Cell screening device and cell screening kit

Abstract

This cell screening device (1) comprises a bottom plate (2), a cell placement membrane (6), a pair of fluid injection parts (8), and a flow channel (10), said flow channel (10) being formed between the bottom plate (2) and the cell placement membrane (6) and extending in such a manner that the flow channel end parts (12) respectively reach the fluid injection parts (8). In the cell placement membrane (6), a plurality of wells, each having a size capable of housing a single cell C, and through holes are formed. In the back side of each lid (14), i.e., the ceiling face of the flow channel end part (12), a debubbling surface (17), which rises in an inclined or step-like manner as getting closer to a fluid injection hole (16A), is formed.

Description

Cell screening device and cell screening kit

[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. 2019-178992 filed in Japan on September 30, 2019, the content of which is incorporated herein by reference.

[0002] In recent years, especially in the field of drug discovery, the target of cell analysis has been refined from the cell group level to the single cell level. Using a cell screening device, cells are captured one by one in fine wells, and then a screening test is performed on a large number of cells at once to select cells having the desired characteristics. As a method for screening cells, for example, a method is adopted in which cells captured in a large number of wells are brought into contact 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 bound to the catcher are searched for and recovered from the wells.

[0003] Patent Document 1 shows a cell screening device previously proposed by the present applicant. This cell screening device has a first substrate and a second substrate arranged horizontally and stacked, and a large number of wells capable of accommodating each cell are formed on the surface of the first substrate, and communication holes reaching from the bottom surface of each well to the back surface of the first substrate are respectively formed. These communication holes are so thin that cells cannot pass through. On the second substrate, an accumulation part for receiving the secretion flowing out from the well through the communication hole is formed, and the secretion and the reagent are reacted in the accumulation part to identify the target cells.

[0004] International Publication No. WO2017 / 057234A1 pamphlet

[0005] In cell screening devices such as those described in Patent Document 1, a reagent introduction section is necessary to supply liquids such as reagents or dispersions to the accumulation section. For example, if the reagent introduction section is located at the end of the device, small air bubbles may enter the reagent introduction channel when adding or removing reagents or dispersions using instruments such as pipettes, micropipettes, or dispensers, or when the device is tilted and the liquid level in the reagent introduction section drops. When such air bubbles enter, they may move into the channel and block the back of the cell placement membrane, potentially obstructing the fluid flow between the well and the channel and affecting the screening process.

[0006] A cell screening device according to aspect [1] of the present invention comprises a bottom plate, a cell mounting section provided on the bottom plate and constituting a cell mounting surface, a fluid injection section provided on the bottom plate and separated from the cell mounting section, a plurality of wells formed on the cell mounting surface of the cell mounting section and having a size capable of individually accommodating cells to be screened, a flow path provided between the bottom plate and the cell mounting section and having a flow path end extending to the fluid injection section, a through hole connecting from the inner bottom surface of the well to the flow path and having an inner diameter that prevents the cells to be screened from passing through, a lid provided on the fluid injection section and closing the end of the flow path, and a fluid injection hole provided on the lid and connecting to the flow path, wherein at least a part of the ceiling surface of the end of the flow path has a foam discharge surface formed thereon that rises in an inclined or stepped manner as it approaches the fluid injection hole.

[0007] With this type of cell screening device, even if air bubbles enter the end of the flow path when reagents or dispersions are added or removed using instruments such as pipettes, micropipettes, or dispensers through the fluid injection port, or when the device tilts and the liquid level in the flow path drops, a bubble discharge surface is formed on the underside of the lid, i.e., on at least a part of the ceiling surface of the flow path end, which rises in an inclined or stepped manner as it approaches the fluid injection port. As a result, air bubbles move along the bubble discharge surface towards the fluid outlet and are discharged from the fluid outlet. Therefore, it is difficult for air bubbles to enter the flow path, and the risk of them affecting the screening is reduced.

[0008] The cell screening device of embodiment [2] is configured such that, in embodiment [1], the bubble discharge surface is formed over the entire ceiling surface of the end of the flow channel in the lid. In this case, regardless of the position of the bubble within the end of the flow channel, the bubble moves along the bubble discharge surface toward the fluid outlet and is discharged from the fluid outlet.

[0009] In the cell screening device of embodiment [3], the foam discharge surface is partially formed around the fluid injection hole on the ceiling surface of the end of the flow path in the lid, as in embodiment [1]. In this case, bubbles entering from the fluid injection hole move along the foam discharge surface formed around the fluid injection hole toward the fluid outlet and are discharged from the fluid outlet. Furthermore, partially forming the foam discharge surface has the advantage of increasing the design freedom of the cell screening device.

[0010] In the cell screening device of embodiment [4], in any of embodiments [1] to [3], the bubble discharge surface is an inclined surface with a straight or curved cross-section that is inclined at 5° to 45° with respect to the lower surface of the bottom plate. In this case, since the inclination angle is set as described above, bubbles move smoothly along the inclined surface of the bubble discharge surface toward the fluid outlet, increasing the certainty of discharge from the fluid outlet. Furthermore, while effectively discharging bubbles, the amount of protrusion of the lid portion at the fluid outlet can be kept relatively small.

[0011] The cell screening device of embodiment [5] is configured such that, in any of embodiments [1] to [4], the base plate has a rectangular shape with a long side and a short side, and the fluid injection section, the lid section, the fluid injection hole, and the bubble discharge surface are formed on both sides of the longitudinal direction of the base plate, and the cell placement section is positioned between the two lid sections. In this case, a wide range of uses are possible, such as injecting fluid into the flow path from one of the two fluid injection holes while discharging excess fluid from the other fluid injection hole. Moreover, in this process, bubbles move along the bubble discharge surface toward the fluid outlet and are discharged from the fluid outlet, thus suppressing problems caused by bubbles. However, the present invention is not limited to a configuration with two fluid injection holes; if necessary, only one side may be used, or three or more fluid injection holes may be provided.

[0012] The cell screening device of embodiment [6] is as described in embodiments [1] to [5], wherein the cell placement portion is a membrane, a plurality of wells are formed in a grid pattern on the upper surface of the membrane, through holes are formed on the lower surface of the membrane that lead to the inner bottom surface of each of the wells, a frame is provided that supports the periphery of the membrane and surrounds the cell placement surface, and the bottom plate is provided with an engaging portion for detachably fixing the frame. In this case, when the frame is engaged with the engaging portion of the bottom plate, the membrane having wells is positioned in the correct position on the bottom plate, and the frame can be removed from the bottom plate after screening.

[0013] In embodiment [7], the cell screening device has a peripheral wall portion formed upright from the periphery of the lid portion, as in any of embodiments [1] to [6], and the lid portion and the peripheral wall portion are formed as an outer frame that can be attached to and detached from the bottom plate portion. In this case, the bottom plate portion and the outer frame portion are molded separately, and by simply attaching the outer frame portion to the bottom plate portion, accurate positioning of the lid portion and the peripheral wall portion is possible, and assembly is easy. Furthermore, after use, the outer frame portion can be removed from the bottom plate portion for disassembly, making maintenance easy.

[0014] In the cell screening device of embodiment [8], as in embodiment [6], an upright peripheral wall is formed on the periphery of the lid, the lid and the peripheral wall are formed as an outer frame that can be attached to the base plate, and the frame that supports the membrane is detachably fixed to the outer frame. In this case, the base plate, the outer frame and the frame are molded as separate parts, and by simply attaching the frame and the outer frame to the base plate, accurate positioning of the membrane, lid and peripheral wall can be achieved, improving ease of assembly. In addition, the frame and the outer frame can be removed from the base plate after use, making maintenance easier.

[0015] The cell screening device of embodiment [9], in any of embodiments [1] to [8], has a rectangular base plate with a long side and a short side, and the fluid injection section, the lid, the fluid injection hole, and the bubble discharge surface are formed on both sides of the longitudinal direction of the base plate, the cell placement section is positioned between the two lids, and the lid, the peripheral wall sections rising from the periphery of each lid, and the peripheral wall section surrounding the cell placement section are formed as an outer frame that can be attached to and detached from the base plate. In this case, the base plate and the outer frame are molded separately, and by simply attaching the outer frame to the base plate, accurate positioning of the cell placement section, the lid, the peripheral wall, and the two fluid injection holes can be achieved, improving ease of assembly. Furthermore, the outer frame can be removed from the base plate and disassembled after use, making maintenance easier.

[0016] In the cell screening device of embodiment

[10] , in any of embodiments [1] to [9], a cylindrical port is formed in the lid portion, rising from the fluid injection hole. In this case, since the fluid injection port is cylindrical, it is easy to inject and inject fluid by placing the pointed tip of a pipette or dispenser against the tip of the fluid injection port.

[0017] The cell screening kit of embodiment

[11] comprises a cell screening device as described in any of embodiments [1] to

[10] , and detection particles which are carrier particles on which a substance binding to the secretions of cells to be screened is immobilized. With this cell screening kit, cells are taken into individual wells, and the detection particles are reacted with the secretions of the cells in the wells, and screening can be performed by luminescence from the detection particles. Furthermore, even if air bubbles enter the end of the flow path when reagents or dispersions are added or removed using instruments such as pipettes, micropipettes, or dispensers from the fluid injection port, or when the device is tilted and the liquid level in the end of the flow path drops, an air bubble discharge surface is formed on the underside of the lid, i.e., the ceiling surface of the end of the flow path, which rises in an inclined or stepped manner as it approaches the fluid injection port. As a result, air bubbles move along the air bubble discharge surface towards the fluid outlet and are discharged from the fluid outlet. Therefore, air bubbles are less likely to enter the flow path, and there is less risk of them affecting the screening.

[0018] As described above, with the cell screening device or cell screening kit of the present invention, even if air bubbles enter the end of the flow path when reagents or dispersions are added or removed using an instrument from the fluid injection port, or when the device is tilted and the liquid level in the end of the flow path drops, a bubble discharge surface is formed on the underside of the lid, i.e., on at least a part of the ceiling surface of the end of the flow path, which rises as it approaches the fluid injection port. Therefore, air bubbles are discharged from the fluid outlet along the bubble discharge surface. Consequently, it is less likely for air bubbles to enter the flow path, reducing the risk of them affecting the screening process.

[0019] This is a plan view showing a cell screening device according to one embodiment of the present invention. This is a front view of the same embodiment. This is a perspective view showing a cross-section of the same embodiment. This is a cross-sectional view of the central part of the same embodiment. This is a cross-sectional view of the end of the same embodiment. This is a side cross-sectional view of the same embodiment. This is a perspective view showing the assembly method of the same embodiment. This is an enlarged perspective view showing the cell placement membrane (cell placement section) and wells of the same embodiment. This is a perspective view showing a cross-section of a cell screening device according to another embodiment of the present invention. This is a plan view of the foam discharge surface

[0020] Figures 1 to 6 all show the completed state of a cell screening device 1 according to one embodiment of the present invention, and Figure 7 shows the assembly method of the cell screening device 1. The cell screening device 1 of this embodiment has a bottom plate portion 2, a cell mounting membrane (cell mounting portion) 6 provided on the bottom plate portion 2 and constituting a cell mounting surface 4, a pair of fluid injection portions 8 provided on the bottom plate portion 2 separated from the cell mounting membrane 6, and a flow path 10 provided between the bottom plate portion 2 and the cell mounting membrane 6, with a flow path end 12 extending to the fluid injection portion 8. On the cell mounting surface 4 of the cell mounting membrane 6, for example as shown in Figure 8, a plurality of wells 50 having a size capable of individually accommodating cells C to be screened are formed, and through holes 52 leading from the inner bottom surface of the wells 50 to the flow path 10 are formed, and the through holes 52 have an inner diameter that prevents cells C to be screened from passing through. Furthermore, on the back side of each lid portion 14, that is, on the ceiling surface of the flow path end 12, a bubble discharge surface 17 is formed that rises in an inclined shape as it approaches the fluid injection hole 16A.

[0021] The structure of the cell screening device 1 will be described in detail below. In this example, the bottom plate portion 2 is an elongated rectangular plate with a constant thickness, and its four corners are rounded. A ridge 2B of a constant height is formed around the entire circumference of the bottom plate portion 2 along the outer edge of the bottom surface. The shape of the bottom plate portion 2 is not limited to that shown in the figure, and it may be any shape such as a disc, ellipse, or square, and the thickness of the bottom plate portion 2 does not have to be constant as long as a horizontal channel 10 can be formed. Generally, it is desirable for the bottom plate portion 2 to be formed from various plastics that are harmless to target cells in terms of molding accuracy and cost, but it may be formed from any material such as ceramic, glass, or metal if necessary. The surface of the bottom plate portion 2 may be coated with some kind of coating to mitigate the effect on cells.

[0022] As shown in Figure 3, an engagement wall 18 is integrally formed on the upper surface of the base plate 2, surrounding the central part. The engagement wall 18 is rectangular in shape when viewed from above, with semicircular ends at both ends, and stands vertically upright from the base plate 2. The engagement wall 18 is not limited to the shape shown, and may be a simple rectangle, circle, ellipse, etc. In this example, the height of the engagement wall 18 is equal around its entire circumference. An outer frame 20 is detachably attached to the engagement wall 18 so as to cover the engagement wall 18 from above around its entire circumference.

[0023] The outer frame 20 has a rectangular peripheral wall portion 22 with semicircular ends when viewed from above, and a pair of partition portions 28 provided parallel to each other on the inner circumference of the peripheral wall portion 22, and the entire structure is integrally formed. The material of the outer frame 20 is not limited, and it is generally desirable to form it from various plastics that are harmless to cells for reasons of molding accuracy and cost, but it may be formed from any material such as ceramic, glass, or metal if necessary. A rectangular parallelepiped space is left between the partition portions 28, and the cell mounting membrane 6 is placed in this space.

[0024] The upper end of the peripheral wall portion 22 of the outer frame body 20 has a cross-sectional shape that is folded outward all around, and a narrow engagement groove 24 that opens downward is formed on the inside of this folded portion to 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 tightened by the folded portion of the peripheral wall portion 22, thereby fixing the outer frame body 20 to the engagement wall 18 in a detachable manner. Projections 26 that protrude horizontally are formed at both ends in the longitudinal direction of the outer frame body 20, and by lifting these projections 26 with a fingertip, the engagement wall 18 can be removed from the engagement groove 24, and the bottom plate portion 2 and the outer frame body 20 can be separated.

[0025] The semicircular regions enclosed by the peripheral wall portion 22 of the outer frame 20 and each partition portion 28 are each designated as fluid injection sections 8. In these fluid injection sections 8, a lid portion 14 is formed so as to connect the lower end of the peripheral wall portion 22 and the lower end of each partition portion 28, forming a three-dimensional shape that is semicircular in plan view and has a central part that rises upward.

[0026] Between the lid portion 14 and the bottom plate portion 2, channel ends 12, which are the ends of the channel 10, are formed, and the lid portion 14 is structured to airtightly seal the channel ends 12. Because the lid portion 14 is formed in this way, the channel ends 12, which are the ends of the channel 10, are sealed, and even if the cell screening device 1 is tilted or shaken, excessive fluid flow from side to side through the channel 10 and the channel ends 12 is prevented.

[0027] In this embodiment, a circular fluid injection hole 16A is formed approximately in the center of each lid portion 14, and cylindrical fluid injection ports 16 are formed upright from the lid portion 14 corresponding to these fluid injection holes 16A. The surface of the lid portion 14 around the fluid injection ports 16 serves as a fluid reservoir, and as shown in Figure 5, fluid L overflowing from the fluid injection ports 16 is collected there. A recess may be actively formed around the fluid injection ports 16 on the upper surface of the lid portion 14 to serve as a fluid reservoir.

[0028] On the underside of each lid portion 14, that is, on the ceiling surface of the flow path end portion 12, a bubble discharge surface 17 is formed that rises in an inclined manner as it approaches the fluid injection hole 16A, and the bubble discharge surface 17 is frustoconical in shape with the fluid injection hole 16A as its apex. As a result, when the flow path end portion 12 is filled with a liquid such as a dispersion and contains bubbles G, the bubbles G move smoothly along the bubble discharge surface 17 toward the fluid injection hole 16A by buoyancy and are discharged from the fluid injection hole 16A.

[0029] The angle α between the bubble discharge surface 17 and the horizontal plane is not constant around the fluid injection hole 16A because the distance from the fluid injection hole 16A to the periphery of the lid portion 14 is not constant. However, from the viewpoint of the bubble discharge effect, it is preferably 5° to 45°, and more preferably 10° to 30°. Within this range, bubbles G do not adhere to and remain on the bubble discharge surface 17, but roll or slide along the bubble discharge surface 17, and can be discharged almost certainly from the fluid injection hole 16A and the fluid injection port 16. Furthermore, if the inclination angle α of the bubble discharge surface 17 is too large than the above range, the volume of the flow path end 12 becomes too large, which may lead to problems such as increased reagent consumption.

[0030] In this embodiment, since the thickness of the lid portion 14 is almost constant, the upper surface of the lid portion 14, i.e., the upper surface of the fluid storage portion, is also inclined in a frustoconical shape. Therefore, the surface of the fluid storage portion also slopes downward as it moves away from the fluid injection port 16, and the fluid that spills from the fluid injection port 16 collects in the peripheral area of ​​the lid portion 14 away from the fluid injection port 16, thus reducing the risk of contamination from re-inflow from the fluid injection port 16. However, the present invention is not limited to this configuration, and it is also possible to make the upper surface of the lid portion 14, i.e., the upper surface of the fluid storage portion, horizontal by increasing the thickness of the lid portion 14 as it moves away from the fluid injection port 16.

[0031] In this embodiment, the fluid injection port 16 is raised from the lid portion 14, but instead, the raised fluid injection port 16 may not be formed in the lid portion 14, and the fluid injection hole 16A may be left open as is.

[0032] The upper inner edge of the fluid injection port 16 has a rounded cross-sectional shape all around, forming an annular chamfered portion 16B. The chamfered portion 16B is shaped to fit well with the tip of an instrument such as a pipette, micropipette, or dispenser used to inject fluid (reagents or dispersions) from the fluid injection port 16, allowing the pointed tip of the instrument to be airtightly contacted with the fluid injection port 16. The shape of the fluid injection port 16 is not limited to a cylindrical shape; it may be a rectangular tube or a polygonal tube as needed, or it may be an annular shape with only a slight raised area around the fluid injection hole 16A, or as described above, a simple opening. The fluid injection port 16 may also be a conical opening recessed in the surface of the lid portion 14.

[0033] At the lower end of the partition portion 28 on the fluid injection portion 8 side, a stepped portion 34 is formed along the entire length of the partition portion 28, having a certain height from the lower surface. Above this stepped portion 34, two ribs 30 are formed, each extending vertically to the upper end of the partition portion 28. The ribs 30 increase the bending strength of the partition portion 28. In addition, the partition portion 28 has an engagement groove 32 of a certain depth that opens to the lower surface of the stepped portion 34, and an engagement projection 42 adjacent to the cell mounting membrane 6 side of the engagement groove 32.

[0034] As shown in Figure 6, two linearly extending portions of the peripheral wall 22 of the outer frame 20 are also formed, each having an engagement groove 32 of a certain depth that opens to the lower surface of the peripheral wall 22, and an engagement ridge 42 adjacent to the cell mounting membrane 6 side of the engagement groove 32. 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 portion 28, respectively, forming a rectangular shape when viewed from above.

[0035] A rectangular tubular frame 36, which forms a rectangular shape when viewed from above, is detachably housed in the square space enclosed by two linearly extending portions of the peripheral wall 22 and two partition portions 28. A cell mounting membrane 6 is stretched across the entire surface of the lower end of the frame 36 with the wells 50 facing upward, as shown in Figure 8, and the lower end of the frame 36 and the cell mounting membrane 6 are joined together without any gaps around the entire circumference. The frame 36 is made of a flexible plastic or the like, and when an outward force is applied, the four walls spread slightly outward, applying tension to the cell mounting membrane 6 and preventing it from sagging.

[0036] The thickness of the cell placement membrane 6 is not limited, but from the viewpoint of forming wells 50 in which each target cell can be placed, and forming fine through-holes 52 that allow fluid to flow from the bottom of the wells 50 to the back side, it is preferably about 5 to 100 μm, and more preferably about 10 to 50 μm. The cell placement membrane 6 may be a multilayer membrane of two or more layers. In that case, through-holes that will become wells 50 may be formed in the upper layer, and through-holes that will become through-holes 52 may be made in the lower layer, and these two layers may be bonded together to form the wells 50 and through-holes 52.

[0037] The material of the cell mounting membrane 6 is not limited, and it is generally desirable for molding accuracy and cost to be formed from various plastics that are harmless to cells, but if necessary, it may be formed from any material such as ceramic, polycrystalline or single-crystal silicon, inorganic compounds such as glass, or metal. The wells 50 and through holes 52 can also be formed by etching or photolithography.

[0038] The planar shape of the well 50 is preferably hexagonal or circular, as shown in Figure 8, from the viewpoint of increasing the arrangement density to match the average shape of the cells C to be captured. However, if necessary, it may also be a square or other quadrilateral, other polygons, or an ellipse. 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 it is generally preferable that the maximum diameter of the circle entering the well opening be about 1 to 100 μm and the depth be about 1 to 100 μm so that one cell C fits in each well 50, but it is difficult for two or more cells C to fit in. Multiple types of frame bodies 36 having cell placement membranes 6 with different well sizes 50 may be prepared and combined with a common bottom plate 2 and outer frame body 20 to constitute a cell screening kit.

[0039] Regarding the size of the cells C, the maximum diameter of the circle that fits inside the well 50 when viewed from above is preferably about 0.5 to 2 times the maximum diameter of the cells C to be collected, and more preferably about 0.8 to 1.9 times. The depth of the well 50 is preferably about 0.5 to 4 times the maximum diameter of the cells C to be collected, and more preferably about 0.8 to 1.9 times. The shortest distance between adjacent wells 50 is preferably about 1 to 10 μm, as this increases the density of the wells 50, but it is not limited to this range.

[0040] In this example, two through-holes 52 are formed in the center of the bottom surface of each well 50. While having multiple through-holes in this way is advantageous because it prevents clogging, the present invention is not limited to this, and one or three or more through-holes may be formed in the center, or multiple through-holes 52 may be formed randomly or at grid point positions on the bottom surface of the well 50. The inner diameter of the through-holes 52 is not limited, but it is preferable that the minimum inner diameter is in the range of about 10 nm to 20 μm so that 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.

[0041] As shown in Figures 4 and 6, the outer circumferential surface of the lower end of the frame 36 is folded upward, forming an upward-opening engagement groove 44 and a downward-protruding engagement ridge 40 along its entire circumference. The depth of the engagement groove 44 and the vertical width of the engagement ridge 40 are substantially constant along the entire circumference of the frame 36. The engagement ridge 40 is inserted into an engagement groove 32 formed on the lower surface of the outer frame 20, and the engagement ridge 42 of the outer frame 20 is inserted into the engagement groove 44 of the frame 36. Through this fitting, the frame 36 is fixed to the outer frame 20 with the frame 36 housed within the central space of the outer frame 20.

[0042] 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 thickness of the spacer 46 accurately determines the distance of the cell mounting membrane 6 from the bottom plate 2, i.e., the thickness of the flow channel 10. In this embodiment, as shown in Figure 7, a pair of spacers 46 that form a U-shape in plan view are formed inside the engagement wall 18 along the shape of the lower end of the frame 36, and notches 47 are formed between the spacers 46. When the frame 36 is fixed on the bottom plate 2, fluid flows from the flow channel 10 to each flow channel end 12 through these notches 47. The spacers 46 do not have to be in the shape shown, and it is sufficient that they abut 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 spacers 46.

[0043] An inclined surface 38 with a constant width is formed on the inner circumferential surface of the lower end of the frame 36, and the inclined surface 38 protrudes toward the cell mounting membrane 6 as it goes downwards. As shown in Figure 4, when aspirating target cells 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 aspirated with the instrument P even if the wells 50 containing the collected target cells are located very close to the inner circumferential edge of the frame 36. Furthermore, the inclined surface 38 increases the adhesion area of ​​the cell mounting membrane 6 to the frame 36, thereby increasing the bonding strength of the cell mounting membrane 6, and also contributes to increasing the strength of the frame 36.

[0044] When manufacturing the cell screening device having the above-described configuration, as shown in FIG. 7, the bottom plate portion 2, the frame body 36, and the outer frame body 20 are each formed separately. First, the frame body 36 is fitted into the lower surface 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, whereby the completed state as shown in FIGS. 1 to 6 is obtained.

[0045] In the process of fitting the frame body 36 into the lower surface of the outer frame body 20, as shown in FIGS. 4 and FIG. 6, the engaging protrusion 42 of the outer frame body 20 fits into the engaging groove 44 of the frame body 36, and the engaging protrusion 40 of the frame body 36 fits into the engaging groove 32 of the outer frame body, so that the two are firmly fixed by the elasticity of each engaging portion. At the same time, at least one of the inner peripheral surface of the engaging protrusion 40 and the outer peripheral surface of the engaging protrusion 42 is shaped to incline slightly outward as it goes upward. Thereby, when the engagement progresses, the engaging protrusions 40 on the four sides of the frame body 36 are pulled outward by the engaging protrusions 42 of the outer frame body 20, the lower end portion of the frame body 36 is slightly widened in all directions, a tension for pulling the four sides of the cell placement membrane 6 is generated, and a uniform tension is applied to the cell placement membrane 6

[0046] Therefore, even if there is a slight slack in the cell placement membrane 6 when the frame body is in a free state, when the frame body 36 is fixed to the outer frame body 20, the slack in the cell placement membrane 6 is eliminated, the flatness of the cell placement surface 4 on which a large number of wells 50 of the cell placement membrane 6 are formed is improved, and a dispersion liquid in which cells are dispersed is filled on the cell placement surface 4, and the cell screening device is shaken, etc., so that the cells can be easily flowed without bias and captured one by one in the wells 50.

[0047] The cell screening kit using this embodiment includes a cell screening device 1 and beads (detection particles) B which are carrier particles on which a substance having binding property to the secretion of the cells to be screened is immobilized. The cell screening kit is a product that houses, in an outer container such as a box, one or a plurality of cell screening devices 1 and an inner container in which a large number of fine beads B as detection particles are enclosed in a powder state or suspended in a dispersion liquid.

[0048] The type of the binding particles is not limited. For example, it may be carrier particles composed of beads (such as magnetic beads, resin beads, etc.), hydrogel particles (such as sodium alginate gel, agarose gel, etc.), metal particles (such as gold nanoparticles), etc., and a catcher that can bind to an antibody secreted from an antibody-producing cell serving as a target and attached to the carrier particles. The particle size of the binding particles is not limited, but generally, it is preferably about 100 nm to 50 μm in terms of the maximum diameter of the particles, and more preferably about 500 nm to 30 μm.

[0049] When actually performing screening, the cells and the beads B serving as detection particles are dispersed in a dispersion liquid and filled on the cell placement membrane 6. The cell screening device 1 is shaken or the cells C are put into the wells 50 one by one by discharging them into the flow path 10 through the through holes 52, etc. At the same time, a large number of beads B are put into the wells 50 together with each cell C, and the excess dispersion liquid is discharged into the flow path 10 through the through holes 52. In this state, if the antibody in the secretion of the cells C and the catcher of the beads B are reacted, only the reacted beads B can be visualized with a fluorescently labeled antibody to find the target cells.

[0050] According to the cell screening device 1 having the above configuration, since a bubble discharge surface 17 that rises as it approaches the fluid injection hole 16A is formed on the back side of the lid part 14, that is, on the ceiling surface of the flow path end part 12, the bubbles G that have entered the flow path end part 12 rise along the bubble discharge surface 17 and are discharged from the fluid injection port 16. Therefore, it is possible to suppress screening troubles such as the bubbles G entering the flow path 10 and contacting the back surface of the cell placement membrane 6, resulting in the blockage of the through holes 52 and the wells 50 or the inability to uniformly allow the fluid to enter and exit each well 50.

[0051] Further, in this embodiment, since the bubble discharge surface 17 is formed over the entire ceiling surface of the flow path end part 12 in the lid part 14, no matter where the bubbles G are located in the flow path end part 12, the bubbles G have the merit of moving along the bubble discharge surface 17 toward the fluid injection hole 16A and being discharged from the fluid injection port 16.

[0052] However, the present invention is not limited to a configuration in which the foam discharge surface is formed over the entire surface; it may also be formed partially, for example, in an annular shape, only around the fluid injection hole 16A on the ceiling surface of the flow channel end 12. In this case as well, bubbles G that enter through the fluid injection hole 16A move towards the fluid outlet 16A along the foam discharge surface formed around the fluid injection hole 16A immediately after entry and are discharged, so the foam discharge effect can be generally sufficiently obtained. Furthermore, by partially forming the foam discharge surface, the amount of protrusion of the lid portion 14 can be reduced, which has the advantage of increasing the design freedom of the cell screening device.

[0053] Furthermore, in this embodiment, the flow path ends 12 located at both ends of the flow path 10 are closed with lids 14, and fluid injection ports 16 having fluid injection holes 16A that communicate with the flow path 10 are provided in these lids 14. As a result, the movement of fluid in the flow path ends 12 and within the flow path 10 is suppressed by the lids 14, and fluid can be added to and removed from the flow path 10 through the fluid injection ports 16. Therefore, even when the cell screening device 1 is carried or tilted while cells C are captured in the well 50 of the cell screening device 1 and a fluid such as a dispersion is placed in the flow path 10, the fluid is less likely to move along the flow path 10 and the flow path ends 12, thereby suppressing the so-called sloshing phenomenon. Thus, problems such as a portion of the dispersion flowing into the well 50 through the through-hole 52 and releasing cells C and beads B that were captured in the well 50 can also be suppressed.

[0054] Furthermore, in this embodiment, as shown in Figure 5, even if fluid L overflows from the fluid injection hole 16A of the fluid injection port 16, the fluid storage section (the area around the upper surface of the lid 14) receives the fluid L, preventing it from re-entering the flow path 10 from the fluid injection port 16, thereby reducing the risk of contamination from external sources, for example. Since a partition 28 is formed between the cell placement surface 4 and the lid 14, even if fluid accumulates in the fluid storage section 14, it is possible to prevent the fluid from flowing to the cell placement section 6.

[0055] Furthermore, in this embodiment, since two fluid injection ports 16 are formed at both ends of the flow path 10, a wide range of applications are possible, such as injecting fluid into the flow path 10 from one fluid injection port 16 and discharging excess fluid from the other fluid injection port 16.

[0056] Furthermore, in this embodiment, by bringing the lower end of the frame 36 into contact with the spacer 46 of the bottom plate 2, the cell mounting membrane 6 is positioned in the correct location on the bottom plate 2, so that the fluid flow between the well 50 and the flow path 10 is as desired, enabling highly accurate screening.

[0057] Furthermore, in this embodiment, the base plate portion 2 and the outer frame 20 are molded separately, and the lid portion 14 and the peripheral wall portion 22 can be positioned precisely relative to the base plate portion 2 simply by attaching the outer frame 20 to the base plate portion 2, thus simplifying the assembly of the cell screening device 1. The outer frame 20 can also be removed from the base plate portion 2 after use, making maintenance easy.

[0058] Furthermore, in this embodiment, the base plate 2, the outer frame 20, and the frame 36 are molded as separate parts, and by simply attaching the frame 36 and the outer frame 20 to the base plate 2, accurate positioning of the cell mounting membrane 6, the lid 14, and the peripheral wall 22 becomes possible, improving ease of assembly. The frame 36 and the outer frame 20 can also be removed from the base plate 2 after use, making maintenance easy.

[0059] Furthermore, in this embodiment, the base plate portion 2 and the outer frame 20 are molded separately, and by simply attaching the outer frame 20 to the base plate portion 2, the cell placement portion 6, the lid portion 14, the peripheral wall portion 22, and the two fluid injection ports 16 can be precisely positioned, making assembly easy.

[0060] Furthermore, in this embodiment, since the fluid injection port 16 is cylindrical, it is easy to inject and remove fluid by applying a cone-shaped pointed tip of a pipette or dispenser to it in a nearly airtight manner.

[0061] In this embodiment, the cell screening kit allows for screening by reacting detection particles B with cell secretions within each well 50 while cells C are incorporated into the wells 50, and detecting the luminescence from the detection particles B. Furthermore, even if air bubbles enter the flow channel end 12 when reagents or dispersions are added or removed using instruments such as pipettes, micropipettes, or dispensers from the fluid injection port 16, or when the device tilts and the liquid level in the flow channel end 12 drops, the air bubbles move along the bubble discharge surface 17 towards the fluid outlet 16A and are discharged from the fluid injection port 16. Therefore, air bubbles are less likely to enter the flow channel 10, and there is less risk of them affecting the screening.

[0062] In the previous embodiment, the cell screening device 1 was configured with foam discharge surfaces 17 and fluid injection ports 16 on the lid portions 14 at both ends. However, as shown in Figure 9, it is also possible to form the foam discharge surface 17 and fluid injection port 16 on only one side of the lid portion 14 of the cell screening device 1. In this case, fluid can be injected and discharged from only one fluid injection port 16.

[0063] Furthermore, in the above embodiment, the bubble discharge surface 17 was a smooth inclined surface, but as shown in Figure 10, ribs 56 that restrict the movement of bubbles G may be formed on the back surface of the lid portion 14, that is, on the ceiling surface of the flow path end 12. In the example in Figure 10, ribs 56 extending radially from the fluid injection hole 16A are formed to a length that reaches the periphery of the lid portion 14. The cross-sectional shape of the ribs 56 may be rectangular or triangular with a pointed tip. When such ribs 56 are formed, the direction of movement of bubbles G is directed toward the fluid injection hole 16A by the ribs 56, allowing bubbles G to be discharged smoothly. In addition, as the bubbles G approach the fluid injection hole 16A, the spacing between the ribs 56 narrows, causing the bubbles G to break into smaller bubbles G, which makes them easier to discharge from the fluid injection hole 16A. The ribs 56 do not need to reach the periphery of the lid portion 14, and can be formed only around the fluid injection hole 16A. Grooves may be formed instead of ribs 56.

[0064] Furthermore, as shown in Figure 11, the bubble discharge surface 17 may be formed with a number of horizontal stepped surfaces 58 that gradually rise toward the fluid injection hole 16A. In this case as well, the bubbles G will ascend the stepped surfaces 58 one after another toward the fluid injection hole 16A, making it possible to discharge them from the fluid injection port 16. It is desirable that the width of the stepped surfaces 58 be sufficiently small so that the bubbles G can ascend smoothly. When such stepped surfaces 58 are formed, there is an advantage that the bubbles G are less likely to move toward the flow path 10 even if the cell screening device 1 is tilted.

[0065] Although embodiments of the present invention have been described above, the present invention is not limited to these embodiments, and within the scope of the claims, the configurations of each embodiment may be combined with each other, some configurations may be omitted, or known configurations may be added.

[0066] According to the cell screening device or cell screening kit of the present invention, even if air bubbles enter the end of the flow path when fluid is added or removed using an instrument from the fluid injection port, or when the device tilts and the liquid level in the end of the flow path drops, a bubble discharge surface is formed on the underside of the lid, i.e., on at least a part of the ceiling surface of the end of the flow path, which rises in an inclined or stepped manner as it approaches the fluid injection port. As a result, air bubbles move along the bubble discharge surface toward the fluid outlet and are discharged from the fluid outlet. Therefore, it is difficult for air bubbles to enter the flow path, and the risk of affecting screening is reduced, making the present invention industrially applicable.

[0067] 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: Flow channel end, 14: Lid (fluid storage section), 16: Fluid injection port, 16A: Opening, 17: Bubble discharge surface, 18: Engaging wall, 20: Outer frame, 22: Peripheral wall, 24: Engaging groove, 26: Protrusion, 28: Partition, 30: Rib, 32: Engaging groove, 34: Step, 36: Frame, 36A: Wall, 38: Inclined surface, 40: Engaging ridge, 42: Engaging ridge, 44: Engaging groove, 46: Spacer, 47: Notch, 50: Well, 52: Through hole, 56: Rib, 58: Stepped surface, C: Cell, B: Beads, L: Dispersion, G: Bubble.

Claims

a plurality of wells formed on the cell mounting surface of the cell mounting section, each well having a size large enough to individually accommodate cells to be screened; a flow channel provided between the bottom plate and the cell mounting section, the flow channel end extending to the fluid injection section; through-holes leading from the inner bottom surfaces of the wells to the flow channel and having an inner diameter that does not allow the cells to be screened to pass through; a lid provided in the fluid injection section and closing the flow channel end; and a fluid injection hole provided in the lid and leading to the flow channel, wherein at least a portion of the ceiling surface of the flow channel end of the lid is formed with a foam discharge surface that rises in an inclined or stepped manner as it approaches the fluid injection hole.

2. A cell screening device as described in claim 1, wherein the bubble discharge surface is formed over the entire ceiling surface of the end of the flow channel in the lid portion.

3. A cell screening device as described in claim 1, wherein the bubble discharge surface is partially formed around the fluid injection hole on the ceiling surface of the end of the flow path in the lid portion.

4. A cell screening device described in any one of claims 1 to 3, wherein the bubble discharge surface is an inclined surface having a linear or curved cross section that is inclined at an angle of 5° to 45° relative to the lower surface of the bottom plate portion.

5. A cell screening device as described in any one of claims 1 to 4, wherein the bottom plate portion is rectangular with long and short sides, and the fluid injection portion, the lid portion, the fluid injection hole, and the foam discharge surface 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.

6. A cell screening device as described in any one of claims 1 to 5, wherein the cell mounting portion is a membrane body, the wells are formed on the upper surface of the membrane body in a grid pattern, and the through holes leading to the inner bottom surfaces of each 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 the bottom plate portion is provided with an engagement portion for detachably fixing the frame body.

7. A cell screening device described in any one of claims 1 to 6, wherein a peripheral wall portion is formed standing 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.

8. A cell screening device as described in claim 6, wherein an upright peripheral wall portion is formed on the periphery of the lid portion, the lid portion and the peripheral wall portion are formed as an outer frame body that is detachable from the bottom plate portion, and the frame body that supports the membrane body is detachably fixed to the outer frame body.

9. A cell screening device as described in any one of claims 1 to 8, wherein the bottom plate portion is rectangular with long and short sides, and the fluid injection portion, the lid portion, the fluid injection hole, and the foam discharge surface are formed on both longitudinal sides of the bottom plate portion, respectively, the cell placement portion is disposed between the two lid portions, and the lid portions, the peripheral wall portions standing from the periphery of each 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.

10. A cell screening device according to any one of claims 1 to 9, wherein the lid portion has a cylindrical port formed therein that stands up from the fluid injection hole.

11. A cell screening kit comprising the cell screening device according to any one of claims 1 to 10 and detection particles, which are carrier particles onto which a substance capable of binding to secretions of the cells to be screened is immobilized.

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