Screening method for secretion-producing cells, and screening kit for secretion-producing cells

Abstract

A method for screening secretion-producing cells by screening cells producing target secretions from a plurality of cells. The method comprises: step A, capturing the cells and detection particles capable of capturing the target secretions by using a plurality of holes having through holes at the bottom of the holes that are too large for the cells to pass through; step B, causing the cells captured in the plurality of holes to produce secretions; step C, detecting the target secretions captured by the detection particles; and step D: using the detection results as an indicator, determining the hole that captures the cell producing the target secretion from the plurality of holes. The hole has a size that can capture the cell in a unit of one cell when one or more detection particles are captured.

Description

Technical Field

[0001] The present invention relates to a method for screening secretion-producing cells and a screening kit for secretion-producing cells. This application claims priority based on Japanese Patent Application No. 2019-178577 filed in Japan on September 30, 2019, and the contents thereof are incorporated into this application. Background Art

[0002] In recent years, especially in the field of innovative drugs, the object of cell analysis has been subdivided from the cell group level to the single cell level. Attempts have been made to capture cells at the level of one by one, identify, screen, recover, culture, and perform genetic analysis, and use the selected cells. As a method for identifying and screening cells, a method is adopted in which the secretions secreted by the cells are separated, the target secretions are detected, and then the cells that secrete the target secretions are screened. In such single cell analysis, a technology that analyzes multiple cells at once is useful.

[0003] As a technique for analyzing cells together, for example, a method has been reported in which a mixture of antibody-producing cells, antigen-binding beads, and fluorescently labeled anti-antibody-antibody is spotted on a slide and incubated, and B cells producing the target antibody are identified by the local increase in fluorescence surrounding the antibody-producing cells (Patent Document 1).

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent No. 4603894 Summary of the invention

[0007] Problems to be solved by the invention

[0008] However, in the method described in Patent Document 1, since the antibody-producing cells are not fixed, there is a possibility that the antibody-producing cells move during the culturing operation, fluorescence detection operation, cell collection operation, etc. Therefore, accurate screening may not be performed.

[0009] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for screening secretion-producing cells capable of accurately obtaining cells producing a target secretion, and a screening kit for the secretion-producing cells.

[0010] Means for solving problems

[0011] In order to solve the above-mentioned problems, the present invention adopts the following configuration.

[0012] The first embodiment of the present invention is a method for screening secretion-producing cells, which screens out cells that produce target secretions from multiple cells, and the method includes: step A, capturing the cells and detection particles in multiple holes having through holes at the bottom that are of a size that the cells cannot pass through, the detection particles being able to capture the target secretions and having a size that cannot pass through the through holes; step B, causing the cells captured in the multiple holes to produce secretions; step C, detecting the target secretions captured by the detection particles; and step D, using the detection results as an indicator, determining the holes that have captured the cells that produce the target secretions from the multiple holes, the holes having a size that can capture the cells in units of one cell when one or more of the detection particles are captured.

[0013] A second embodiment of the present invention is a screening kit for secretion-producing cells, which screens out cells that produce target secretions from multiple cells. The screening kit for secretion-producing cells comprises a device including multiple holes, and carrier particles, wherein the holes have a size that can capture the cells in units of one cell when one or more of the detection particles are captured, and have through holes at the bottom that are of a size that the cells cannot pass through, and the carrier particles have a size that cannot pass through the through holes.

[0014] Effects of the Invention

[0015] According to the present invention, a method for screening secretion-producing cells capable of accurately obtaining cells producing a target secretion, and a screening kit for the secretion-producing cells can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] [ Figure 1 ] is a diagram illustrating an example of step A of the method for screening secretion-producing cells.

[0017] [ Figure 2 ] is a diagram illustrating an example of step B of the method for screening secretion-producing cells.

[0018] [ Figure 3 ] is a diagram illustrating an example of step C of the method for screening secretion-producing cells.

[0019] [ Figure 4 ] is a diagram illustrating an example of step D of the method for screening secretion-producing cells.

[0020] [ Figure 5 ] is a diagram illustrating a variation of step A of the method for screening secretion-producing cells.

[0021] [ Figure 6] is a diagram illustrating an example of a method for screening secretion-producing cells when the detection particles are cells.

[0022] [ Fig. 7A ] is a diagram illustrating an example of a washing step in a method for screening secretion-producing cells.

[0023] [ Figure 7B ] is a diagram illustrating an example of a washing step in a method for screening secretion-producing cells.

[0024] [ Figure 8 ] is a top view showing one embodiment of a device included in a screening kit.

[0025] [ Fig. 9 ] is a front view of the device of the same embodiment.

[0026] [ Fig.10 ] is a stereoscopic view showing a front cross section of a device of the same embodiment.

[0027] [ Fig.11 ] is a front cross-sectional view of the central portion of the device of the same embodiment.

[0028] [ Fig.12 ] is a side sectional view of the device of the same embodiment.

[0029] [ Fig.13 ] is a stereoscopic diagram showing the assembly method of the same embodiment of the device of the same embodiment.

[0030] [ Fig.14 ] are fluorescence microscopic photographs of wells in an example of a screening method according to one embodiment. (A) is a fluorescence microscopic photograph of a well without a washing step. (B) is a fluorescence microscopic photograph of a well after a washing step. DETAILED DESCRIPTION

[0031] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings as appropriate. In the accompanying drawings, identical or corresponding symbols are used to mark identical or corresponding parts, and repeated descriptions are omitted. The dimensional ratios in the drawings are exaggerated for the sake of convenience of description and are not necessarily consistent with the actual dimensional ratios.

[0032] (Screening method for secretion-producing cells)

[0033] The first mode of the present invention is a method for screening secretion-producing cells for screening cells producing target secretions from a plurality of cells. The screening method comprises: step A, capturing the cells and detection particles at the bottom of a plurality of holes having through holes of a size that the cells cannot pass through, the detection particles being able to capture the target secretions and having a size that cannot pass through the through holes; step B, causing the cells captured in the plurality of holes to produce secretions; step C, detecting the target secretions captured by the detection particles; and step D, using the detection results as an indicator, determining the hole that captures the cell producing the target secretion from the plurality of holes. The hole has a size that can capture the cell in a cell unit when one or more detection particles are captured.

[0034] In the screening method of this embodiment, first, a plurality of holes 50 having through holes 52 of a size that the screened cells C cannot pass through are used to capture the cells C and the detection particles B that can capture the target secretion (step A; Figure 1 ). The detection particle B has a size that cannot pass through the through hole 52. In addition, the hole 50 has a size that can capture the cell C in a single cell unit in a state where one or more detection particles B are captured.

[0035] Next, the cells C captured in the wells 50 are incubated to allow the cells C to produce secretions A (step B; Figure 2 ).

[0036] Next, the target secretion A captured by the detection particle B is detected using a detection reagent D or the like (step C; Figure 3 ).

[0037] Finally, using the detection result obtained in step C as an indicator, the well 50 (well 50-1) in which the cell producing the target secretion A (secretion A-1) is captured is determined from the plurality of wells 50 (wells 50-1, 50-2, 50-3) (step D; Figure 4 ).

[0038] <Cell Screening>

[0039] The plurality of cells (hereinafter also referred to as "screening cells") provided for the screening method of the present embodiment are a cell group predicted to contain the secretion-producing cells as the target. The plurality of cells are preferably a group of cells that produce secretions. Examples of cells include animal cells such as mammalian (human, mouse, rat, rabbit, horse, camel, monkey, etc.) cells, bird (chicken, etc.) cells, insect (ostracod, etc.) cells, plant cells, fungi such as yeast, bacteria such as Escherichia coli, etc. The screening cells may be genetically recombinant cells into which genes encoding secretory proteins or secretory peptides are introduced. The secretory proteins and secretory peptides may be chimeric proteins obtained by linking secretory signal peptides to non-secretory proteins or non-secretory peptides.

[0040] The target secretion (hereinafter also referred to as "target secretion") may be of natural origin or may be a non-natural secretion produced by genetic engineering. The target secretion is not limited, but is preferably a single secretion. Examples of target secretions include immunoglobulins (immunoglobulin G (IgG), immunoglobulin M (IgM), etc.); interleukins (IL-2, IL-7, IL-12, IL-15, etc.), chemokines, interferons (IFN-γ, etc.), hematopoietic factors (colony stimulating factor, granulocyte colony stimulating factor, erythropoietin, etc.), cell growth factors (epithelial growth factor, fibroblast growth factor, platelet-derived growth factor, hepatocyte growth factor, transforming growth factor, etc.), cytotoxic factors (tumor necrosis factor, lymphotoxin), adipokines (leptin and tumor necrosis factor secreted by adipose tissue, etc.), neurotrophic factors (nerve growth factor, etc.), antibiotics; metabolites of microorganisms such as pigments; hormones (peptide hormones, steroid hormones, microbial hormones, etc.); chimeric proteins formed by secretory signal peptides and non-secretory proteins, etc., but are not limited to these. Target secretions are not limited to natural proteins or natural peptides, and may also be proteins or peptides obtained by genetically modifying them.

[0041] In one embodiment, the secretion is preferably an antibody. As natural antibodies, immunoglobulin G (IgG), immunoglobulin M (IgM), etc. can be cited. As non-natural antibodies, antibody fragments such as Fab, scFv, Diabody, etc. can be cited; single domain antibodies (Single Domain Antibodies, Methods in Molecular Biology Volume, 911, 2012); artificial protein molecules with antibody-like properties (Skrlec K, Strukelj B, Berlec A. Non-immunoglobulin scaffolds: a focus on their targets., Trends Biotechnol., 2015, Apr 27), etc.

[0042] Examples of secretion-producing cells that produce a target secretion include antibody-producing cells, cytokine-producing cells, hormone-secreting cells, and the like.

[0043] Antibody molecule production cells are not limited, and examples thereof include B cells, hybridomas obtained by fusing B cells with myeloma cells, and gene recombinant cells obtained by introducing polynucleotides encoding antibody molecules into cells. Cells into which antibody genes are introduced include, for example, animal cells, fungi such as yeast, bacteria such as Escherichia coli, etc. Cells into which antibody genes are introduced include, for example, NSO cells, CHO cells, COS cells, 293FT cells, etc.

[0044] Examples of cytokine-producing cells include, but are not limited to, macrophages, B cells, T cells, NK cells, NKT cells, dendritic cells, Kupffer cells, stromal cells, fibroblasts, and vascular endothelial cells.

[0045] Examples of hormone-secreting cells include, but are not limited to, anterior pituitary cells, somatotropin-producing cells, prolactin-producing cells, thyroid-stimulating hormone-producing cells, gonadotropin-producing cells, adrenocorticotropic hormone-producing cells, middle pituitary cells, cells that secrete melanocyte-stimulating hormone, oxytocin-secreting cells, vasopressin-secreting cells, serotonin-secreting cells, endorphin-secreting cells, somatostatin-secreting cells, gastrin-secreting cells, secretin-secreting cells, cholecystokinin-secreting cells, insulin-secreting cells, glucagon-secreting cells, bombesin-secreting cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, aerobic cells, adrenal cells, chromaffin cells, steroid hormone (mineralocorticoid or glucocorticoid)-producing cells, testosterone-secreting cells, estrogen-secreting cells, progesterone-secreting cells, cells of the juxtaglomerular apparatus, macula densa cells of the kidney, pericytes of the kidney, mesangial cells of the kidney, and the like.

[0046] Among them, the target secretion is preferably an antibody, and the secretion-producing cell is preferably an antibody-producing cell.

[0047] <Step A>

[0048] In step A, the cells and detection particles capable of capturing target secretions and having a size that is too large to pass through the through holes are captured in a plurality of wells having through holes at the bottom thereof.

[0049] Figure 1 : is a figure for explaining an example of step A. The device 100 has a plurality of holes 50. The hole 50 has a through hole 52 at the bottom 50a. The device 100 can be composed of, for example, a substrate or a film having a first surface and a second surface. The plurality of holes 50 can be, for example, a recess having an opening on the first surface of the substrate or the film. The through hole 52 can be a through hole that passes from the first surface of the substrate or the film to the second surface. Preferably, the material of the device 100 is not a material that is harmful to cells. As the material of the device 100, there can be cited: for example, glass; common resins such as polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), cycloolefin polymer (COP), epoxy resin, etc.

[0050] The planar shape of the opening of the hole 50 is not particularly limited as long as it is a shape that can capture and screen the cells C in 1 cell unit. As the planar shape of the hole 50, for example, a circle, an ellipse, a polygon (a quadrilateral such as a square, a hexagon, an octagon, etc.) and the like can be cited. From the perspective of increasing the configuration density, a regular hexagon is preferably used. The bottom 50a of the hole 50 can be a flat bottom or a round bottom. From the perspective of being easy to form a through hole 52 and being able to accommodate a large amount of the detection particles B, a flat bottom is preferably used.

[0051] The hole 50 has a size that can capture the screening cell C in 1 cell unit in a state where one or more detection particles B described later are captured. 1 cell unit means, for example, a single cell. The size of the hole 50 can be appropriately selected according to the size of the screening cell C. The size of the hole 50 is not limited, and can usually be set to a maximum diameter of about 1 to 100 μm and a depth of about 1 to 100 μm for the circle entering the hole opening. The maximum diameter of the circle entering the hole opening is more preferably 2 to 50 μm, and more preferably 3 to 25 μm. The depth of the hole 50 is more preferably 2 to 70 μm, more preferably 3 to 50 μm, and particularly preferably 4 to 30 μm.

[0052] In relation to the size of the selected cells C, the maximum diameter of a circle that enters the hole 50 when viewed from above can be set to about 0.5 to 2 times, preferably 0.8 to 1.9 times, the maximum diameter of the selected cells C. The depth of the hole 50 can be set to about 0.5 to 4 times, more preferably 0.8 to 1.9 times, the maximum diameter of the selected cells C.

[0053] The through hole 52 is provided at the bottom 50a of the hole 50, and connects the internal space of the hole 50 with the external space. The through hole 52 is a through hole that penetrates the member forming the bottom 50a. The position, shape, size, etc. of the through hole 52 are not particularly limited as long as the screening cells C cannot pass through and the liquid containing the secretion secreted by the screening cells C can pass through. As the planar shape of the through hole 52, for example, a circle, an ellipse, a polygon (a quadrilateral such as a square, a hexagon, an octagon, etc.) and the like can be cited. From the perspective of easy formation, a circle is preferred.

[0054] The size of the through hole 52 is not limited, but the minimum inner diameter of the through hole 52 can generally be set to 10 nm to 20 μm. The minimum inner diameter of the through hole 52 is preferably 50 nm to 15 μm, and more preferably 100 nm to 10 μm.

[0055] In relation to the size of the selection cells C, the minimum inner diameter of the through hole 52 is preferably 0.5 times or less of the maximum diameter of the selection cells C, more preferably 0.1 times or less, and even more preferably 0.05 times or less.

[0056] The detection particle B is a particle that can capture the target secretion. The detection particle B has a size that can be captured in the hole 50 and cannot pass through the through hole 52. The size of the detection particle B can be appropriately selected according to the size of the hole 50 and the through hole 52. The size of the detection particle B can be set to a particle diameter of 50nm to 80μm, for example. The particle diameter of the detection particle B is preferably 100nm to 50μm, more preferably 500nm to 30μm, and further preferably 1 to 20μm.

[0057] In terms of the relationship with the size of the through hole 52 , the particle diameter of the detection particle B is preferably 1.5 times or more, more preferably 2 times or more, and even more preferably 3 times or more, the minimum inner diameter of the through hole 52 .

[0058] The number of through holes 52 is not particularly limited, and can be appropriately set according to the size of the hole 50 and the through hole 52. The through hole 52 only needs to be one or more, and can be set in the range of 1 to 100, for example. From the perspective of the outflow efficiency of the liquid in the hole 50, the number of through holes 52 is preferably two or more. Specific examples of the number of through holes 52 include, for example, 2 to 10, 2 to 5, 2 or 3.

[0059] The position of the through hole 52 is not particularly limited as long as it is the bottom 50a of the hole 50. From the perspective of facilitating the storage of the screening cells C in the hole 50 and the outflow efficiency of the liquid in the hole 50, it is preferred to arrange one or more through holes 52 near the center of the bottom 50a. When there are multiple through holes 52, the arrangement of the multiple through holes 52 is not particularly limited. For example, the multiple through holes 52 may be arranged together near the center of the bottom 50a, or the multiple through holes 52 may be arranged randomly or in a grid pattern on the bottom 50a.

[0060] There are no particular limitations on the detection particles B as long as they can capture the target secretion. Examples of the detection particles B include carrier particles on which a substance that binds to the target secretion is immobilized, and cells containing a cell membrane protein that binds to the target secretion. Figure 1 In the example, the detection particle B is composed of a carrier particle B1 on which a substance (hereinafter also referred to as a "binding substance") B2 that has binding properties to the target secretion is immobilized.

[0061] When the detection particle B is composed of the carrier particle B1 and the binding substance B2, the material of the carrier particle B1 is not particularly limited, and materials commonly used for detecting antibodies, etc. can be used. Examples of the carrier particle B1 include beads (magnetic beads, resin beads, etc.), hydrogel particles (sodium alginate gel, agarose gel, etc.), metal particles (gold nanoparticles), etc., but are not limited to these.

[0062] The binding substance B2 is a substance that specifically binds to the target secretion. When the target secretion is an antibody, an antigen (antigenic peptide, antigenic protein, etc.) of the antibody can be used as the target secretion. When the target secretion is a cytokine or a hormone, for example, an antibody that binds to the cytokine or hormone can be used as the binding substance B2.

[0063] The binding substance B2 can be immobilized on the carrier particles B1 by a known method, and examples of the immobilization method include, but are not limited to, a method using a binding pair such as biotin-avidin binding and a passive adsorption method.

[0064] In step A, multiple holes 50 are made to capture screening cells C and detection particles B. At this time, preferably one screening cell C is accommodated in one hole 50. The operation of making the hole 50 capture the screening cell C (hereinafter also referred to as "cell capture operation") and the operation of making it capture the detection particles B (hereinafter also referred to as "detection particle capture operation") can be performed separately or simultaneously. When each capture operation is performed separately, the order of the cell capture operation and the detection particle capture operation is not particularly limited. From the perspective of making it easy to evenly arrange the detection particles B in the hole 50, it is preferred to perform the detection particle capture operation first.

[0065] When the cell capture operation and the detection particle capture operation are performed separately, the detection particle capture operation can be performed by suspending the detection particles B in a suitable liquid such as a buffer solution (PBS, physiological saline, etc.) or a culture medium, and supplying the above suspension to a plurality of holes 50. It is not necessary to perform the operation of supplying the suspension to each hole 50, and the suspension can be supplied to the opening surface of the plurality of holes 50. The detection particles B in the suspension supplied to the hole 50 are received in each hole 50 due to their own weight. The detection particles B can be received in the hole 50 while the liquid in the hole 50 is sucked from a suction hole connected to the through hole 52. In this case, the detection particles B can be received in the hole 50 more quickly. The storage amount of the detection particles B in each hole 50 can be adjusted by the density of the detection particles B in the above suspension. The density of the detection particles B in the suspension is not particularly limited as long as it does not impair the fluidity of the suspension, and can be appropriately selected according to the size of the detection particles B. Usually, the density of the detection particles B in the suspension can be set to, for example, 10 5 ~10 12 About pcs / mL.

[0066] When a medium is used as the liquid for suspending the suspended particles, the medium can be appropriately selected according to the type of the screening cells C. When the screening cells C are mammalian cells, examples of the medium include MEM medium, MEMα medium, and RPMI medium.

[0067] In this specification, "appropriate liquid" means a liquid in which the screening cell C can survive. When the screening cell C is a secretion-producing cell, the appropriate liquid is preferably a liquid that can produce secretions. Examples of appropriate liquids include the buffer solution and culture medium described above.

[0068] The cell capture operation can be performed by suspending the screening cells C in a suitable liquid and supplying the above-mentioned cell suspension to multiple holes 50. It is not necessary to perform the operation of adding the screening cells C to each hole 50. It is sufficient to supply the cell suspension to the surface of the opening portion provided with multiple holes 50 in the component (culture plate, membrane, etc.) having multiple holes 50. The screening cells C in the cell suspension supplied to the hole 50 are received in each hole 50 as a unit of 1 cell due to their own weight. The screening cells C can be received in the hole 50 while sucking the liquid in the hole 50 from the suction hole connected to the through hole 52. In this case, the screening cells C can be received in the hole 50 more quickly. As long as it is to a degree that does not damage the fluidity of the cell suspension, the density of the screening cells C in the cell suspension is not particularly limited and can be appropriately selected according to the size of the screening cells C. Usually, the density of the screening cells C in the cell suspension can be set to 10, for example. 5 ~10 9 About pcs / mL.

[0069] When a culture medium is used as the liquid for suspending the cells, examples of the culture medium include the same ones as described above.

[0070] When the cell capturing operation is performed after the detection particle capturing operation, the liquid in the well 50 may be discharged from the through hole 52 after the detection particle capturing operation, and then the cell suspension containing the screening cells C may be supplied to the well 50. Thus, since the excess medium in the well 50 is removed, the efficiency of capturing the screening cells C in the well 50 can be improved. The liquid in the well 50 may be discharged while being sucked from a suction hole or the like connected to the through hole 52. In this case, the liquid can be efficiently discharged from the through hole 52.

[0071] When the cell capture operation and the detection particle capture operation are performed simultaneously, it is sufficient to suspend the detection particles B and the screening cells C in a suitable liquid and supply the above suspension to a plurality of holes 50. The detection particles B and the screening cells C in the suspension supplied to the holes 50 are contained in each hole 50 due to their own weight. The detection particles B and the screening cells C can be contained in the holes 50 while the liquid in the holes 50 is sucked from the suction hole connected to the through hole 52. In this case, the detection particles B and the screening cells C can be contained in the holes 50 more quickly. The amount of detection particles B contained in each hole 50 can be adjusted by the density of the detection particles B in the above suspension. The density of the detection particles B in the suspension can be set to the same as above. The screening cells C are contained in each hole 50 in units of 1 cell. The density of the screening cells C in the cell suspension can be set to the same as above.

[0072] <Step B>

[0073] In step B, the selected cells captured in the plurality of wells are allowed to produce secretions.

[0074] Figure 2 : is a diagram for explaining an example of step B. When the screening cells C captured in the well 50 are secretion-producing cells, the screening cells C can be made to secrete secretion A by incubating the screening cells C in the well 50. The incubation conditions can be appropriately set according to the type of screening cells C. Generally, 25 to 38°C and 0.03 to 5% CO can be used. 2 Specific examples of incubation conditions include 37°C, 5% CO 2 .

[0075] The incubation is performed in a state where the selection cells C captured in the wells 50 are present in a liquid such as a buffer or a culture medium. It is preferred that the inside of the wells 50 is filled with a liquid such as a buffer or a culture medium.

[0076] When the secretion A secreted by the screening cell C is the target secretion, the secretion A binds to the binding substance B2 and is captured by the detection particle B. In addition, a portion of the secretion A moves from the through hole 52 to the outside of the well 50 together with the liquid (buffer, culture medium, etc.) in the well 50 .

[0077] The incubation time can be set to a time sufficient for the secretion A to be captured by the detection particles B. The incubation time may be appropriately set depending on the type of the cells C to be screened, and may be set to, for example, about 0.5 to 24 hours.

[0078] <Step C>

[0079] In step C, the target secretion captured by the detection particles is detected.

[0080] Figure 3 : is a diagram for explaining an example of step C. The detection of the target secretion can be performed, for example, using a detection reagent D. The detection reagent D is composed of, for example, a substance (binding substance) D1 that specifically binds to the target secretion and a labeling substance D2.

[0081] As the binding substance D1, for example, an antibody that specifically binds to the target secretion can be used. The antibody used as the binding substance D1 can be a natural antibody or a non-natural antibody (for example, an antibody fragment such as Fab or scFv). The site of the target secretion to which the binding substance D1 binds is preferably different from the site to which the binding substance B2 binds. For example, when the target secretion is an antibody, the binding substance D1 can be an antibody that specifically binds to the constant region of the antibody that is the target secretion.

[0082] The labeling substance D2 is a substance that generates a signal that can be detected by any detection device or the like. The labeling substance D2 is not particularly limited, and a labeling substance commonly used in the biochemical field can be used. As the labeling substance D2, for example, enzyme labels, fluorescent labels, radioactive substances, etc. can be cited. As enzyme labels, HRP (Horse Radish Peroxidase), AP (Alkaline Phosphatase), etc. can be cited. As fluorescent labels, (FAM (carboxyfluorescein), JOE (6-carboxy-4', 5'-dichloro-2', 7'-dimethoxyfluorescein), FITC (fluorescein isothiocyanate), TET (tetrachlorofluorescein), HEX (5'-hexachloro-fluorescein-CE phosphoramidite) and other fluoresceins can be cited; Cy pigment-labeled carboxylic acids such as Cy3 and Cy5; Alexa Fluor such as Alexa568 and Alexa488, etc. For the labeling substance D2, fluorescent labels are preferred from the point of view that the signal is easy to detect.

[0083] The detection reagent D can be dissolved in a suitable liquid (buffer, culture medium, etc.) and supplied to the hole 50 so as to contact the target secretion A in the hole 50. Before supplying the reagent solution as the detection liquid to the hole 50, the liquid in the hole 50 can be discharged from the through hole 52. For example, the liquid can be discharged by removing the liquid from the flow path arranged below the through hole 52. The liquid can also be removed from the flow path by suction from a suction hole connected to the flow path. By removing the liquid in the hole 50 before supplying the detection reagent D, the detection reagent D can be efficiently supplied to the hole 50.

[0084] The detection reagent D supplied to the hole 50 contacts the secretion A in the hole 50. When the secretion A is the target secretion, the detection reagent D binds to the secretion A via the binding substance D1. When the secretion A is the target secretion, most of the secretion A in the hole 50 is captured by the detection particles B. In this case, the detection reagent D is captured by the detection particles B via the secretion A according to the amount of the secretion A captured by the detection particles B. Therefore, by detecting the signal of the labeling substance D2, the secretion A captured by the detection particles B can be detected.

[0085] The signal of the labeling substance D2 can be detected by a method corresponding to the type of the labeling substance D2. For example, when the labeling substance D2 is an enzyme label, a color-developing substrate of the enzyme can be used to develop color, and the color development signal can be detected using an optical microscope. When the labeling substance D2 is a fluorescent label, a fluorescent signal can be detected using a fluorescent microscope. When the labeling substance D2 is a radioactive substance, an X-ray microscope can be used to detect the radioactive signal.

[0086] <Step D>

[0087] In step D, the wells in which the cells producing the target secretion are captured are identified from the plurality of wells using the detection results in step C as an indicator.

[0088] Figure 4 This is a diagram for explaining an example of step D. At the time point when step C is completed, screening cells C, detection particles B, secretions A secreted by screening cells C, and detection reagent D are present in each of the plurality of wells 50. However, the amount of detection reagent D present in the well 50 varies depending on the type of secretion A.

[0089] The well 50-1 contains the screening cell C-1 that secretes the secretion A-1. The secretion A-1 is the target secretion and is captured by the detection particle B. Therefore, the detection reagent D is captured by the detection particle B via the secretion A-1 that is the target secretion. Thus, the detection reagent D accumulates in the well 50-1 according to the amount of the secretion A-1 captured by the detection particle B.

[0090] The well 50-2 contains the screening cell C-2 that secretes the secretion A-2. The secretion A-2 is not the target secretion and is not captured by the detection particle B. Therefore, the detection reagent D is not captured by the detection particle B, but flows out of the through hole 52 together with the liquid in the well 50-2.

[0091] The well 50-3 contains the screening cells C-3 secreting the secretion A-3. The secretion A-3 is not the target secretion and is not captured by the detection particles B. Therefore, the detection reagent D is not captured by the detection particles B, but flows out of the through hole 52 together with the liquid in the well 50-3.

[0092] As described above, in well 50-1, since the detection reagent D is captured by the detection particles B, the amount of detection reagent D in the well is greater than that in wells 50-2 and 50-3. Therefore, when the signal of the marker substance D2 is detected, the signal intensity detected in well 50-1 is stronger than that in wells 50-2 and 50-3. In other words, it can also be said that the wells with a higher signal intensity of the marker substance D2 than in other wells contain the screening cells C that produce the target secretion. Therefore, the wells with a higher signal intensity of the marker substance D2 than in other wells can be determined as the wells that have captured the secretion-producing cells that produce the target secretion.

[0093] For example, Fig.14 (A) shows a fluorescence microscopic photograph of a well subjected to steps A to C in the example. Fig.14 In (A), Alexa488 was used as the labeling substance D2, and it was confirmed that the fluorescence signal of the well indicated by the arrow was stronger than that of the other wells ( Fig.14 (A) Alexa). Therefore, the pores indicated by arrows can be identified as pores in which secretion-producing cells producing the target secretion are captured.

[0094] <Variation 1>

[0095] The detection reagent D and the screening cells C and / or the detection particles B may be supplied to the well 50 simultaneously. Figure 5 This is a diagram for explaining step A in the case where the detection reagent D, the screening cells C, and the detection particles B are supplied to the well 50 simultaneously.

[0096] When the detection reagent D is supplied to the well 50 simultaneously with the screening cells C and the detection particles B, the detection reagent D may be added to the suspension obtained by suspending the screening cells C and the detection particles B. Alternatively, the screening cells C and the detection particles B may be suspended in a liquid (buffer, culture medium, etc.) to which the detection reagent D is added. Then, the suspension containing the detection reagent D, the screening cells C, and the detection particles B may be supplied to the well 50.

[0097] The detection reagent D may be supplied to the hole 50 simultaneously with either the detection particle capture operation or the cell capture operation. For example, when the detection reagent D is supplied to the hole 50 together with the detection particles B, the detection reagent D may be added to a suitable liquid (buffer, culture medium, etc.) in which the detection particles B are suspended. Alternatively, the detection particles B may be suspended in a suitable liquid (buffer, culture medium, etc.) to which the detection reagent D is added. Then, the suspension containing the detection reagent D and the detection particles B may be supplied to the hole 50.

[0098] When the detection reagent D is supplied to the well 50 together with the screening cells C, the detection reagent D may be added to a suitable liquid (buffer, culture medium, etc.) in which the screening cells C are suspended. Alternatively, the screening cells C may be suspended in a suitable liquid (buffer, culture medium, etc.) to which the detection reagent D is added. Then, the suspension containing the detection reagent D and the screening cells C may be supplied to the well 50.

[0099] When the operation of discharging the liquid in the well 50 from the through-hole 52 is performed between the detection particle capturing operation and the cell capturing operation, it is preferable that the detection reagent D is added to the suspension in the capturing operation performed after the discharging operation.

[0100] In step A, after the detection reagent D is supplied to the hole 50 simultaneously with the cell capture operation and / or the detection particle capture operation, step B is performed in the same manner as described above. When the secretion A secreted by the screening cell C is the target secretion, the secretion A is captured by the detection particle B via the binding substance B2. In addition, the detection reagent D is bound to the secretion A via the binding substance D1. Therefore, when the secretion A is the target secretion, the detection reagent D is captured by the detection particle B via the secretion A.

[0101] Since the detection reagent D already exists in the well 50 and reacts with the secretion A, it is not necessary to supply the detection reagent D to the well 50 in step C. Therefore, in step C, it is sufficient to detect the signal of the labeling substance D2. The signal of the labeling substance D2 can be detected in the same manner as described above.

[0102] Step D can be performed in the same manner as described above based on the detection results in step C.

[0103] <Variation 2>

[0104] The detection particle B may be a cell containing a cell membrane protein that has binding properties to the target secretion. Figure 6 The state of step C is shown when the detection particle B is a cell containing a cell membrane protein B2' that has binding properties to a target secretion (hereinafter also referred to as a "target cell").

[0105] "Membrane protein" means a protein that is locally present in the cell membrane. The membrane protein may be an intrinsic membrane protein or a peripheral membrane protein, preferably an intrinsic membrane protein. The intrinsic membrane protein may be a multi-transmembrane type that passes through the cell membrane multiple times or a single-transmembrane type that passes through the cell membrane once. Examples of membrane proteins include GPCRs (G protein-coupled receptors), ligand-gated ion channels, voltage-gated ion channels, transporters, and the like.

[0106] The target cell can be a natural cell or a genetically recombinant cell into which a membrane protein gene has been introduced. The target secretion binds to the region where the membrane protein B2' is exposed on the cell membrane surface. When the target secretion is an antibody against a specific membrane protein of a diseased cell (e.g., a cancer cell), the diseased cell can be used as a target cell.

[0107] The target cell has a size that allows it to be captured in the hole 50 and cannot pass through the through hole 52. The size of the target cell is, for example, about 1 μm to 80 μm. When the target cell is used as the detection particle B, the size of the hole 50 can be set according to the combined size of the screening cell C and the target cell.

[0108] When the target cells are larger than the screening cells C, the maximum diameter of the circle entering the opening of the well 50 is set to, for example, a size larger than the maximum diameter of the target cells and smaller than twice the maximum diameter of the screening cells C. In addition, the depth of the well 50 can be set to a size of about 0.5 to 2 times the total value of the maximum diameter of the target cells and the maximum diameter of the screening cells C, preferably 0.8 to 1.9 times, and more preferably 0.9 to 1.5 times.

[0109] When the target cells are smaller than the screening cells C, the size of the pore 50 can be set in the same manner as in the description of the detection particles B described above.

[0110] In step A, the operation of capturing target cells in the well 50 (hereinafter also referred to as "target cell capturing operation") can be performed simultaneously with the cell capturing operation or separately, preferably separately. When the target cells are larger than the screening cells C, the target cell capturing operation is preferably performed before the cell capturing operation.

[0111] The target cell capture operation can be performed by suspending the target cells in a suitable liquid (buffer, culture medium, etc.) and supplying the target cell suspension to a plurality of wells 50. The target cells C in the target cell suspension supplied to the wells 50 are contained in each well 50 due to their own weight. The density of the target cells in the target cell suspension is not particularly limited as long as the fluidity of the target cell suspension is not impaired, and can be appropriately selected according to the size of the target cells. Generally, the density of the target cells in the target cell suspension can be set to, for example, 10 5 ~10 9 About pcs / mL.

[0112] Steps B to D can be performed in the same manner as described above. When the secretion A secreted by the screening cell C is the target secretion, the secretion A binds to the cell membrane protein B2' of the target cell as the detection particle B. In addition, the detection reagent D binds to the secretion A. Therefore, the detection reagent D is captured by the cell membrane protein B2' of the target cell via the secretion A. Therefore, based on the detection result of the signal of the marker substance D2, the wells with high signal intensity can be determined as the wells that have captured the secretion-producing cells that produce the target secretion.

[0113] According to the screening method of the present embodiment described above, the screening cells are stored in the wells in units of 1 cell and the secretion measurement is performed, so the cells do not move during the measurement operation. In addition, since the target secretion is detected in each well using the detection particles, the distance and time until the target secretion secreted by the screening cells is captured by the detection particles can be shortened. Therefore, compared with the case where the target secretion is detected outside the well, the diffusion of the target secretion can be suppressed, and the detection particles can reliably capture the target secretion. Therefore, the cells producing the target secretion can be correctly identified and obtained.

[0114] In addition, since the well 50 has the through hole 52, the detection reagent not captured by the detection particle flows out from the through hole 52 together with the liquid in the well 50. Therefore, the secretion-producing cells producing the target secretion can be identified based on the difference in signal intensity of the marker substance D2.

[0115] <Other steps>

[0116] The screening method of this embodiment may include other steps in addition to the above steps A to D. Examples of other steps include a washing step (step E) and a secretion-producing cell recovery step (step F), but are not limited thereto.

[0117] 《Washing step (step E)》

[0118] In the washing step, after the liquid in the well 50 is drained from the through hole 52 , an appropriate liquid (buffer, culture medium, etc.) is supplied to the well 50 to replace the liquid in the well 50 .

[0119] Fig. 7A and 7B This is a diagram for explaining an example of a washing step. Fig. 7A An example in which a washing step is performed after step C is shown. Figure 7B The state after the washing step is shown.

[0120] After step C, when the secretion A secreted by the screening cell C is the target secretion, the secretion A is mostly captured by the detection particles B, and the detection reagent D is captured by the detection particles B via the secretion A. However, it is considered that a portion of the detection reagent D is present in the hole 50 in a free state (a portion is in a state bound to the secretion A). In addition, when the secretion A is not the target secretion, all the detection reagent D in the hole 50 is present in the hole 50 in a free state. In this state, when the liquid in the hole 50 is discharged from the through hole 52, the free detection reagent D is also discharged to the outside of the hole 50 together with the liquid. In this way, the detection reagent D in the hole 50 that is present in a free state can be removed.

[0121] Then, an appropriate liquid (buffer, culture medium, etc.) is supplied to the wells 50 to prevent the selected cells C from drying out.

[0122] By performing a washing step after step C, such as Figure 7B As shown, the free detection reagent D (the detection reagent D not captured by the detection particle B) is almost absent in the well 50. For the well 50 in which the secretion A secreted by the screening cell C is not the target secretion (for example, Figure 4 For wells 50-2 and 50-3), there is almost no detection reagent D in well 50. Therefore, the difference in signal intensity between the well where secretion A is the target secretion and the other wells of the marker D2 is more obvious.

[0123] For example, Fig.14 (B) shows a fluorescence microscopic photograph of a well subjected to a washing step after steps A to C in the example. Fig.14 In (B), it can be confirmed that the fluorescence signal in the wells other than the wells indicated by the arrows almost disappears. Fig.14 (A) More clear identification of pores that capture secretion-producing cells that produce the secretion of interest.

[0124] In the washing step, the liquid in the well 50 may be discharged multiple times, and the liquid may be supplied to the well 50 multiple times. By discharging and supplying the liquid multiple times, the free detection reagent D remaining in the well 50 can be almost completely removed.

[0125] The washing step may be performed after step B. By performing the washing step after step B, the free secretion A that is not captured by the detection particles B can be removed from the well 50. Thus, when the detection reagent D is supplied to the well 50, the detection reagent D that reacts with the free secretion A decreases, and thus the amount of detection reagent D used can be reduced.

[0126] The discharge of the liquid from the through hole 52 can be performed, for example, by removing the liquid from a flow path disposed below the through hole 52. For example, if the flow path is filled with liquid, the hole 50 is also kept filled with liquid. Here, when the liquid is removed from the flow path, the liquid in the hole 50 flows out of the through hole 52 due to gravity, and most of the liquid in the hole 50 can be removed. The removal of the liquid from the flow path can be performed by sucking the liquid from a suction hole connected to the flow path.

[0127] 《Secretion-producing cell recovery step (step F)》

[0128] In the secretion-producing cell recovery step, the screening cells C are recovered as secretion-producing cells producing the target secretion from the wells determined in step D. The screening cells C can be recovered using a known single cell recovery means. Examples of single cell recovery means include methods for recovering cells using a robot, a microcapillary, or a micropipette.

[0129] (Screening kit for secretion-producing cells)

[0130] The second aspect of the present invention is a screening kit for secretion-producing cells for screening cells that produce target secretions from a plurality of cells. The screening kit comprises a device comprising a plurality of holes and carrier particles. The holes have a size that allows the cells to be captured in a unit of one cell when more than one detection particle is captured, and have through holes at the bottom that are so large that the cells cannot pass through. The carrier particles have a size that prevents them from passing through the through holes.

[0131] The screening kit of this embodiment can be used in the screening method of the above-mentioned first embodiment.

[0132] <Device>

[0133] The device includes a plurality of holes having through holes at the bottom. Examples of the plurality of holes and through holes include the same holes and through holes as those mentioned in the above-mentioned (method for screening secretion-producing cells). Examples of the device including a plurality of holes include, for example, Figure 1 A device having a structure similar to device 100.

[0134] Preferably, the device is connected to the through hole and has a flow path arranged below the through hole. The flow path arranged below the through hole is preferably a flow path arranged along the outer bottom surface of the plurality of holes. By having a flow path below the through hole, the liquid in the hole can be discharged via the flow path and the through hole. In addition, by providing a suction hole connected to the flow path, the liquid in the hole can be discharged more smoothly by suction from the suction hole.

[0135] 《Specific example of device》

[0136] Figure 8 to Figure 13 An example of a device having the above-described structure is shown. The device 1 has: a bottom plate portion 2; a cell-carrying membrane (cell-carrying portion) 6, which is provided on the bottom plate portion 2 and constitutes a cell-carrying surface 4; a pair of liquid inlet and outlet portions 8, which are provided on the bottom plate portion 2 in a manner partitioned from the cell-carrying membrane 6; and a flow path 10, which is provided between the bottom plate portion 2 and the cell-carrying membrane 6, and the flow path end 12 extends to the liquid inlet and outlet portions 8. A plurality of holes 50 and through holes 52 leading from the inner bottom surface of the hole 50 to the flow path 10 are formed on the cell-carrying surface 4 of the cell-carrying membrane 6. In addition, a bubble discharge surface 17 that rises in an inclined shape as it approaches the suction hole 16A is formed on the back side of each cover portion 14, that is, on the top surface of the flow path end 12.

[0137] The bottom plate 2 is formed into a thin and long rectangular plate with constant wall thickness, and four corners are chamfered into an arc. The whole week of the bottom plate 2 is formed with a projection 2B of a certain height along the outer peripheral edge of the bottom surface. The shape of the bottom plate 2 is not limited to the shape shown in the figure, and can be any shape such as a disc, an ellipse, a square, as long as a horizontal flow path 10 can be formed, and the wall thickness of the bottom plate 2 may not be constant. When the step C and step D of the screening method of the first mode are carried out under a microscope, it is preferred that the bottom plate 2 is formed into a shape (for example, a shape similar to a slide) that can be placed on the stage of a microscope. Usually, from the aspect of molding accuracy and cost, it is expected that the bottom plate 2 is formed with various plastics that are harmless to cells, and as required, it can also be formed with any material such as ceramics, glass, metals. It is also possible to implement any coating that can ease the influence on cells on the surface of the bottom plate 2.

[0138] like Fig.10 As shown, a snap-fitting wall 18 is integrally formed on the upper surface of the bottom plate 2 in a state of vertically standing from the bottom plate 2, and the snap-fitting wall 18 is formed into a rectangular shape with semicircular ends when viewed from above in a manner that surrounds the central portion. The snap-fitting wall 18 is not limited to the shape shown in the figure, and can be a simple rectangular, circular, elliptical, etc. The height of the snap-fitting wall 18 of this example is equal over the entire circumference. An outer frame 20 is detachably mounted on the snap-fitting wall 18 so that the outer frame 20 covers the snap-fitting wall 18 from above over the entire circumference.

[0139] The outer frame 20 has: a rectangular peripheral wall portion 22 with semicircular ends when viewed from above, and a pair of partitions 28 arranged parallel to each other on the inner peripheral side of the peripheral wall portion 22, and the whole is integrated. The material of the outer frame 20 is not limited. Generally, from the aspects of molding accuracy and cost, it is expected to be formed with various plastics that are harmless to cells. It can also be formed with any material such as ceramics, glass, metal, etc. as needed. A rectangular space is opened between the partitions 28 and the partitions 28, in which the cell-carrying membrane 6 is arranged.

[0140] The upper end of the peripheral wall portion 22 of the outer frame 20 is formed into a cross-sectional shape that is folded outward over the entire circumference, and a narrow engagement groove 24 that opens downward is formed at a certain depth over the entire circumference on the inner side of the folded portion. By inserting the upper end of the engagement wall 18 into the engagement groove 24 over the entire circumference and elastically fastening it with the folded portion of the peripheral wall portion 22, the outer frame 20 is fixed to the engagement wall 18 in a detachable manner. Horizontally protruding protrusions 26 are formed at each of the two front ends in the longitudinal direction of the outer frame 20, and by lifting these protrusions 26 with fingertips, the engagement wall 18 is disengaged from the engagement groove 24, so that the bottom plate portion 2 can be separated from the outer frame 20.

[0141] The semicircular areas surrounded by the peripheral wall portion 22 of the outer frame 20 and the partitions 28 are each formed as a liquid inlet and outlet portion 8. In these liquid inlet and outlet portions 8, a cover portion 14 is formed in a manner connecting the lower end of the peripheral wall portion 22 and the lower end of each partition 28, and the cover portion 14 is formed in a three-dimensional shape that is semicircular in plan view and bulges upward in the center.

[0142] The flow path ends 12 as the two ends of the flow path 10 are formed between the cover 14 and the bottom plate 2, and the cover 14 is configured to hermetically block the flow path ends 12. By forming the cover 14 in this way, the flow path ends 12 as the two ends of the flow path 10 are sealed, and even when the device 1 is tilted or shaken, it is possible to prevent the liquid from excessively flowing to the left and right through the flow path 10 and the flow path ends 12.

[0143] In this embodiment, a circular suction hole 16A is formed substantially in the center of each cover 14, and corresponding to these suction holes 16A, a cylindrical suction port 16 is formed upright from the cover 14. The surface of the cover 14 around the suction port 16 serves as a liquid storage portion, and the liquid overflowing from the suction port 16 is accumulated. As the liquid storage portion, a recessed portion may be actively formed around the suction port 16 on the upper surface of the cover 14.

[0144] A bubble discharge surface 17 is formed on the back side of each cover 14, i.e., the top surface of the flow path end 12, which rises in an inclined plane as it approaches the suction hole 16A. The bubble discharge surface 17 is formed in a truncated cone shape with the suction hole 16A as the vertex. Therefore, when the flow path end 12 is filled with liquid such as a dispersion liquid and contains bubbles, the bubbles move smoothly toward the suction hole 16A along the bubble discharge surface 17 under buoyancy and are discharged from the suction hole 16A.

[0145] In this embodiment, since the wall thickness of the cover 14 is substantially constant, the upper surface of the cover 14, i.e., the upper surface of the liquid storage portion, is also inclined in a truncated cone shape. Therefore, the liquid storage portion also becomes a surface that is inclined downward as it moves away from the suction port 16, and the liquid overflowing from the suction port 16 is concentrated in the peripheral portion of the cover 14 away from the suction port 16, thereby reducing the possibility of contamination caused by re-inflow from the suction port 16. However, the present invention is not limited to this configuration, and the upper surface of the cover 14, i.e., the upper surface of the liquid storage portion, may be horizontal by making the wall thickness of the cover 14 thicker as it moves away from the suction port 16.

[0146] In this embodiment, the suction port 16 stands upright from the cover portion 14 . However, instead of forming the suction port 16 standing upright on the cover portion 14 , the suction hole 16A may be directly opened.

[0147] At the lower end of the partition 28 on the liquid inlet and outlet 8 side, a step 34 having a certain height from the lower surface is formed over the entire length of the partition 28, and above the step 34, two ribs 30 are formed in the upper and lower directions to reach the upper end of the partition 28. The ribs 30 can improve the bending strength of the partition 28. In addition, each of the partitions 28 is formed with an engagement groove 32 of a certain depth that opens toward the lower side of the step 34, and an engagement protrusion 42 that is adjacent to the cell-carrying membrane 6 side of the engagement groove 32.

[0148] In the quadrilateral space surrounded by two places where the peripheral wall portion 22 extends linearly and two partitions 28, a square cylindrical frame 36 having a rectangular shape when viewed from above is detachably accommodated. At the lower end of the frame 36, the cell-carrying membrane 6 is spread over the entire surface so that the hole 50 faces upward, and the lower end of the frame 36 is joined to the cell-carrying membrane 6 without a gap around the entire circumference. The frame 36 is formed of a flexible plastic or the like, and when an outward expansion force is applied, the four walls expand slightly outward, applying tension to the cell-carrying membrane 6, thereby preventing the cell-carrying membrane 6 from loosening.

[0149] The thickness of the cell-carrying membrane 6 is not limited, but is preferably about 5 to 100 μm, and more preferably about 10 to 50 μm, from the perspective of forming a hole 50 that can capture and select cells in units of one cell, and forming a fine through hole 52 for the liquid to flow from the bottom of the hole 50 to the back side. The cell-carrying membrane 6 can be a multilayer membrane with more than two layers. In this case, a through hole that becomes the hole 50 can be formed in the upper layer, and a through hole that becomes the through hole 52 can be opened in the lower layer, and these two layers are bonded together to form the hole 50 and the through hole 52.

[0150] The material of the cell-carrying membrane 6 is not limited. Generally, it is desirable to use various plastics that are harmless to cells from the perspective of molding accuracy and cost. It can also be made of any material such as ceramics, polycrystalline or single-crystalline silicon, inorganic compounds such as glass, metals, etc. as needed. The holes 50 and through holes 52 can also be formed by etching, photolithography, etc.

[0151] The planar shape and size of the hole 50 may be the planar shape and size exemplified in the above-mentioned (method for screening secretion-producing cells). A plurality of frames 36 having cell-carrying membranes 6 with holes 50 of different sizes may be prepared in advance and combined with a common bottom plate 2 and outer frame 20 to constitute the device 1. The shape and size of the through hole 52 may be the shape and size exemplified in the above-mentioned (method for screening secretion-producing cells).

[0152] like Fig.11 and Fig.12 As shown, the outer peripheral surface of the lower end of the frame 36 is formed with: a snap-fit ​​groove 44 that opens upward by being formed into an upwardly folded shape; and a snap-fit ​​protrusion 40 that protrudes downward. The depth of the snap-fit ​​groove 44 and the upper and lower widths of the snap-fit ​​protrusion 40 are substantially constant over the entire circumference of the frame 36. The snap-fit ​​protrusion 40 is inserted into the snap-fit ​​groove 32 formed on the lower surface of the outer frame 20, and the snap-fit ​​protrusion 42 of the outer frame 20 is inserted into the snap-fit ​​groove 44 of the frame 36. By their engagement, the frame 36 is fixed to the outer frame 20 in a state where the frame 36 is accommodated in the central space of the outer frame 20.

[0153] 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 spacing between the cell-carrying membrane 6 and the bottom plate 2, that is, the thickness of the flow path 10, can be accurately determined by the thickness of the spacer 46. Fig.13 As shown, a pair of U-shaped spacers 46 are formed along the lower end shape of the frame 36 in a top view on the inner side of the engagement wall 18, and a notch 47 is formed between the spacers 46. When the frame 36 is fixed to the bottom plate 2, the liquid flows from the flow path 10 to each flow path end 12 through these notches 47. The spacer 46 may not be in a shape as shown in the figure, as long as it abuts against the lower surface of the frame 36 at multiple locations. Depending on the situation, the spacer 46 may not be formed, and the height of the frame 36 from the bottom plate 2 may be accurately specified by engaging with the outer frame 20.

[0154] The inner peripheral surface of the lower end of the frame 36 is formed with a slope 38 having a certain width throughout the entire circumference, and the slope 38 protrudes toward the side of the cell-carrying membrane 6 as it goes downward. By forming such a slope 38, when the secretion-producing cells are recovered from the holes 50 of the cell-carrying membrane 6 using instruments such as a manipulator, a micropipette, and a microcapillary, the cells can be easily recovered even if the holes 50 containing the secretion-producing cells are located near the inner peripheral edge of the frame 36. In addition, the slope 38 also has the effect of increasing the area of ​​the cell-carrying membrane 6 attached to the frame 36 and improving the bonding strength of the cell-carrying membrane 6, and also helps to improve the strength of the frame 36.

[0155] When manufacturing a cell screening device formed by the above constitution, Fig.13 As shown, after the bottom plate 2, the frame 36, and the outer frame 20 are formed, first, the frame 36 is embedded in the lower surface of the outer frame 20, and then the outer frame 20 is embedded in the engagement wall 18 of the bottom plate 2, thereby forming Figure 8 to Figure 12 The completion status shown.

[0156] In the process of embedding the frame 36 into the lower surface of the outer frame 20, as shown in FIG. Fig.11 and Fig.12 As shown, by embedding the engaging protrusion 42 of the outer frame 20 into the engaging groove 44 of the frame 36, and embedding the engaging protrusion 40 of the frame 36 into the engaging groove 32 of the outer frame 20, the elasticity of each engaging portion is utilized to firmly fix the two. At the same time, for the inner peripheral surface of the engaging protrusion 40 and the outer peripheral surface of the engaging protrusion 42, at least one side becomes a shape that is slightly inclined to the outside as it moves upward. Thus, when the engagement is carried out, the engaging protrusion 40 of the four sides of the frame 36 is pulled outward by the engaging protrusion 42 of the outer frame 20, and the lower end of the frame 36 is slightly expanded to the surroundings, generating tension for stretching the four sides of the cell-carrying membrane 6, and applying uniform tension to the cell-carrying membrane 6.

[0157] Therefore, it has the following advantages: even if the cell-carrying membrane 6 is slightly loose when the frame 36 is in a free state, the looseness of the cell-carrying membrane 6 can be eliminated when the frame 36 is fixed to the outer frame 20, thereby improving the flatness of the cell-carrying surface 4 of the cell-carrying membrane 6 on which a plurality of holes 50 are formed, filling the cell-carrying surface 4 with a dispersion containing dispersed cells, and shaking the cell screening device to allow the screened cells to flow without bias, thereby making it easy to capture the screened cells in the hole 50 in units of 1 cell.

[0158] In this embodiment, the flow path ends 12 located at both ends of the flow path 10 are blocked by the cover 14, and the suction ports 16 having the suction holes 16A connected to the flow path 10 are provided on these cover 14, so that the movement of the flow path ends 12 and the liquid in the flow path 10 is suppressed by the cover 14, and the fluid can enter and exit the flow path 10 through the suction ports 16. Therefore, even if the screening cells C are captured in the hole 50 of the device 1 and the device 1 is transported or tilted in a state where the flow path 10 is filled with liquid, the fluid is not easy to move along the flow path 10 and the flow path ends 12, and the so-called sloshing phenomenon can be suppressed. As a result, it is also possible to suppress the problem that part of the liquid flows into the hole 50 through the through hole 52, causing the screening cells C and the detection particles B accommodated in the hole 50 to separate.

[0159] In addition, in this embodiment, even if the liquid overflows from the suction hole 16A of the suction port 16, the liquid storage portion (the upper surface peripheral portion of the cover portion 14) receives the liquid, and it is possible to suppress the liquid from entering the flow path 10 again from the suction port 16, and the possibility of contamination from the outside can be reduced. In addition, since the partition 28 is formed between the cell mounting surface 4 and the cover portion 14, even if the liquid accumulates in the liquid storage portion 14, the liquid can be suppressed from flowing to the cell mounting portion 6.

[0160] In addition, in this embodiment, by making the lower end of the frame 36 abut against the spacer 46 of the bottom plate 2, the cell-carrying membrane 6 can be positioned at the correct position on the bottom plate 2, so that the flow of the liquid between the hole 50 and the flow path 10 becomes the desired state, enabling high-precision screening.

[0161] In addition, in this embodiment, the bottom plate portion 2 and the outer frame 20 are formed in a separate manner, and the cover portion 14 and the peripheral wall portion 22 can be arranged at the correct position of the bottom plate portion 2 by simply installing the outer frame 20 on the bottom plate portion 2, so it is easy to assemble the device 1. In addition, the outer frame 20 can also be removed from the bottom plate portion 2 after use, which is easy to maintain.

[0162] In addition, in this embodiment, the bottom plate 2, the outer frame 20 and the frame 36 are separately formed, and the cell-carrying membrane 6, the cover 14 and the peripheral wall 22 can be correctly positioned by simply attaching the frame 36 and the outer frame 20 to the bottom plate 2, thereby improving the ease of assembly. In addition, the frame 36 and the outer frame 20 can be removed from the bottom plate 2 after use, making maintenance easy.

[0163] In addition, in this embodiment, the bottom plate portion 2 and the outer frame 20 are molded separately. As long as the outer frame 20 is installed on the bottom plate portion 2, the cell mounting portion 6, the cover portion 14, the peripheral wall portion 22, and the two suction ports 16 can be correctly positioned, so it is easy to assemble.

[0164] In the device 1, the screening cells C, the detection particles B, and the detection reagent D are supplied to the hole 50 from the upper side of the cell-carrying surface 4 of the cell-carrying membrane 6. The liquid in the hole 50 can be discharged from the through hole 52. By suction from the suction hole 16A, the discharge can be performed more effectively. The suction from the suction hole 16A can be performed by sucking the liquid from the suction hole 16A using a micropipette or the like. Alternatively, a suction pump can be connected to the suction port 16, and suction can be performed from the suction hole 16A using the suction pump.

[0165] <Carrier particles>

[0166] The carrier particle is the same as the carrier particle B1 described in the above section (screening method for secretion-producing cells). The carrier particle may be a carrier particle (detection particle B in the above first embodiment) to which a substance (binding substance) having binding properties to the target secretion is immobilized. The substance having binding properties to the target secretion is the same as the binding substance B2 described in the above section (screening method for secretion-producing cells).

[0167] The carrier particles may be carrier particles to which no binding substance is fixed. In this case, the carrier particles are in a state where the binding substance can be fixed. The carrier particles may be surface-modified in a manner that enables the binding substance to be fixed, or may be formed of a material that enables the binding substance to be fixed.

[0168] For example, when a binding substance is immobilized using a binding pair such as biotin-avidin binding, the surface of the carrier particle may be modified with one of the binding pairs. As a specific example, carrier particles coated with streptavidin can be mentioned.

[0169] In addition, when the binding substance is fixed by passive adsorption, the carrier particles can be made of a material having the property of adsorbing the binding substance. When the binding substance is a peptide or protein, the carrier particles can be polystyrene particles, gold nanoparticles, etc.

[0170] When the carrier particles do not have a binding substance fixed thereto, the user may fix any binding substance to the carrier particles.

[0171] <Other components>

[0172] The screening kit of this embodiment may also have other components in addition to the above-mentioned device and carrier particles. Examples of other components include a detection reagent for the target secretion, a culture medium or a buffer, a labeling reagent for the binding substance, and instructions for use.

[0173] The detection reagent for the target secretion is the same as the detection reagent D described in the above (screening method for secretion-producing cells). The culture medium or buffer can be exemplified by the culture medium or buffer exemplified in the above (screening method for secretion-producing cells). The labeling reagent for the binding substance is used to modify the binding substance so that it can be fixed to the carrier particles. For example, when the carrier particles are coated with streptavidin, a biotin labeling reagent can be cited as the labeling reagent.

[0174] <How to use>

[0175] The screening kit of this embodiment can be used to carry out the screening method of the first embodiment, and can be used in the manner described in the above section (Screening method for secretion-producing cells).

[0176] Example

[0177] Hereinafter, the present invention will be described in further detail by way of examples, but the present invention is not limited to these examples.

[0178] <Cell preparation>

[0179] A gene encoding mouse anti-mouse RANKL monoclonal antibody (hereinafter also referred to as "mouse monoclonal antibody") was introduced into 293FT cells. The transgenic cells were cultured in MEMα medium supplemented with fetal bovine serum (FBS) for 48 hours and used as cells for secretion assay.

[0180] <Preparation of test particles>

[0181] Add 5 μL of goat anti-mouse antibody (Jackson ImmunoResearch Inc. Code: 115-005-003) to 500 μL of Polybead polystyrene microspheres (particle size 6.0 μm; 07312, Polysciences; hereinafter also referred to as "beads") washed with phosphate buffered saline (PBS), and react overnight at 4°C to bind the goat anti-mouse antibody to the beads. Wash the beads with PBS to remove unreacted antibodies. Next, add 1% BSA / PBS to the beads, react at 4°C for 1 hour, and then wash the beads with PBS. Use them as detection particles.

[0182] <Secretion assay>

[0183] [Step A]

[0184] The secretion assay was performed using a device having a plurality of wells having through holes with a pore size of 2 μm at the bottom. The detection particles prepared in the above <Preparation of detection particles> were added to the wells pre-wetted with phosphate buffered saline (PBS) to fill the wells with detection particles.

[0185] Next, the culture solution of the cells prepared in the above <Preparation of Cells> is inoculated into the wells to capture the cells in the wells.

[0186] [Step B]

[0187] Next, Hoechst 33342 (H342, Dojin Chemical Research Institute) and Alexa488-labeled goat anti-mouse IgG antibody (model #A11001, Life Technologies) were diluted 500-fold in MEMα medium supplemented with fetal bovine serum (FBS) and added to the wells.

[0188] To detect particles, cells, and the addition of Hoechst 33342 and Alexa488 labeled goat anti-mouse IgG antibodies into the wells, the device was placed in a CO 2 5% CO at 37°C 2 The cells were left to stand for 3 hours in an incubator. Thus, the cells secreted mouse monoclonal antibodies. The secreted mouse monoclonal antibodies were captured by the goat anti-mouse antibodies of the detection particles, and then combined with the Alexa488-labeled goat anti-mouse IgG antibodies. Thus, an antigen-antibody complex consisting of goat anti-mouse antibodies-mouse monoclonal antibodies-Alexa488-labeled goat anti-mouse IgG antibodies was formed.

[0189] [Step C(1)]

[0190] The CO 2 The device was observed after being placed in the incubator for 3 hours. Fig.14 (A) is a representative fluorescence microscope photograph taken from the bottom side of the well. As a result, the nucleus of the cell was detected based on the blue fluorescence of Hoechst 33342 (Hoechst; arrow). In addition, the mouse monoclonal antibody bound to the detection particle was detected based on the green fluorescence of Alexa488 (Alexa; arrow).

[0191] [Step D(1)]

[0192] Based on the fluorescence signal detected in step C(1), the wells in which the secretion-producing cells secreting the mouse monoclonal antibody were successfully captured were determined (merge; arrow).

[0193] [Step E (Washing Step)]

[0194] The solution in the well was drained from the through-hole, and PBS was added to the well to wash the detection particles, thereby removing the unreacted Alexa488-labeled goat anti-mouse IgG antibody.

[0195] [Step C(2)]

[0196] The washed devices were observed using a fluorescence microscope. Fig.14 (B) is a representative fluorescence microscope photo taken from the bottom side of the hole. As a result, the nucleus of the cell was detected based on the blue fluorescence of Hoechst 33342 (Hoechst; arrow). In addition, the mouse monoclonal antibody bound to the detection particles was detected based on the green fluorescence of Alexa488 (Alexa; arrow). In addition, the unreacted Alexa488-labeled goat anti-mouse IgG antibody was removed by the washing step, and the background was reduced. As a result, the fluorescent signal of the mouse monoclonal antibody captured by the detection particles became obvious.

[0197] [Step D(2)]

[0198] Based on the fluorescence signal detected in step C(2), the wells in which the secretion-producing cells secreting the mouse monoclonal antibody were successfully captured were determined (merge; arrow).

[0199] The above results confirm that the screening method of the present invention can screen for secretion-producing cells that produce target secretions. In addition, by performing a washing step, the background is reduced, and the signal of the target secretion secreted by the secretion-producing cells can be made distinct.

[0200] Description of Reference Numerals

[0201] 1 Device

[0202] 2 Bottom plate

[0203] 4 Cell loading surface

[0204] 6. Cell-carrying membrane (cell-carrying part)

[0205] 8 Liquid inlet and outlet

[0206] 10 flow path

[0207] 12 Flow path end

[0208] 14. Cover (fluid storage part)

[0209] 16 Suction port

[0210] 16A Suction hole

[0211] 17 Instant Noodles

[0212] 18 Snap-on wall

[0213] 20 Outer frame

[0214] 22 Wall

[0215] 24 snap-in slots

[0216] 26 Protrusion

[0217] 28 Divider

[0218] 30 ribs

[0219] 32 snap-in slots

[0220] 34 Section

[0221] 36 Frame

[0222] 36A Wall

[0223] 38 bevel

[0224] 40 snap-fit ​​protrusion

[0225] 42 snap-fit ​​protrusion

[0226] 44 snap-in slot

[0227] 46 Spacer

[0228] 47 Gap

[0229] 50 holes

[0230] 50a Bottom

[0231] 52 Through hole

[0232] A, A-1, A-2, A-3 secretions

[0233] C, C-1, C-2, C-3 screening cells

[0234] B Bead

[0235] B1 Carrier particles

[0236] B2 Binding substances

[0237] B2' cell membrane protein

[0238] D Detection Reagents

[0239] D1 Binding substance

[0240] D2 Marking substance

Claims

1. A method for screening secretion-producing cells, wherein cells producing target secretions are screened out from a plurality of cells, the method comprising: Step A, capturing the cells and detection particles by making a plurality of holes having through holes at the bottom thereof of a size that the cells cannot pass through, wherein the detection particles can capture the target secretion and have a size that cannot pass through the through holes; Step B, causing the cells captured in the plurality of wells to produce secretions; Step C, using a detection reagent that can specifically bind to the target secretion to detect the target secretion captured by the detection particles, wherein the detection reagent is captured by the detection particles through the target secretion, and the detection reagent that is not bound to the target secretion can flow out of the through hole; and Step D, using the detection result as an indicator, determining the wells that capture the cells producing the target secretion from the plurality of wells, The pore has a size capable of capturing the cell in units of one cell when one or more detection particles are captured. The detection particles are carrier particles on which a substance capable of binding to the target secretion is immobilized, or cells containing cell membrane proteins capable of binding to the target secretion.

2. The method for screening secretion-producing cells according to claim 1, wherein: The target secretion is an antibody.

3. A screening kit for secretion-producing cells, which screens cells producing target secretions from multiple cells, The screening kit for secretion-producing cells comprises a device comprising a plurality of holes, carrier particles, and a detection reagent capable of specifically binding to the target secretion. The hole has a size that allows the cell to be captured in a unit of one cell when one or more of the carrier particles are captured, and has a through hole at the bottom that is too large for the cell to pass through. The carrier particles have a size that cannot pass through the through hole, and are carrier particles on which a substance that binds to the target secretion is fixed, or carrier particles that can fix a substance that binds to the target secretion, The detection reagent is captured by the carrier particles via the target secretion and the substance that binds to the target secretion, and the detection reagent that is not bound to the target secretion can flow out from the through hole.

4. The screening kit for secretion-producing cells according to claim 3, wherein: The device further includes a flow path connected to the through hole and disposed below the through hole.

5. The screening kit for secretion-producing cells according to claim 4, wherein The device also includes a suction hole in communication with the flow path.

6. The screening kit for secretion-producing cells according to any one of claims 3 to 5, wherein The secretion is an antibody.

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