, since a partition section is formed between the cell placement surface and the lid section, even if the fluid is stored in the fluid storage section, the fluid can be prevented from flowing to the cell placement section.

[0009] The cell screening device of aspect [3] is the above aspect [1] or [2], in which the bottom plate part is rectangular with long sides and short sides, the fluid injection part, the lid part, and the fluid injection port are formed on both sides of the bottom plate part in the longitudinal direction, and the cell placement part is disposed between the two lid parts. In this case, a wide range of usage methods are possible, such as flowing a fluid into a flow path from one of the two fluid injection ports while discharging an excess amount of fluid from the other fluid injection port. However, the present invention is not limited to a configuration in which two fluid injection ports are provided, and if necessary, it is possible to provide only one side, or three or more fluid injection ports.

[0010] The cell screening device of aspect [4] is any one of the aspects [1] to [3], in which the cell mounting portion is a membrane body, the wells are formed on the upper surface of the membrane body in a grid pattern, the through-holes leading to the inner bottom surfaces of the wells are formed on the lower surface of the membrane body, a frame body is provided that supports the periphery of the membrane body and surrounds the cell mounting surface, and an engagement portion is provided on the bottom plate portion for removably fixing the frame body. In this case, when the frame body is engaged with the engagement portion of the bottom plate portion, the membrane body having the wells is positioned at the correct position on the bottom plate portion, and the frame body can be removed from the bottom plate portion after screening.

[0011] The cell screening device of aspect [5] is any one of the aspects [1] to [4], in which a peripheral wall is formed standing from the periphery of the lid, and the lid and the peripheral wall are formed as an outer frame that is detachable from the bottom plate. In this case, the bottom plate and the outer frame are molded separately, and by simply attaching the outer frame to the bottom plate, the lid and the peripheral wall can be accurately positioned, making assembly easy. In addition, the outer frame can be removed from the bottom plate after use, making maintenance easy.

[0012] The cell screening device of embodiment [6] is the same as embodiment [4], except that a peripheral wall is formed on the periphery of the lid, and the lid and the peripheral wall are formed as an outer frame that is detachable from the bottom plate, and the frame that supports the membrane is detachably fixed to the outer frame. In this case, the bottom plate, the outer frame, and the frame are each molded separately, and simply by attaching the frame and the outer frame to the bottom plate, the membrane, the lid, and the peripheral wall can be accurately positioned, improving ease of assembly. In addition, the frame and the outer frame can be removed from the bottom plate after use, making maintenance easier.

[0013] The cell screening device of aspect [7] is any one of the aspects [1] to [5], in which the bottom plate is rectangular having a long side and a short side, the fluid injection part, the lid part, and the fluid injection port are formed on both sides of the longitudinal direction of the bottom plate, respectively, the cell placement part is disposed between the two lid parts, and the lid part, the peripheral wall part standing from the periphery of the lid part, and the peripheral wall part surrounding the periphery of the cell placement part are formed as an outer frame body that can be detached from the bottom plate part. In this case, the bottom plate part and the outer frame body are molded separately, and by simply attaching the outer frame body to the bottom plate part, the cell placement part, the lid part, the peripheral wall part, and the two fluid injection ports can be accurately positioned, and the ease of assembly can be improved. In addition, the outer frame body can be removed from the bottom plate part and disassembled after use, making maintenance easier.

[0014] The cell screening device of embodiment [8] is any one of the embodiments [1] to [7], in which the fluid injection port is cylindrical and stands up from the upper surface of the lid. In this case, since the fluid injection port is cylindrical, it is easy to inject and eject fluid by applying a conically pointed tip of a pipette or dispenser to the fluid injection port in an almost airtight manner.

[0015] The cell screening device of embodiment [9] is any of the embodiments [1] to [8], in which the cover has a foam discharge surface that rises in an inclined or stepped manner as it approaches the fluid injection port on at least a part of the ceiling surface of the end of the flow channel. In this case, the ceiling surface on the back side of the cover has a foam discharge surface that rises in an inclined or stepped manner toward the fluid injection port, so that air bubbles that have entered the flow channel can be easily discharged from the fluid injection port, and problems caused by air bubbles can be suppressed.

[0016] The cell screening kit of aspect

[10] includes the cell screening device according to any one of aspects [1] to [9], and detection particles, which are carrier particles to which a substance capable of binding to a secretion product of a cell to be screened is immobilized. According to this cell screening kit, in a state where cells are taken into each well, the detection particles are reacted with the secretion product of the cell in the well, and screening can be performed by light emission from the detection particles, etc., and even if the cell screening device is carried or tilted with a fluid such as a dispersion liquid in the flow path, the fluid is less likely to move along the flow path, and the sloshing phenomenon can be suppressed. Therefore, it is possible to suppress a problem in which a part of the dispersion liquid flows into the well through the through-hole, causing the cells and detection particles captured in the well to detach from the well. Effect of the Invention

[0017] As described above, according to the cell screening device or cell screening kit of the present invention, even when cells are captured in the wells of the cell screening device and the cell screening device is carried or tilted with a fluid such as a dispersion liquid in the flow path, the fluid is less likely to move along the flow path, suppressing the sloshing phenomenon and preventing problems such as part of the dispersion liquid flowing into the well through the through holes and causing the cells or detection particles captured in the well to become detached, providing the excellent effect of preventing such problems. [Brief description of the drawings]

[0018] [Figure 1]FIG. 1 is a plan view showing a cell screening device according to one embodiment of the present invention. [Diagram 2] FIG. [Diagram 3] FIG. 2 is a perspective view showing a front cross section of the embodiment. [Figure 4] FIG. 2 is a front cross-sectional view of the center portion of the embodiment. [Diagram 5] FIG. 4 is a front cross-sectional view of an end portion of the embodiment. [Figure 6] FIG. [Figure 7] FIG. 4 is a perspective view showing an assembly method of the embodiment. [Figure 8] FIG. 2 is an enlarged perspective view showing the cell-mounting membrane (cell-mounting portion) and wells of the embodiment. [Figure 9] FIG. 11 is a perspective view showing a front cross section of a cell screening device according to another embodiment of the present invention. [Figure 10] FIG. 11 is a front cross-sectional view of an end portion of a cell screening device according to another embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] 1 to 6 show a completed state of a cell screening device 1 according to an embodiment of the present invention, and FIG. 7 shows a method of assembling the cell screening device 1. The cell screening device 1 of this embodiment has a bottom plate 2, a cell mounting membrane (cell mounting portion) 6 provided on the bottom plate 2 and constituting a cell mounting surface 4, a pair of fluid injection portions 8 provided on the bottom plate 2 separately from the cell mounting membrane 6, and a flow channel 10 provided between the bottom plate 2 and the cell mounting membrane 6, the flow channel 10 having a flow channel end portion 12 extending to the fluid injection portion 8. As shown in FIG. 8, for example, the cell mounting surface 4 of the cell mounting membrane 6 is provided with a plurality of wells 50 having a size capable of individually accommodating cells C to be screened, and a through hole 52 leading from the inner bottom surface of the well 50 to the flow channel 10. The through hole 52 has an inner diameter that does not allow the cells C to be screened to pass through.

[0020] The structure of the cell screening device 1 will be described in detail below. The bottom plate 2 in this example is a long and narrow rectangular plate with a constant thickness, and the four corners are rounded. A slight protrusion 2B is formed around the entire circumference of the bottom surface of the bottom plate 2. The shape of the bottom plate 2 is not limited to that shown in the figure, and may be any shape such as a disk, an ellipse, or a square, and the thickness of the bottom plate 2 does not need to be constant as long as a horizontal flow channel 10 can be formed. In general, it is desirable for the bottom plate 2 to be made of various plastics that are harmless to target cells in terms of molding accuracy and cost, but if necessary, it may be made of any material such as ceramic, glass, or metal. The surface of the bottom plate 2 may be coated with some kind of coating to reduce the effect on the cells.

[0021] As shown in Fig. 3, an engagement wall 18 having a rectangular shape with both ends semicircular in plan view is integrally formed on the upper surface of the bottom plate portion 2 so as to surround the center portion and stand vertically from the bottom plate portion 2. The shape of the engagement wall 18 is not limited to that shown in the figure, and it may be a simple rectangular shape, a circular shape, an elliptical shape, or the like. The height of the engagement wall 18 in this example is made uniform around the entire circumference. An outer frame body 20 is removably attached to the engagement wall 18 so as to cover the engagement wall 18 from above around the entire circumference.

[0022] The outer frame 20 has a rectangular peripheral wall 22 with semicircular ends in plan view, and a pair of partitions 28 arranged parallel to each other on the inner peripheral side of the peripheral wall 22, and is integrally formed as a whole. There are no limitations on the material of the outer frame 20, and it is generally preferable to form it from various plastics that are harmless to cells in terms of molding accuracy and cost, but it may be formed from any material such as ceramic, glass, metal, etc., as necessary. A rectangular parallelepiped space is provided between the partitions 28, and the cell-mounting membrane 6 is disposed therein.

[0023] The upper end of the peripheral wall 22 of the outer frame 20 has a cross-sectional shape that is folded back outward all around, and a narrow engagement groove 24 that opens downward is formed on the inside of this folded back portion at a constant depth all around. The upper end of the engagement wall 18 is inserted into this engagement groove 24 all around, and is elastically fastened by the folded back portion of the peripheral wall 22, thereby removably fixing the outer frame 20 to the engagement wall 18. Horizontally protruding projections 26 are formed on both ends of the outer frame 20 in the longitudinal direction, and by lifting these projections 26 with fingertips, the engagement wall 18 comes out of the engagement groove 24, allowing the bottom plate 2 and the outer frame 20 to be separated.

[0024] Each semicircular region surrounded by the peripheral wall 22 of the outer frame 20 and each partition 28 is a fluid injection section 8. In each of the fluid injection sections 8, a semicircular lid 14 is formed parallel to the bottom plate 2 so as to connect the lower end of the peripheral wall 22 and the lower end of each partition 28. Between the lid 14 and the bottom plate 2, channel ends 12, which are both ends of the channel 10, are formed, and the lid 14 is structured to airtightly close the channel ends 12. By forming the lid 14 in this manner, the channel ends 12, which are both ends of the channel 10, are sealed, and even if the cell screening device 1 is tilted or shaken, excessive flow of fluid to the left and right through the channel 10 and the channel ends 12 is prevented. In this example, the thickness of the channel ends 12 in the vertical direction is constant, but it may be partially changed as necessary, as in the embodiment of FIG. 10 described later.

[0025] In this embodiment, a circular opening 16A is formed in each of the lids 14 approximately at the center, and cylindrical fluid injection ports 16 are formed upright from the lids 14 corresponding to these openings 16A. The surface of the lids 14 around the fluid injection ports 16 serves as a fluid storage section, where fluid L spilling from the fluid injection ports 16 is stored, as shown in Fig. 5. A recess may be intentionally formed around the fluid injection port 16 on the upper surface of the lids 14 as the fluid storage section.

[0026] In this embodiment, the fluid injection port 16 is raised from the lid portion 14, but instead, it is possible for the fluid injection port to be a flat opening formed in the lid portion 14 that does not protrude upward, while a recess, such as a ring-shaped recess, surrounding the opening can be formed in the surface of the lid portion 14, and the recess can serve as a fluid storage portion.

[0027] The opening 16A of the fluid injection port 16 is formed with a diameter that is compatible with the tip of an instrument such as a pipette, micropipette, or dispenser for injecting a fluid (reagent or dispersion liquid) from the fluid injection port 16, and the sharp tip of the instrument can be abutted airtightly against the opening 16A of the fluid injection port 16. The shape of the fluid injection port 16 is not limited to a cylindrical shape, and may be a square or polygonal cylindrical shape as necessary, a ring shape with the periphery of the opening 16A slightly raised, or a simple opening as described above. The fluid injection port 16 may be an inverted cone-shaped opening formed by recessing the surface of the cover 14.

[0028] At the lower end of the partition 28 on the fluid injection section 8 side, a step 34 having a constant height from the lower surface is formed over the entire length of the partition 28, and above this step 34, two ribs 30 are formed extending in the vertical direction and reaching the upper end of the partition 28. The ribs 30 increase the bending strength of the partition 28. In addition, the partition 28 is formed with an engagement groove 32 of a constant depth that opens onto the lower surface of the step 34, and an engagement ridge 42 adjacent to the cell mounting membrane 6 side of this engagement groove 32.

[0029] 6, engagement grooves 32 of a certain depth that open to the underside of the peripheral wall 22 and engagement ridges 42 adjacent to the cell-mounting membrane 6 side of the engagement grooves 32 are formed in two linearly extending locations on the peripheral wall 22 of the outer frame body 20. These two linearly extending engagement grooves 32 and engagement ridges 42 are connected to the engagement grooves 32 and engagement ridges 42 of the partition section 28, respectively, and form a rectangular shape in plan view.

[0030] A square cylindrical frame 36, which is rectangular in plan view, is removably housed in a square space surrounded by the two linearly extending portions of the peripheral wall 22 and the two partitions 28. At the lower end of the frame 36, the cell mounting membrane 6 is stretched over the entire surface with the wells 50 facing upward as shown in Fig. 8, and the lower end of the frame 36 and the cell mounting membrane 6 are joined without any gaps around the entire circumference. The frame 36 is made of flexible plastic or the like, and when a force is applied to spread it outward, the four walls spread outward slightly, applying tension to the cell mounting membrane 6 and preventing the cell mounting membrane 6 from sagging.

[0031] The thickness of the cell-mounting membrane 6 is not limited, but from the viewpoint of forming wells 50 into which target cells are placed one by one and forming minute through-holes 52 through which fluid flows from the bottom of the wells 50 to the back side, the thickness is preferably about 5 to 100 μm, and more preferably about 10 to 50 μm. The cell-mounting membrane 6 may be a multi-layer membrane of two or more layers. In this case, through-holes that will become the wells 50 may be formed in the upper layer, and through-holes that will become the through-holes 52 may be opened in the lower layer, and these two layers may be attached together to form the wells 50 and the through-holes 52.

[0032] The material of the cell-mounting membrane 6 is not limited, and it is generally desirable to form it from various plastics that are harmless to cells in terms of molding accuracy and cost, but if necessary, it may be formed from any material such as ceramic, polycrystalline or single crystal silicon, inorganic compounds such as glass, metal, etc. The wells 50 and through-holes 52 can also be formed by etching, photolithography, etc.

[0033] The planar shape of the well 50 is preferably hexagonal or circular as shown in FIG. 8 in order to increase the arrangement density according to the average shape of the cells C to be captured, but may be quadrangular such as square, other polygonal, elliptical, etc., if necessary. The size of the well 50 should be selected according to the size of the cells C to be captured and is not limited, but generally, the maximum diameter of the circle that fits into the well opening is about 1 to 100 μm and the depth is about 1 to 100 μm so that one cell C fits into one well 50 but two or more cells C are unlikely to fit into one well 50. A cell screening kit may be constructed by preparing a plurality of types of frame bodies 36 having cell mounting membranes 6 with different sizes of wells 50 and combining them with a common bottom plate portion 2 and outer frame body 20.

[0034] In terms of the size of the cells C, the maximum diameter of a circle that fits within the well 50 in a plan view is preferably about 0.5 to 2 times, and more preferably 0.8 to 1.9 times, the maximum diameter of the cells C to be collected. The depth of the well 50 is preferably about 0.5 to 4 times, and more preferably 0.8 to 1.9 times, the maximum diameter of the cells C to be collected. The shortest distance between adjacent wells 50 is preferably about 1 to 10 μm, since this increases the density of the wells 50, but is not limited to this range.

[0035] In this example, two through-holes 52 are formed in the center of the bottom surface of each well 50. Forming a plurality of through-holes in this manner is advantageous since clogging is less likely to occur, but the present invention is not limited to this, and one or three or more through-holes may be formed in the center, or a plurality of through-holes 52 may be formed randomly or at lattice points on the bottom surface of the well 50. The inner diameter of the through-hole 52 is not limited, but the minimum inner diameter is preferably in the range of about 10 nm to 20 μm so that the cells C do not pass through, and is preferably 0.5 times or less the average diameter of the cells C to be captured in the well 50.

[0036] As shown in Figures 4 and 6, the outer peripheral surface of the lower end of the frame body 36 is folded back upward to form an upwardly opening engagement groove 44 and a downwardly protruding engagement rib 40 around the entire circumference. The depth of the engagement groove 44 and the vertical width of the engagement rib 40 are approximately constant around the entire circumference of the frame body 36. The engagement rib 40 is inserted into the engagement groove 32 formed on the lower surface of the outer frame body 20, and the engagement rib 42 of the outer frame body 20 is inserted into the engagement groove 44 of the frame body 36. By fitting these together, the frame body 36 is fixed to the outer frame body 20 with the frame body 36 housed in the central space of the outer frame body 20.

[0037] At this time, the lower end surface of the frame 36 abuts against the upper surface of the spacer 46 formed on the bottom plate 2, and the amount of separation of the cell mounting membrane 6 from the bottom plate 2, i.e., the thickness of the flow path 10, is accurately determined by the thickness of the spacer 46. In this embodiment, as shown in FIG. 7, a pair of spacers 46 having a U-shape in plan view are formed on the inside of the engagement wall 18 along the shape of the lower end of the frame 36, and a notch 47 is formed between the spacers 46. When the frame 36 is fixed on the bottom plate 2, the fluid flows from the flow path 10 to each flow path end 12 through these notches 47. The spacer 46 does not have to have the shape shown in the figure, and it is sufficient that it abuts against the lower surface of the frame 36 at several points. In some cases, the height of the frame 36 from the bottom plate 2 may be accurately determined by engagement with the outer frame 20 without forming the spacer 46.

[0038] An inclined surface 38 having a constant width is formed on the inner peripheral surface of the lower end of the frame 36, and the inclined surface 38 protrudes toward the cell-mounting membrane 6 as it goes downward. As a result of the formation of such an inclined surface 38, as shown in FIG. 4, when target cells are sucked up from the wells 50 of the cell-mounting membrane 6 with an instrument P such as a pipette, micropipette, or dispenser, the cells can be easily sucked up with the instrument P even if the wells 50 in which the target cells are collected are located close to the inner peripheral edge of the frame 36. The inclined surface 38 also increases the adhesion area of ​​the cell-mounting membrane 6 to the frame 36, thereby achieving the effect of increasing the bonding strength of the cell-mounting membrane 6, and also serving to increase the strength of the frame 36.

[0039] When manufacturing a cell screening device having the above configuration, the bottom plate portion 2, frame body 36, and outer frame body 20 are each formed separately as shown in Figure 7, and then the frame body 36 is first fitted onto the underside of the outer frame body 20, and then the outer frame body 20 is fitted into the engaging wall 18 of the bottom plate portion 2, resulting in the completed state as shown in Figures 1 to 6.

[0040] In the process of fitting the frame body 36 to the bottom surface of the outer frame body 20, as shown in Figures 4 and 6, the engaging ridges 42 of the outer frame body 20 fit into the engaging grooves 44 of the frame body 36, and the engaging ridges 40 of the frame body 36 fit into the engaging grooves 32 of the outer frame body 20, so that the two are firmly fixed together by the elasticity of the respective engaging portions. At the same time, the inner peripheral surface of the engaging ridges 40 and the outer peripheral surface of the engaging ridges 42 are shaped so that at least one of them is slightly inclined outward as it goes upward. As a result, as the engagement progresses, the engaging ridges 40 on the four sides of the frame body 36 are pulled outward by the engaging ridges 42 of the outer frame body 20, the bottom end of the frame body 36 is slightly spread out in all directions, generating tension that pulls the four sides of the cell-mounting membrane 6, and a uniform tension is applied to the cell-mounting membrane 6.

[0041] Therefore, even if there is slight slack in the cell-mounting membrane 6 when the frame body 36 is in a free state, when the frame body 36 is fixed to the outer frame body 20, the slack in the cell-mounting membrane 6 is eliminated, which has the advantage of increasing the flatness of the cell-mounting surface 4 on which the numerous wells 50 of the cell-mounting membrane 6 are formed, filling the cell-mounting surface 4 with a dispersion liquid in which the cells are dispersed, and rocking the cell screening device, for example, to cause the cells to flow evenly, making it easy to capture the cells one by one in the wells 50.

[0042] A cell screening kit using this embodiment includes a cell screening device 1 and beads (detection particles) B, which are carrier particles to which a substance capable of binding to a secretion of a cell to be screened is immobilized. The cell screening kit is a product that contains one or more cell screening devices 1 and an inner container in which a large number of fine beads B, which are detection particles, are enclosed in a powder state or suspended in a dispersion liquid, in an outer container such as a box.

[0043] The type of binding particle is not limited, but may be, for example, a carrier particle made of beads (magnetic beads, resin beads, etc.), hydrogel particles (sodium alginate gel, agarose gel, etc.), metal particles (gold nanoparticles), etc., and a catcher that is attached to the carrier particle and can bind to an antibody secreted from a target antibody-producing cell. The particle size of the binding particle is not limited, but generally, the maximum particle size is preferably 100 nm to 50 μm, and more preferably about 500 nm to 30 μm.

[0044] When actually performing screening, cells and beads B, which are detection particles, are dispersed in a dispersion liquid and filled onto the cell-mounting membrane 6, and cells C are placed one by one into the well 50 by shaking the cell screening device 1 or discharging the dispersion liquid into the flow channel 10 through the through-holes 52, while a large number of beads B are placed into the well 50 together with each cell C, and excess dispersion liquid is discharged into the flow channel 10 through the through-holes 52. In this state, if the antibody in the secretion of the cells C is reacted with the catcher of the beads B, only the reacted beads B can be visualized with a fluorescently labeled antibody, and the target cells can be found.

[0045] According to the cell screening device 1 having the above-mentioned configuration, the flow channel ends 12 located at both ends of the flow channel 10 are closed with the lids 14, and the lids 14 are provided with the fluid injection ports 16 having the openings 16A communicating with the flow channel 10, so that the movement of the fluid in the flow channel ends 12 and the flow channel 10 is suppressed by the lids 14, and the fluid can be introduced into and removed from the flow channel 10 through the fluid injection ports 16. Therefore, even if the cell screening device 1 is carried or tilted with the cell C captured in the well 50 of the cell screening device 1 and a fluid such as a dispersion liquid is placed in the flow channel 10, the fluid is less likely to move along the flow channel 10 and the flow channel ends 12, and the sloshing phenomenon can be suppressed. Therefore, the problem of part of the dispersion liquid flowing into the well 50 through the through-holes 52 and causing the cell C and beads B captured in the well 50 to detach can be suppressed.

[0046] In this embodiment, even if the fluid overflows from the opening 16A of the fluid injection port 16, the fluid storage section (the upper surface of the lid section 14) receives the fluid and prevents the fluid from re-entering the flow channel 10 from the fluid injection port 16, thereby reducing the risk of contamination from the outside, for example. In addition, since the partition section 28 is formed between the cell mounting surface 4 and the lid section 14, even if the fluid accumulates in the fluid storage section 14, the fluid can be prevented from flowing into the cell mounting section 6.

[0047] In addition, in this embodiment, two fluid injection ports 16 are formed on both ends of the flow path 10, enabling a wide range of uses, such as flowing fluid into the flow path 10 from one fluid injection port 16 and discharging excess fluid from the other fluid injection port 16.

[0048] Furthermore, in this embodiment, the cell-mounting membrane 6 is positioned in the correct position on the bottom plate portion 2 by abutting the lower end of the frame body 36 against the spacer 46 of the bottom plate portion 2, thereby ensuring the desired flow of fluid between the well 50 and the flow path 10, enabling highly accurate screening.

[0049] Furthermore, in this embodiment, the bottom plate 2 and the outer frame 20 are molded as separate bodies, and simply attaching the outer frame 20 to the bottom plate 2 allows the lid 14 and the peripheral wall 22 to be positioned accurately relative to the bottom plate 2, facilitating assembly of the cell screening device 1. The outer frame 20 can also be removed from the bottom plate 2 after use, facilitating maintenance.

[0050] In this embodiment, the bottom plate 2, outer frame 20, and frame 36 are each molded as separate bodies, and simply attaching the frame 36 and outer frame 20 to the bottom plate 2 enables accurate positioning of the cell mounting membrane 6, lid 14, and peripheral wall 22, improving ease of assembly. The frame 36 and outer frame 20 can also be removed from the bottom plate 2 after use, facilitating maintenance.

[0051] Furthermore, in this embodiment, the bottom plate portion 2 and the outer frame body 20 are molded as separate bodies, and simply by attaching the outer frame body 20 to the bottom plate portion 2, accurate positioning of the cell placement portion 6, the lid portion 14, the peripheral wall portion 22, and the two fluid injection ports 16 is possible, making assembly easy.

[0052] In addition, in this embodiment, since the fluid injection port 16 is cylindrical, it is easy to inject and expel fluid by applying the conically pointed tip of a pipette, dispenser, or the like to the opening 16A in an airtight manner.

[0053] In this embodiment of the cell screening kit, cells C are captured in individual wells 50, and detection particles B are reacted with cell secretions within the wells 50, allowing screening to be performed by light emission from the detection particles B, etc. Furthermore, even if the cell screening device 1 is carried or tilted with a fluid such as a dispersion liquid contained in the flow path 10, the fluid will not easily move along the flow path 10, and part of the dispersion liquid will flow into the well 50 through the through hole 52, thereby preventing the problem of cells C and detection particles B captured in the well 50 being detached from the well 50.

[0054] In the previous embodiment, the fluid injection ports 16 were provided on both ends of the cell screening device 1, but as shown in Fig. 9, it is also possible to form a fluid injection port 16 only on the cover part 14 on one side of the cell screening device 1. In this case, fluid can be injected and discharged from only one fluid injection port 16.

[0055] In the previous embodiment, the lid 14 was flat, but as shown in Fig. 10, it is also possible to form a foam discharge surface 54 that rises in an inclined or stepped manner as it approaches the fluid injection port 16 on at least a part of the ceiling surface of the flow path end 12 of the lid 14. In this case, the ceiling surface on the back side of the lid 14 rises toward the fluid injection port, so that air bubbles G that have entered the flow path end 12 rise along the inclined surface 54 and are easily discharged from the fluid injection port 16, thereby preventing problems such as the blocking of the through-hole 52 by air bubbles G. [Industrial Applicability]

[0056] According to the cell screening device or cell screening kit of the present invention, even when cells are captured in the wells of the cell screening device and the cell screening device is carried or tilted with a fluid such as a dispersion liquid in the flow path, the fluid is less likely to move along the flow path, suppressing the sloshing phenomenon and preventing problems such as part of the dispersion liquid flowing into the wells through the through-holes and causing the cells or detection particles captured in the wells to break away. Thus, the present invention is industrially applicable. [Explanation of symbols]

[0057] 1: cell screening device, 2: bottom plate, 4: cell placement surface, 6: cell placement membrane (cell placement section), 8: fluid injection section, 10: flow channel, 12: end of flow channel, 14: lid portion (fluid storage portion), 16: fluid injection port, 16A: opening portion, 18: engagement wall, 20: outer frame body, 22: peripheral wall portion, 24: engagement groove, 26: protrusion, 28: Partition portion, 30: Rib, 32: Engagement groove, 34: Step portion, 36: Frame body, 36A: Wall portion, 38: Inclined surface, 40: Engagement protrusion, 42: Engagement protrusion, 44: Engagement groove, 46: spacer, 47: notch, 50: well, 52: through hole, 54: Inclined surface (bubble discharge surface), C: cells, B: beads, L: dispersion, G: bubbles.

Claims

1. A bottom plate portion; a cell placement part provided on the bottom plate part and constituting a cell placement surface; a fluid injection section provided on the bottom plate section and separated from the cell placement section; a plurality of wells formed on the cell placement surface of the cell placement part, the wells having a size capable of individually accommodating cells to be screened; a flow channel provided between the bottom plate portion and the cell placement portion, the flow channel end portion extending to the fluid injection portion; a through hole that communicates with the flow channel from the inner bottom surface of the well and has an inner diameter that does not allow the cells to be screened to pass through; a cover portion provided in the fluid injection portion and airtightly closing an end portion of the flow channel; a fluid injection port provided on an upper surface of the lid portion and having an opening communicating with the flow channel; a fluid reservoir portion capable of storing fluid overflowing from the opening of the fluid injection port is formed on the upper surface of the lid portion around the fluid injection port; A cell screening device characterized in that a partition is provided between the cell placement surface and the lid portion to prevent the fluid from flowing into the cell placement portion when the fluid accumulates in the fluid storage portion.

2. The cell screening device of claim 1, wherein the bottom plate portion is rectangular having long and short sides, the fluid injection portion, the lid portion, and the fluid injection port are formed on both sides of the longitudinal direction of the bottom plate portion, and the cell placement portion is arranged between the two lid portions.

3. The cell placement portion is a membrane body, A plurality of the wells are formed in a lattice pattern on the upper surface of the membrane body, and the through holes are formed in the lower surface of the membrane body, the through holes communicating with the inner bottom surfaces of the wells, A frame is provided to support the periphery of the membrane body and surround the cell mounting surface, The cell screening device according to claim 1 , wherein the bottom plate portion is provided with an engagement portion for detachably fixing the frame body.

4. A cell screening device described in any one of claims 1 to 3, wherein a peripheral wall portion is formed rising from the periphery of the lid portion, and the lid portion and the peripheral wall portion are formed as an outer frame body that is detachable from the bottom plate portion.

5. a peripheral wall portion is formed on a peripheral edge of the lid portion, and the lid portion and the peripheral wall portion are formed as an outer frame body that is detachable from the bottom plate portion, The cell screening device according to claim 3 , wherein the frame body supporting the membrane body is removably fixed to the outer frame body.

6. The bottom plate portion has a rectangular shape having long sides and short sides, and the fluid injection portion, the lid portion, and the fluid injection port are formed on both sides of the bottom plate portion in the longitudinal direction, respectively, and the cell placement portion is disposed between the two lid portions; A cell screening device described in any one of claims 1 to 5, wherein the lid portion, the peripheral wall portion standing from the periphery of the lid portion, and the peripheral wall portion surrounding the periphery of the cell placement portion are formed as an outer frame body that is detachable from the bottom plate portion.

7. The cell screening device according to claim 1 , wherein the fluid injection port is cylindrical and stands upright from the upper surface of the lid portion.

8. A cell screening device described in any one of claims 1 to 7, wherein in the lid portion, at least a portion of the ceiling surface of the end of the flow path is formed with a bubble discharge surface that rises in an inclined or stepped manner as it approaches the fluid injection port.

9. A cell screening device according to any one of claims 1 to 8, and detection particles, which are carrier particles having immobilized thereon a substance capable of binding to the secretions of the cells to be screened.

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