Screening method for secretory cells and screening kit for secretory cells

Abstract

This method for screening a secretion producing cell involves screening, from a plurality of cells, cells which produce a secretion of interest. The method comprises: a step A for capturing detection particles capable of capturing the cell and the secretion of interest, in a plurality of wells having, in the bottom section thereof, through-holes sized so that cells do not pass therethrough; a step B for causing the cells captured in the plurality of wells to produce the secretion; a step C for detecting the secretion of interest captured by the detection particles; and a step D for identifying, from the plurality of wells, a well in which the cells that produce the secretion of interest were captured, by using the detection result as an index. The well has a size with which the cells can be captured in units of one cell while capturing one or more of the detection particles.

Description

[Technical field]

[0001] The present invention relates to a method for screening secretory cells and a screening kit for secretory cells. This application claims priority based on Japanese Patent Application No. 2019-178577, filed on September 30, 2019, the contents of which are incorporated herein by reference. [Background technology]

[0002] In recent years, especially in the field of drug discovery, the target of cell analysis has been subdivided from the cell group level to the single cell level. Attempts have been made to capture, identify, select, recover, culture, and genetically analyze cells one by one, and to use the selected cells. A method for identifying and selecting cells is to separate secretions from cells, detect the target secretions, and then screen the cells that secreted the target secretions. In such single cell analysis, a technology that can analyze multiple cells at once is useful.

[0003] As a technique for analyzing cells en masse, 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 onto a glass slide, followed by incubation, and B cells producing the target antibody are identified based on the localized increase in fluorescence surrounding the antibody-producing cells (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 4603894 Summary of the Invention [Problem to be solved by the invention]

[0005] However, in the method described in Patent Document 1, the antibody-producing cells are not fixed, and therefore there is a risk that the antibody-producing cells may move during culturing, fluorescence detection, cell collection, etc. As a result, accurate screening may not be possible.

[0006] The present invention has been made in consideration of the above-mentioned circumstances, and has an objective of providing a screening method for secretion-producing cells, which makes it possible to accurately obtain cells that produce a target secretion, and a screening kit for said secretion-producing cells. [Means for solving the problem]

[0007] In order to solve the above problems, the present invention employs the following configuration.

[0008] A first aspect of the present invention is a method for screening secretion-producing cells, which screens for cells that produce a target secretion from a plurality of cells, comprising the steps of: step A of capturing the cells and detection particles capable of capturing the target secretion in a plurality of wells having through-holes at the bottom that are large enough that the cells cannot pass through the through-holes; step B of causing the cells captured in the plurality of wells to produce a secretion; step C of detecting the target secretion captured by the detection particles; and step D of using the detection result as an indicator to identify a well from the plurality of wells in which a cell producing the target secretion has been captured, wherein the wells have a size that allows the cells to be captured on a single cell basis while capturing one or more of the detection particles.

[0009] A second aspect of the present invention is a secretion-producing cell screening kit for screening cells that produce a target secretion from a plurality of cells, comprising a device including a plurality of wells and carrier particles, the wells being large enough to capture the cells on a single cell basis while capturing one or more of the carrier particles, and having through-holes at the bottom which are large enough that the cells cannot pass through, and the carrier particles being large enough that they cannot pass through the through-holes. Effect of the Invention

[0010] According to the present invention, it is possible to provide a screening method for secretion-producing cells, which makes it possible to accurately obtain cells that produce a target secretion, and a screening kit for said secretion-producing cells. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a diagram illustrating an example of step A of the method for screening secretion-producing cells. [Diagram 2] FIG. 13 is a diagram illustrating an example of step B of the method for screening secretion-producing cells. [Diagram 3] FIG. 13 is a diagram illustrating an example of step C of the method for screening secretion-producing cells. [Figure 4] FIG. 13 is a diagram illustrating an example of step D of the method for screening secretion-producing cells. [Diagram 5] FIG. 13 is a diagram illustrating a modified example of step A of the method for screening secretion-producing cells. [Figure 6] FIG. 1 is a diagram illustrating an example of a method for screening secretory cells when the detection particles are cells. [Figure 7A] FIG. 1 is a diagram illustrating an example of a washing step in a screening method for secretion-producing cells. [Figure 7B] FIG. 1 is a diagram illustrating an example of a washing step in a screening method for secretion-producing cells. [Figure 8] FIG. 2 is a plan view showing one embodiment of a device included in the screening kit. [Figure 9] FIG. 2 is a front view of the device according to the embodiment. [Figure 10] FIG. 2 is a perspective view showing a front cross section of the device according to the embodiment. [Figure 11] FIG. 2 is a front cross-sectional view of the center part of the device according to the embodiment. [Figure 12] FIG. 2 is a side cross-sectional view of the device according to the embodiment. [Figure 13] FIG. 2 is a perspective view showing a method of assembling the device of the embodiment; [Figure 14] Fluorescence micrographs of wells in an example where a screening method according to one embodiment is applied, (A) is a fluorescence micrograph of a well that has not been subjected to a washing step, and (B) is a fluorescence micrograph of a well that has been subjected to a washing step. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings as needed. In the drawings, the same or corresponding parts are denoted by the same or corresponding reference numerals, and duplicate explanations are omitted. The dimensional ratios in each drawing are exaggerated for the purpose of explanation, and do not necessarily correspond to the actual dimensional ratios.

[0013] (Method for screening secretory cells) A first aspect of the present invention is a method for screening secretion-producing cells, which screens for cells that produce a secretion of interest from a plurality of cells. The screening method includes the steps of: capturing the cells and detection particles capable of capturing the secretion of interest in a plurality of wells having through-holes at the bottom that are large enough for the cells to pass through; capturing the cells and detection particles that are large enough not to pass through the through-holes; causing the cells captured in the plurality of wells to produce a secretion; detecting the secretion of interest captured by the detection particles; and using the detection result as an index to identify a well in which a cell producing the secretion of interest has been captured from the plurality of wells. The wells have a size that allows the cells to be captured on a cell-by-cell basis while capturing one or more of the detection particles.

[0014] In the screening method according to this embodiment, first, cells C and detection particles B capable of capturing a target secretion product are captured in a plurality of wells 50 having through-holes 52 at the bottom that are large enough that the cells C to be screened cannot pass through (Step A; FIG. 1). The detection particles B are large enough not to pass through the through-holes 52. The wells 50 are also large enough to capture cells C on a cell-by-cell basis while capturing one or more detection particles B. Next, the cells C captured in the well 50 are incubated to allow the cells C to produce the secretory product A (Step B; FIG. 2). Next, the target secretion product A captured by the detection particles B is detected using a detection reagent D or the like (Step C; Figure 3). Finally, using the detection result obtained in step C as an index, the well 50 (well 50-1) in which cells producing the target secretion A (secretion A-1) are captured is identified from the multiple wells 50 (wells 50-1, 50-2, 50-3) (step D; Figure 4).

[0015] <Screening cells> The plurality of cells (hereinafter, also referred to as "screening cells") subjected to the screening method according to the present embodiment is a cell population predicted to include a target secretory product-producing cell. The plurality of cells is preferably a population of cells that produce a secretory product. Examples of the cells include animal cells such as mammalian (human, mouse, rat, rabbit, horse, camel, monkey, etc.) cells, avian (chicken, etc.) cells, and insect (shellfish, etc.) cells; plant cells; fungi such as yeast; and bacteria such as Escherichia coli. The screening cells may be genetically modified cells obtained by introducing a gene encoding a secretory protein or secretory peptide into these cells. The secretory protein and secretory peptide may be chimeric proteins in which a secretory signal peptide is linked to a non-secretory protein or non-secretory peptide.

[0016] The target secretion product (hereinafter also referred to as "target secretion product") may be naturally derived or may be a non-natural product obtained by using genetic engineering. The target secretion product is not limited, but is preferably a single secretion product. Examples of the target secretory product include, but are not limited to, 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 factors, granulocyte colony stimulating factors, erythropoietin, etc.), cell growth factors (epidermal 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 from adipose tissue, etc.), and neurotrophic factors (nerve growth factor, etc.); antibiotics; metabolic products of microorganisms such as pigments; hormones (peptide hormones, steroid hormones, microbial hormones, etc.); and chimeric proteins of secretory signal peptides and non-secretory proteins. The target secretory product is not limited to natural proteins or natural peptides, and may be genetically modified versions of these.

[0017] In one embodiment, the secreted product is preferably an antibody. Examples of natural antibodies include immunoglobulin G (IgG) and immunoglobulin M (IgM). Examples of non-natural antibodies include antibody fragments such as Fab, scFv, and diabody; single domain antibodies (Single Domain Antibodies, Methods in Molecular Biology Volume, 911, 2012); and 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).

[0018] Examples of secretion-producing cells that produce a target secretion include antibody-producing cells, cytokine-producing cells, and hormone-secreting cells.

[0019] Examples of antibody molecule-producing cells include, but are not limited to, B cells, hybridomas formed by fusing B cells with myeloma cells, and genetically modified cells into which a polynucleotide encoding an antibody molecule has been introduced. Examples of cells into which an antibody gene is introduced include animal cells, fungi such as yeast, and bacteria such as Escherichia coli. Specific examples of cells into which an antibody gene can be introduced include NSO cells, CHO cells, COS cells, and 293FT cells.

[0020] Examples of cytokine-producing cells include, but are not limited to, macrophages, B cells, T cells, NK cells, NKT cells, dendritic cells, hepatic Kupffer cells, stromal cells, fibroblasts, vascular endothelial cells, and the like. Hormone-secreting cells include, but are not limited to, anterior pituitary cells, somatotrope cells, lactotrope cells, thyrotrope cells, gonadotrope cells, corticotrope cells, intermediate pituitary cells, melanocyte-stimulating hormone-secreting cells, oxytocin-secreting cells, vasopressin-secreting cells, serotonin-secreting cells, endorphin-secreting cells, somatostatin-secreting cells, gastrin-secreting cells, secretin-secreting cells, cholecystokinin-secreting cells, and the like. Examples of such cells include insulin-secreting cells, glucagon-secreting cells, bombesin-secreting cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, oxic cells, adrenal gland cells, chromaffin cells, steroid hormone (mineralocorticoid or glucocorticoid) producing cells, testosterone-secreting cells, estrogen-secreting cells, progesterone-secreting cells, cells of the juxtaglomerular apparatus of the kidney, macula densa cells of the kidney, perivascular pole cells of the kidney, and mesangial cells of the kidney.

[0021] Among these, the target secretion product is preferably an antibody, and the secretion product-producing cells are preferably antibody-producing cells.

[0022] <Process A> In step A, the cells and detection particles capable of capturing the target secretion product and having a size that does not allow the cells to pass through are captured in multiple wells having through-holes at the bottom that are large enough to prevent the cells from passing through the through-holes.

[0023] FIG. 1 is a diagram for explaining an example of step A. The device 100 includes a plurality of wells 50. The wells 50 have a through-hole 52 in the bottom 50a. The device 100 may be composed of, for example, a substrate or a membrane having a first surface and a second surface. The plurality of wells 50 may be, for example, a recess having an opening in the first surface of the substrate or membrane. The through-hole 52 may be a through-hole penetrating from the first surface to the second surface of the substrate or membrane. It is preferable that the material of the device 100 is not harmful to cells. Examples of the material of the device 100 include glass; polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), cycloolefin polymer (COP), and general resins such as epoxy.

[0024] The planar shape of the opening of the well 50 is not particularly limited as long as it is a shape that allows for capturing the screening cells C on a cell-by-cell basis. Examples of the planar shape of the well 50 include a circle, an ellipse, a polygon (a quadrangle such as a square, a hexagon, an octagon, etc.). From the viewpoint of increasing the arrangement density, a regular hexagon is preferable. The bottom 50a of the well 50 may be a flat bottom or a round bottom. From the viewpoint of ease of forming the through-hole 52 and from the viewpoint of being able to store a large amount of detection particles B, a flat bottom is preferable. The well 50 has a size that allows the screening cell C to be captured in units of one cell while capturing one or more detection particles B described below. A unit of one cell means, for example, a single cell. The size of the well 50 can be appropriately selected according to the size of the screening cell C. The size of the well 50 is not limited, but generally, the maximum diameter of a circle that fits into the well opening can be about 1 to 100 μm, and the depth can be about 1 to 100 μm. The maximum diameter of a circle that fits into the well opening is more preferably 2 to 50 μm, and even more preferably 3 to 25 μm. The depth of the well 50 is more preferably 2 to 70 μm, even more preferably 3 to 50 μm, and particularly preferably 4 to 30 μm. In relation to the size of the screening cell C, the maximum diameter of a circle that fits within the well 50 in a plan view can be about 0.5 to 2 times, and preferably 0.8 to 1.9 times, the maximum diameter of the screening cell C. The depth of the well 50 can be about 0.5 to 4 times, and more preferably 0.8 to 1.9 times, the maximum diameter of the screening cell C.

[0025] The through-hole 52 is provided in the bottom 50a of the well 50, and communicates the internal space of the well 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 do not pass through it and liquid including secretions secreted by the screening cells C can pass through it. Examples of the planar shape of the through-hole 52 include a circle, an ellipse, a polygon (a quadrangle such as a square, a hexagon, an octagon, etc.), etc. From the viewpoint of ease of formation, a circle is preferable. Although the size of the through hole 52 is not limited, 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. In relation to the size of the screening cell C, the minimum inner diameter of the through hole 52 is preferably 0.5 times or less, more preferably 0.1 times or less, and even more preferably 0.05 times or less, the maximum diameter of the screening cell C.

[0026] Detection particles B are particles capable of capturing a target secretion. Detection particles B have a size that allows them to be captured in well 50 and not pass through through-hole 52. The size of detection particles B can be appropriately selected according to the size of well 50 and through-hole 52. The size of detection particles B can be, for example, a particle size of 50 nm to 80 μm. The particle size of detection particles B is, for example, preferably 100 nm to 50 μm, more preferably 500 nm to 30 μm, and even more preferably 1 to 20 μm. In relation to the size of the through-hole 52, the particle size 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.

[0027] The number of through-holes 52 is not particularly limited and can be appropriately set depending on the size of the well 50 and the through-hole 52. The number of through-holes 52 may be one or more, and can be in the range of, for example, 1 to 100. From the viewpoint of the outflow efficiency of the liquid in the well 50, the number of through-holes 52 is preferably two or more. Specific examples of the number of through-holes 52 include 2 to 10, 2 to 5, 2, or 3, etc. The position of the through-hole 52 is not particularly limited as long as it is located on the bottom 50a of the well 50. From the viewpoint of facilitating storage of the screening cells C in the well 50 and of the outflow efficiency of the liquid in the well 50, it is preferable that one or more through-holes 52 are arranged 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 lattice pattern on the bottom 50a.

[0028] The detection particle B is not particularly limited as long as it can capture the target secretion. Examples of the detection particle B include carrier particles to which a substance capable of binding to the target secretion is immobilized, and cells containing cell membrane proteins capable of binding to the target secretion. In the example of FIG. 1, the detection particle B is composed of a carrier particle B1 to which a substance (hereinafter also referred to as a "binding substance") B2 capable of binding to the target secretion is immobilized. 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 it is possible to use a material that is usually used for detecting antibodies, etc. Examples of the carrier particle B1 include, but are not limited to, beads (magnetic beads, resin beads, etc.), hydrogel particles (sodium alginate gel, agarose gel, etc.), metal particles (gold nanoparticles), etc. The binding substance B2 is a substance that specifically binds to the target secretion product. When the target secretion product is an antibody, the antigen of the antibody (antigen peptide, antigen protein, etc.) can be used as the target secretion product. When the target secretion product is a cytokine or hormone, for example, an antibody that binds to the cytokine or hormone can be used as the binding substance B2. The binding substance B2 can be immobilized on the carrier particle B1 by a known method, such as, but not limited to, a method using a binding pair such as biotin-avidin binding, a passive adsorption method, etc.

[0029] In step A, screening cells C and detection particles B are captured in multiple wells 50. At this time, it is preferable that one screening cell C is stored in one well 50. The operation of capturing screening cells C in the well 50 (hereinafter also referred to as the "cell capturing operation") and the operation of capturing detection particles B (hereinafter also referred to as the "detection particle capturing operation") may be performed separately or simultaneously. When each capturing operation is performed separately, the order of the cell capturing operation and the detection particle capturing operation is not particularly limited. From the viewpoint of making it easier for detection particles B to be evenly arranged in the well 50, it is preferable to perform the detection particle capturing operation first.

[0030] When the cell capturing operation and the detection particle capturing operation are performed separately, the detection particle capturing operation can be performed by suspending the detection particles B in an appropriate liquid such as a buffer solution (PBS, physiological saline, etc.) or a culture medium, and supplying the suspension to the multiple wells 50. There is no need to perform an operation of introducing the suspension into each well 50, and the suspension can be supplied to the opening surface of the multiple wells 50. The detection particles B in the suspension supplied to the wells 50 are stored in each well 50 by their own weight. The storage of the detection particles B in the wells 50 may be performed while the liquid in the wells 50 is sucked from a suction hole or the like communicating with the through hole 52. In this case, the storage of the detection particles B in the wells 50 can be performed more quickly. The amount of the detection particles B stored in each well 50 can be adjusted by the density of the detection particles B in the 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. The density of the detection particles B in the suspension is generally, for example, 10 5 ~10 12 The concentration can be approximately 1 / mL. When a culture medium is used as the liquid for suspending the suspended particles, the culture medium can be appropriately selected from culture media for the cells depending on the type of the screening cells C. When the screening cells C are mammalian cells, examples of the culture medium include MEM medium, MEMα medium, and RPMI medium. As used herein, the term "suitable liquid" refers to a liquid in which the screening cell C can survive. When the screening cell C is a secretory product-producing cell, the suitable liquid is preferably a liquid in which the cell C can produce a secretory product. Examples of suitable liquids include the above-mentioned buffer solutions and culture media.

[0031] The cell capture operation can be performed by suspending the screening cells C in an appropriate liquid and supplying the cell suspension to a plurality of wells 50. There is no need to perform an operation of introducing the screening cells C into each well 50, and the cell suspension may be supplied to a surface of a member (plate, membrane, etc.) having a plurality of wells 50, on which openings of the plurality of wells 50 are provided. The screening cells C in the cell suspension supplied to the wells 50 are stored in each well 50 on a cell-by-cell basis due to their own weight. The storage of the screening cells C in the wells 50 may be performed while sucking the liquid in the wells 50 through a suction hole or the like communicating with the through-hole 52. In this case, the storage of the screening cells C in the wells 50 can be performed more quickly. The density of the screening cells C in the cell suspension is not particularly limited as long as the fluidity of the cell suspension is not impaired, and can be appropriately selected according to the size of the screening cells C. The density of the screening cells C in the cell suspension is generally, for example, 10 5 ~10 9 The concentration can be approximately 1 / mL. When a culture medium is used as the liquid for suspending the cells, examples of the culture medium include those similar to those described above.

[0032] When a cell capture operation is performed after a detection particle capture operation, the liquid in the well 50 may be discharged from the through-hole 52 after the detection particle capture operation, and then a cell suspension containing the screening cells C may be supplied to the well 50. This removes excess medium from the well 50, thereby improving the efficiency of trapping the screening cells C in the well 50. The liquid in the well 50 may be discharged while being sucked from a suction hole or the like communicating with the through-hole 52. In this case, the liquid can be efficiently discharged from the through-hole 52.

[0033] When the cell capture operation and the detection particle capture operation are performed simultaneously, the detection particles B and the screening cells C may be suspended in an appropriate liquid, and the suspension may be supplied to a plurality of wells 50. The detection particles B and the screening cells C in the suspension supplied to the wells 50 are stored in each well 50 by their own weight. The detection particles B and the screening cells C may be stored in the wells 50 while the liquid in the wells 50 is sucked through a suction hole or the like communicating with the through hole 52. In this case, the detection particles B and the screening cells C can be stored in the wells 50 more quickly. The amount of the detection particles B stored in each well 50 can be adjusted by the density of the detection particles B in the suspension. The density of the detection particles B in the suspension can be the same as above. The screening cells C are stored in each well 50 in units of one cell. The density of the screening cells C in the cell suspension can be the same as above.

[0034] <Process B> In step B, the screening cells captured in the multiple wells are allowed to produce secretions.

[0035] FIG. 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 the secretion A by incubating the screening cells C in the well 50. The incubation conditions can be appropriately set depending on the type of the screening cells C. In general, incubation conditions such as 25 to 38° C. and 0.03 to 5% CO2 can be used. Specific examples of the incubation conditions include 37° C. and 5% CO2. The incubation is performed in a state in which the screening cells C captured in the well 50 are present in a liquid such as a buffer solution or a medium. Preferably, the inside of the well 50 is filled with a liquid such as a buffer solution or a medium.

[0036] When secretion A secreted from screening cell C is the target secretion, secretion A binds to binding substance B2 and is captured by detection particle B. In addition, a portion of secretion A moves from through-hole 52 to the outside of well 50 together with the liquid in well 50 (buffer solution, medium, etc.). The incubation time can be a time sufficient for the secretion product A to be captured by the detection particles B. The incubation time can be appropriately set depending on the type of the screening cells C, and can be, for example, about 0.5 to 24 hours.

[0037] <Process C> In step C, the target secretion product captured by the detection particles is detected.

[0038] 3 is a diagram illustrating an example of step C. The target secretion product can be detected, for example, by 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 product and a labeling substance D2. As the binding substance D1, for example, an antibody that specifically binds to the target secretion product can be used. The antibody as the binding substance D1 may be a natural antibody or a non-natural antibody (e.g., an antibody fragment such as Fab or scFv). It is preferable that the site of the target secretion product to which the binding substance D1 binds is different from the site to which the binding substance B2 binds. For example, when the target secretion product is an antibody, the binding substance D1 may be an antibody that specifically binds to the constant region of the antibody that is the target secretion product. 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 generally used in the field of biochemistry can be used. Examples of the labeling substance D2 include enzyme labels, fluorescent labels, radioactive substances, and the like. Examples of the enzyme labels include HRP (Horse Radish Peroxidase), AP (Alkaline Phosphatase), and the like. Examples of the fluorescent label include fluoresceins such as FAM (carboxyfluorescein), JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), FITC (fluorescein isothiocyanate), TET (tetrachlorofluorescein), and HEX (5'-hexachloro-fluorescein-CE phosphoramidite); Cy dye-labeled carboxylic acids such as Cy3 and Cy5; and Alexa Fluors such as Alexa568 and Alexa488. The labeling substance D2 is preferably a fluorescent label because the signal can be easily detected.

[0039] The detection reagent D can be dissolved in an appropriate liquid (buffer solution, medium, etc.) and brought into contact with the target secretion A in the well 50 by supplying the detection reagent solution to the well 50. Before supplying the detection reagent solution to the well 50, the liquid in the well 50 may be drained from the through-hole 52. The liquid may be drained, for example, by removing the liquid from a flow path disposed below the through-hole 52. The liquid may be removed from the flow path by suction from a suction hole or the like communicating with the flow path. By removing the liquid in the well 50 before supplying the detection reagent D, the detection reagent D can be efficiently supplied into the well 50.

[0040] The detection reagent D supplied into the well 50 comes into contact with the secretion A in the well 50. If the secretion A is the target secretion, the detection reagent D binds to the secretion A via the binding substance D1. If the secretion A is the target secretion, most of the secretion A in the well 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 depending on 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.

[0041] The detection of the signal of the labeling substance D2 may be performed by a method according 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 is used to develop color, and the color 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, a radioactive signal can be detected using an X-ray microscope.

[0042] <Process D> In step D, the detection result in step C is used as an index to identify wells in which cells producing the target secretion product are captured from among a plurality of wells.

[0043] 4 is a diagram illustrating an example of step D. At the time when step C is completed, screening cells C, detection particles B, secretion substance A secreted from screening cells C, and detection reagent D are present in each of the multiple wells 50. However, the amount of detection reagent D present in wells 50 differs depending on the type of secretion substance A.

[0044] Well 50-1 stores screening cell C-1 that secretes secretion A-1. Secretion A-1 is a target secretion and is captured by detection particle B. Therefore, detection reagent D is captured by detection particle B via secretion A-1, which is a target secretion. As a result, detection reagent D is accumulated in well 50-1 according to the amount of secretion A-1 captured by detection particle B. Well 50-2 stores screening cell C-2 that secretes secretion A-2. Secretion A-2 is not the target secretion, and is not captured by detection particles B. Therefore, detection reagent D is not captured by detection particles B and flows out of through-hole 52 together with the liquid in well 50-2. Well 50-3 stores screening cell C-3 that secretes secretion A-3. Secretion A-3 is not the target secretion, and is not captured by detection particles B. Therefore, detection reagent D is not captured by detection particles B, and flows out of through-hole 52 together with the liquid in well 50-3.

[0045] 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 present in the well is greater than in wells 50-2 and 50-3. Therefore, when the signal of the labeling substance D2 is detected, the signal strength detected in well 50-1 is stronger than in wells 50-2 and 50-3. In other words, it can be said that the screening cells C producing the target secretion are stored in the wells in which the signal strength of the labeling substance D2 is higher than in other wells. Therefore, the wells in which the signal strength of the labeling substance D2 is higher than in other wells can be identified as wells in which secretion-producing cells producing the target secretion are captured.

[0046] For example, Figure 14(A) shows a fluorescence microscope photograph of a well in which steps A to C were performed in the example. In Figure 14(A), Alexa488 was used as the labeling substance D2, and it can be seen that the fluorescent signal of the well indicated by the arrow is stronger than that of the other wells (Figure 14(A) Alexa). Therefore, the well indicated by the arrow can be identified as the well in which the secretion-producing cells that produce the target secretion product were captured.

[0047] <Variation 1> The detection reagent D may be supplied to the well 50 simultaneously with the screening cells C and / or the detection particles B. Fig. 5 is a diagram illustrating step A in the case where the detection reagent D is supplied to the well 50 simultaneously with the screening cells C and the detection particles B.

[0048] 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 can be added to a suspension in which the screening cells C and the detection particles B are suspended. Alternatively, the screening cells C and the detection particles B may be suspended in a liquid (buffer solution, culture medium, etc.) to which the detection reagent D has been added. Then, the suspension containing the detection reagent D, the screening cells C, and the detection particles B can be supplied to the well 50.

[0049] The supply of the detection reagent D to the well 50 can also be performed simultaneously with either the detection particle capture operation or the cell capture operation. For example, when the detection reagent D is supplied to the well 50 together with the detection particles B, the detection reagent D can be added to an appropriate liquid (buffer solution, culture medium, etc.) in which the detection particles B are suspended. Alternatively, the detection particles B may be suspended in an appropriate liquid (buffer solution, culture medium, etc.) to which the detection reagent D has been added. Then, the suspension containing the detection reagent D and the detection particles B can be supplied to the well 50. When the detection reagent D is supplied to the well 50 together with the screening cells C, the detection reagent D can be added to an appropriate liquid (buffer, medium, etc.) in which the screening cells C are suspended. Alternatively, the screening cells C may be suspended in an appropriate liquid (buffer, medium, etc.) to which the detection reagent D has been added. Then, the suspension containing the detection reagent D and the screening cells C can be supplied to the well 50. If an operation of discharging the liquid in the well 50 from the through-hole 52 is performed between the detection particle capture operation and the cell capture operation, it is preferable that the detection reagent D is added to the suspension for the capture operation performed after the discharge operation.

[0050] In step A, detection reagent D is supplied to well 50 simultaneously with the cell capture operation and / or detection particle capture operation, and then step B can be carried out in the same manner as described above. When secretion A secreted from screening cell C is the target secretion, secretion A is captured by detection particle B via binding substance B2. Furthermore, detection reagent D binds to secretion A via binding substance D1. Therefore, when secretion A is the target secretion, detection reagent D is captured by detection particle B via secretion A.

[0051] Since the detection reagent D is already present in the well 50 and has reacted 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 detection of the signal of the labeling substance D2 can be performed in the same manner as described above. Step D can be carried out in the same manner as described above, based on the detection results in step C.

[0052] <Variation 2> The detection particle B may be a cell containing a cell membrane protein capable of binding to the target secretion product. Figure 6 shows the state of step C when the detection particle B is a cell containing a cell membrane protein B2' capable of binding to the target secretion product (hereinafter also referred to as a "target cell").

[0053] "Membrane protein" means a protein localized in a cell membrane. The membrane protein may be an integral membrane protein or a surface membrane protein, but is preferably an integral membrane protein. The integral membrane protein may be a multi-pass type that penetrates the cell membrane multiple times, or a single-pass type that penetrates the cell membrane once. Examples of membrane proteins include GPCRs (G protein-coupled receptors), ligand-gated ion channels, voltage-gated ion channels, transporters, etc.

[0054] The target cell may be a natural cell or a genetically modified cell into which a membrane protein gene has been introduced. The target secretory product binds to a region where the membrane protein B2' is exposed on the cell membrane surface. When the target secretory product is an antibody against a membrane protein specific to diseased cells (e.g., cancer cells), the diseased cells can be used as the target cells.

[0055] The target cells have a size that allows them to be captured in the well 50 and not pass through the through-hole 52. The size of the target cells is, for example, about 1 μm to 80 μm. When target cells are used as the detection particles B, the size of the well 50 can be set based on the total size of the screening cells C and the target cells. When the target cells are larger than the screening cells C, the maximum diameter of the circle that fits into the opening of the well 50 can be, for example, 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 approximately 0.5 to 2 times the sum of the maximum diameter of the target cells and the maximum diameter of the screening cells C, more preferably 0.8 to 1.9 times, and even more preferably 0.9 to 1.5 times. When the target cells are smaller than the screening cells C, the size of the wells 50 can be set in the same manner as described above for the detection particles B.

[0056] In step A, the operation of capturing the target cells in the wells 50 (hereinafter also referred to as the "target cell capturing operation") may be performed simultaneously with or separately from the cell capturing operation, but it is preferable to perform them separately. When the target cells are larger than the screening cells C, it is preferable to perform the target cell capturing operation before the cell capturing operation. The target cell capture operation can be performed by suspending the target cells in an appropriate liquid (buffer, 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 stored 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 depending on the size of the target cells. The density of the target cells in the target cell suspension is generally, for example, 10 5 ~10 9 The concentration can be approximately 1 / mL.

[0057] Steps B to D can be carried out in the same manner as described above. When secretion A secreted from screening cell C is the target secretion, secretion A binds to cell membrane protein B2' of the target cell as detection particle B. Detection reagent D also binds to secretion A. Therefore, detection reagent D is captured by cell membrane protein B2' of the target cell via secretion A. Therefore, based on the detection result of the signal of labeling substance D2, wells with high signal intensity can be identified as wells in which secretion-producing cells that produce the target secretion are captured.

[0058] According to the screening method of the present embodiment described above, the screening cells are stored in the wells one cell at a time, and the secretion assay is performed, so that the cells do not move during the assay operation. In addition, the detection of the target secretion is performed in each well using detection particles, so that the distance and time required for the target secretion secreted from the screening cell to be captured by the detection particles is shortened. Therefore, compared to the case where the target secretion is detected outside the well, the diffusion of the target secretion is suppressed, and the target secretion can be reliably captured by the detection particles. Therefore, it is possible to accurately identify and obtain the cells that produce the target secretion. Furthermore, since the well 50 has a through-hole 52, the detection reagent that is not captured by the detection particles flows out from the through-hole 52 together with the liquid in the well 50. Therefore, the secretion-producing cells that produce the target secretion can be identified based on the difference in signal intensity of the labeling substance D2.

[0059] <Other processes> The screening method of this embodiment may include other steps in addition to the above steps A to D. Examples of other steps include, but are not limited to, a washing step (step E) and a secretion-producing cell recovery step (step F).

[0060] <Cleaning process (Process E)> In the washing step, the liquid in the well 50 is discharged from the through-hole 52, and then an appropriate liquid (buffer solution, culture medium, etc.) is supplied to the well 50 to replace the liquid in the well 50.

[0061] 7A and 7B are diagrams for explaining an example of the cleaning step. Fig. 7A shows an example in which the cleaning step is performed after step C. Fig. 7B shows the state after the cleaning step. After step C, if secretion A secreted from screening cell C is the target secretion, most of secretion A is captured by detection particles B, and detection reagent D is captured by detection particles B via secretion A. However, it is believed that some of detection reagent D remains free (part of which is bound to secretion A) in well 50. Also, if secretion A is not the target secretion, all of detection reagent D remains free in well 50. In this state, when the liquid in well 50 is discharged from through-hole 52, the free detection reagent D is also discharged out of well 50 together with the liquid. This makes it possible to remove detection reagent D that remains free in well 50. Thereafter, an appropriate liquid (buffer, medium, etc.) is supplied to the wells 50 to prevent the screening cells C from drying out.

[0062] By carrying out the washing step after step C, almost no free detection reagent D (detection reagent D not captured by detection particles B) remains in well 50, as shown in FIG. 7B. In wells 50 in which secretion A secreted from screening cells C is not the target secretion (e.g., wells 50-2, 50-3 in FIG. 4), almost no detection reagent D remains in well 50. Therefore, the difference in signal intensity of labeling substance D2 between wells in which secretion A is the target secretion and other wells becomes clearer.

[0063] For example, Figure 14(B) shows a fluorescence microscope photograph of a well in which a washing step was performed after steps A to C in the example. In Figure 14(B), it can be seen that the fluorescent signals in wells other than the well indicated by the arrow have almost disappeared. As a result, it is possible to identify wells in which secretion-producing cells that produce the target secretion are captured more clearly than in Figure 14(A).

[0064] In the washing step, the liquid in the well 50 may be discharged and the liquid may be supplied to the well 50 multiple times. By discharging and supplying the liquid multiple times, it is also possible to almost completely remove the free detection reagent D remaining in the well 50.

[0065] The washing step may be performed after step B. By performing the washing step after step B, free secretion A that is not captured by detection particles B can be removed from inside the well 50. As a result, when the detection reagent D is supplied to the well 50, less detection reagent D reacts with the free secretion A, and therefore the amount of detection reagent D used can be reduced.

[0066] Discharge of liquid from through-hole 52 can be performed, for example, by removing liquid from a flow path disposed below through-hole 52. For example, when the flow path is filled with liquid, the inside of well 50 is also maintained filled with liquid. When liquid is removed from the flow path, gravity causes the liquid in well 50 to flow out of through-hole 52, and ultimately most of the liquid in well 50 can be removed. Removal of liquid from the flow path may be performed by sucking liquid from a suction hole communicating with the flow path.

[0067] <Secretory cell recovery process (process F)> In the secretion-producing cell recovery step, screening cells C are recovered as secretion-producing cells that produce the target secretion from the wells identified in step D. Recovery of screening cells C can be performed using a known single cell recovery means. Examples of single cell recovery means include methods of recovering cells using a manipulator, a microcapillary, a micropipette, or the like.

[0068] (Screening kit for secretory cells) A second aspect of the present invention is a screening kit for secretion-producing cells, which screens for cells that produce a target secretion from a plurality of cells. The screening kit comprises a device including a plurality of wells, and carrier particles. The well has a size that allows the cells to be captured one by one while capturing one or more of the carrier particles, and has a through-hole at the bottom that is large enough that the cells cannot pass through. The carrier particles are large enough that they cannot pass through the through-hole. The screening kit according to this embodiment can be used in the screening method according to the first aspect.

[0069] <Device> The device includes a plurality of wells having through-holes at the bottom. Examples of the plurality of wells and through-holes include those listed in the above section (Screening method for secretory cells). Examples of devices including a plurality of wells include those having a configuration like device 100 in FIG. 1.

[0070] The device preferably has a flow path connected to the through-hole and disposed below the through-hole. The flow path disposed below the through-hole is preferably a flow path provided along the outer bottom surface of the multiple wells. By having a flow path below the through-hole, liquid in the well can be discharged via the flow path and the through-hole. In addition, by providing a suction hole communicating with the flow path, liquid in the well can be discharged more smoothly by suction from the suction hole.

[0071] <Specific device examples> 8 to 13 show an example of a device having the above-mentioned configuration. The device 1 has a bottom plate 2, a cell mounting membrane (cell mounting portion) 6 provided on the bottom plate 2 and constituting a cell mounting surface 4, a pair of liquid inflow / outflow portions 8 provided on the bottom plate 2 separately from the cell mounting membrane 6, and a flow path 10 provided between the bottom plate 2 and the cell mounting membrane 6, the flow path 10 having a flow path end portion 12 extending to the liquid inflow / outflow portion 8. A plurality of wells 50 and through holes 52 leading from the inner bottom surface of the wells 50 to the flow path 10 are formed on the cell mounting surface 4 of the cell mounting membrane 6. Furthermore, a foam discharge surface 17 is formed on the back side of each lid portion 14, i.e., on the ceiling surface of the flow path end portion 12, which rises in an inclined plane as it approaches the suction hole 16A.

[0072] The bottom plate portion 2 is a long and narrow rectangular plate with a constant thickness, and the four corners are rounded. A protrusion 2B of a constant height is formed along the entire circumference of the bottom surface of the bottom plate portion 2. The shape of the bottom plate portion 2 is not limited to that shown in the figure, and may be any shape such as a disk shape, an ellipse shape, or a square shape, and the thickness of the bottom plate portion 2 does not need to be constant as long as a horizontal flow path 10 can be formed. When steps C and D of the screening method according to the first embodiment are performed under a microscope, it is preferable that the bottom plate portion 2 has a shape that can be placed on the stage of the microscope (for example, a shape similar to a slide glass). In general, it is desirable that the bottom plate portion 2 is formed of various plastics that are harmless to cells in terms of molding accuracy and cost, but if necessary, it may be formed of any material such as ceramic, glass, or metal. The surface of the bottom plate portion 2 may be coated with some kind of coating to reduce the effect on the cells.

[0073] As shown in Fig. 10, an engagement wall 18 having a rectangular shape with both ends semicircular in plan view is integrally formed on the upper surface of the bottom plate portion 2 so as to surround the center and stand vertically from the bottom plate portion 2. The shape of the engagement wall 18 is not limited to that shown in the figure, and it may be a simple rectangular shape, a circular shape, an elliptical shape, or the like. The height of the engagement wall 18 in this example is made uniform all around. An outer frame body 20 is removably attached to the engagement wall 18 so as to cover the engagement wall 18 from above all around.

[0074] The outer frame 20 has a rectangular peripheral wall 22 with semicircular ends in plan view, and a pair of partitions 28 arranged parallel to each other on the inner peripheral side of the peripheral wall 22, and is integrally formed as a whole. There are no limitations on the material of the outer frame 20, and it is generally preferable to form it from various plastics that are harmless to cells in terms of molding accuracy and cost, but it may be formed from any material such as ceramic, glass, metal, etc., as necessary. A rectangular parallelepiped space is provided between the partitions 28, and the cell-mounting membrane 6 is disposed therein.

[0075] The upper end of the peripheral wall 22 of the outer frame 20 has a cross-sectional shape that is folded back outward all around, and a narrow engagement groove 24 that opens downward is formed on the inside of this folded back portion at a constant depth all around. The upper end of the engagement wall 18 is inserted into this engagement groove 24 all around, and is elastically fastened by the folded back portion of the peripheral wall 22, thereby removably fixing the outer frame 20 to the engagement wall 18. Horizontally protruding projections 26 are formed on both ends of the outer frame 20 in the longitudinal direction, and by lifting these projections 26 with fingertips, the engagement wall 18 comes out of the engagement groove 24, allowing the bottom plate 2 and the outer frame 20 to be separated.

[0076] The semicircular regions surrounded by the peripheral wall 22 of the outer frame 20 and the partitions 28 each serve as a liquid inlet / outlet section 8. In each of these liquid inlet / outlet sections 8, a lid section 14 is formed that is three-dimensional and semicircular in plan view with the center raised upward so as to connect the lower end of the peripheral wall 22 and the lower ends of the partitions 28.

[0077] Between the lid 14 and the bottom plate 2, channel ends 12, which are both ends of the channel 10, are formed, and the lid 14 is structured to airtightly close the channel ends 12. By forming the lid 14 in this manner, the channel ends 12, which are both ends of the channel 10, are sealed, and even if the device 1 is tilted or shaken, excessive left-right flow of liquid through the channel 10 and the channel ends 12 is prevented.

[0078] In this embodiment, a circular suction hole 16A is formed in each of the lids 14 approximately at the center, and cylindrical suction ports 16 are formed upright in the lids 14 corresponding to the suction holes 16A. The surface of the lids 14 around the suction ports 16 serves as a liquid storage area, where liquid spilling over from the suction ports 16 can be stored. A recess may be intentionally formed around the suction ports 16 on the top surface of the lids 14 to serve as the liquid storage area.

[0079] On the back side of each lid portion 14, i.e., on the ceiling surface of flow path end portion 12, there is formed a foam discharge surface 17 that rises in an inclined manner as it approaches suction hole 16A, and foam discharge surface 17 has a truncated cone shape with suction hole 16A as its apex. As a result, when flow path end portion 12 is filled with liquid such as a dispersion liquid and contains air bubbles, the air bubbles move smoothly by buoyancy along foam discharge surface 17 toward suction hole 16A and are discharged from suction hole 16A.

[0080] In this embodiment, the thickness of the lid 14 is almost constant, so the upper surface of the lid 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 has a surface that inclines downward as it moves away from the suction port 16, so that liquid spilled from the suction port 16 collects on the periphery of the lid 14 away from the suction port 16, reducing the risk of contamination caused by re-flowing from the suction port 16. However, this is not limited to the configuration, and it is also possible to make the upper surface of the lid 14, i.e., the upper surface of the liquid storage portion, horizontal by making the thickness of the lid 14 thicker as it moves away from the suction port 16.

[0081] In this embodiment, the suction port 16 stands upright from the lid portion 14, but instead, the standing suction port 16 may not be formed in the lid portion 14, and the suction hole 16A may be left open as it is.

[0082] At the lower end of the partition 28 on the liquid inflow / outflow section 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 this step 34, two ribs 30 are formed extending in the vertical direction and reaching the upper end of the partition 28. The ribs 30 increase the bending strength of the partition 28. In addition, the partition 28 is formed with an engagement groove 32 of a certain depth that opens onto the lower surface of the step 34, and an engagement ridge 42 adjacent to the cell-mounting membrane 6 side of this engagement groove 32.

[0083] A square cylindrical frame 36, which is rectangular in plan view, is removably housed in a square space surrounded by the two linearly extending portions of the peripheral wall 22 and the two partitions 28. The cell mounting membrane 6 is stretched over the entire surface of the lower end of the frame 36 with the wells 50 facing upward, and the lower end of the frame 36 and the cell mounting membrane 6 are joined without any gaps around the entire circumference. The frame 36 is made of flexible plastic or the like, and when a force is applied to spread it outward, the four walls spread outward slightly, applying tension to the cell mounting membrane 6 and preventing the cell mounting membrane 6 from sagging.

[0084] The thickness of the cell-mounting membrane 6 is not limited, but from the viewpoint of forming wells 50 capable of capturing screening cells on a cell-by-cell basis and forming minute through-holes 52 through which liquid flows from the bottom of the wells 50 to the back side, the thickness is preferably about 5 to 100 μm, and more preferably about 10 to 50 μm. The cell-mounting membrane 6 may be a multi-layer membrane of two or more layers. In this case, a through-hole that will become the well 50 may be formed in the upper layer, and a through-hole that will become the through-hole 52 may be opened in the lower layer, and these two layers may be attached together to form the well 50 and the through-hole 52.

[0085] The material of the cell-mounting membrane 6 is not limited, and it is generally desirable to form it from various plastics that are harmless to cells in terms of molding accuracy and cost, but if necessary, it may be formed from any material such as ceramic, polycrystalline or single crystal silicon, inorganic compounds such as glass, metal, etc. The wells 50 and through-holes 52 can also be formed by etching, photolithography, etc.

[0086] The planar shape and size of the well 50 may be those exemplified above in the section (Method for screening secretion-producing cells). A plurality of types of frames 36 having cell-mounting membranes 6 with wells 50 of different sizes may be prepared and combined with a common bottom plate portion 2 and outer frame 20 to form the device 1. The shape and size of the through-hole 52 may be those exemplified above in the section (Method for screening secretion-producing cells).

[0087] 11 and 12, the outer peripheral surface of the lower end of the frame body 36 is folded back upward to form an upwardly opening engagement groove 44 and a downwardly protruding engagement rib 40 around the entire circumference. The depth of the engagement groove 44 and the vertical width of the engagement rib 40 are substantially constant around the entire circumference of the frame body 36. The engagement rib 40 is inserted into the engagement groove 32 formed on the lower surface of the outer frame body 20, and the engagement rib 42 of the outer frame body 20 is inserted into the engagement groove 44 of the frame body 36. With these fittings, the frame body 36 is fixed to the outer frame body 20 with the frame body 36 accommodated in the central space of the outer frame body 20.

[0088] At this time, the lower end surface of the frame 36 abuts against the upper surface of the spacer 46 formed on the bottom plate 2, and the amount of separation of the cell mounting membrane 6 from the bottom plate 2, i.e., the thickness of the flow path 10, is accurately determined by the thickness of the spacer 46. In this embodiment, as shown in FIG. 13, a pair of spacers 46 having a U-shape in plan view are formed on the inside of the engagement wall 18 along the shape of the lower end of the frame 36, and a notch 47 is formed between the spacers 46. When the frame 36 is fixed on the bottom plate 2, liquid flows from the flow path 10 to each flow path end 12 through these notches 47. The spacer 46 does not have to have the shape shown in the figure, and it is sufficient that it abuts against the lower surface of the frame 36 at several points. In some cases, the height of the frame 36 from the bottom plate 2 may be accurately determined by engagement with the outer frame 20 without forming the spacer 46.

[0089] An inclined surface 38 having a constant width is formed on the inner peripheral surface of the lower end of the frame 36, and the inclined surface 38 protrudes toward the cell-mounting membrane 6 as it goes downward. By forming such an inclined surface 38, when secretion-producing cells are collected from the wells 50 of the cell-mounting membrane 6 with an instrument such as a manipulator, a micropipette, or a microcapillary, the cells can be easily collected even if the wells 50 containing the secretion-producing cells are located close to the inner peripheral edge of the frame 36. In addition, the inclined surface 38 increases the adhesion area of ​​the cell-mounting membrane 6 to the frame 36, and has the effect of increasing the bonding strength of the cell-mounting membrane 6, and is also useful for increasing the strength of the frame 36.

[0090] When manufacturing a cell screening device having the above configuration, the bottom plate portion 2, frame body 36, and outer frame body 20 are each formed separately as shown in Figure 13, and then the frame body 36 is first fitted onto the underside of the outer frame body 20, and then the outer frame body 20 is fitted into the engaging wall 18 of the bottom plate portion 2, resulting in the completed state as shown in Figures 8 to 12.

[0091] In the process of fitting the frame body 36 to the bottom surface of the outer frame body 20, as shown in Figures 11 and 12, the engaging ridges 42 of the outer frame body 20 fit into the engaging grooves 44 of the frame body 36, and the engaging ridges 40 of the frame body 36 fit into the engaging grooves 32 of the outer frame body 20, so that the two are firmly fixed together by the elasticity of the respective engaging portions. At the same time, the inner peripheral surface of the engaging ridges 40 and the outer peripheral surface of the engaging ridges 42 are shaped so that at least one of them is slightly inclined outward as it goes upward. As a result, as the engagement progresses, the engaging ridges 40 on the four sides of the frame body 36 are pulled outward by the engaging ridges 42 of the outer frame body 20, the bottom end of the frame body 36 is slightly spread out in all directions, generating tension that pulls the four sides of the cell-mounting membrane 6, and a uniform tension is applied to the cell-mounting membrane 6.

[0092] Therefore, even if there is slight slack in the cell mounting membrane 6 when the frame body 36 is in a free state, when the frame body 36 is fixed to the outer frame body 20, the slack in the cell mounting membrane 6 is eliminated, and the flatness of the cell mounting surface 4 on which the numerous wells 50 of the cell mounting membrane 6 are formed is increased, and by filling the cell mounting surface 4 with a dispersion liquid in which the cells are dispersed and rocking the cell screening device, the screening cells are caused to flow evenly, making it easy to capture the screening cells one cell at a time within the well 50.

[0093] In this embodiment, the flow channel ends 12 located at both ends of the flow channel 10 are closed with the lids 14, and the lids 14 are provided with the suction ports 16 having the suction holes 16A communicating with the flow channel 10. This allows the lids 14 to suppress the movement of liquid in the flow channel ends 12 and the flow channel 10, and allows the liquid to be introduced into and removed from the flow channel 10 through the suction ports 16. Therefore, even if the device 1 is carried or tilted with the screening cells C captured in the wells 50 of the device 1 and the flow channel 10 filled with liquid, the liquid is less likely to move along the flow channel 10 and the flow channel ends 12, and the so-called sloshing phenomenon can be suppressed. This also prevents a problem in which a part of the liquid flows into the wells 50 through the through holes 52, causing the screening cells C and detection particles B stored in the wells 50 to detach.

[0094] In this embodiment, even if liquid overflows from the suction hole 16A of the suction port 16, the liquid storage portion (the peripheral portion of the upper surface of the lid portion 14) receives the liquid and prevents the liquid from re-entering the flow channel 10 from the suction port 16, thereby reducing the risk of contamination from the outside, for example. In addition, since the partition portion 28 is formed between the cell mounting surface 4 and the lid portion 14, even if liquid accumulates in the liquid storage portion 14, the liquid can be prevented from flowing into the cell mounting portion 6.

[0095] Furthermore, in this embodiment, the cell-mounting membrane 6 is positioned in the correct position on the bottom plate portion 2 by abutting the lower end of the frame body 36 against the spacer 46 of the bottom plate portion 2, thereby ensuring the desired flow of liquid between the well 50 and the flow path 10, enabling highly accurate screening.

[0096] In this embodiment, the bottom plate 2 and the outer frame 20 are molded as separate bodies, and simply attaching the outer frame 20 to the bottom plate 2 allows the lid 14 and the peripheral wall 22 to be positioned accurately relative to the bottom plate 2, facilitating assembly of the device 1. In addition, the outer frame 20 can be removed from the bottom plate 2 after use, facilitating maintenance.

[0097] In this embodiment, the bottom plate 2, outer frame 20, and frame 36 are each molded as separate bodies, and simply attaching the frame 36 and outer frame 20 to the bottom plate 2 enables accurate positioning of the cell mounting membrane 6, lid 14, and peripheral wall 22, improving ease of assembly. Furthermore, the frame 36 and outer frame 20 can be removed from the bottom plate 2 after use, facilitating maintenance.

[0098] Furthermore, in this embodiment, the bottom plate portion 2 and the outer frame body 20 are molded as separate bodies, and simply by attaching the outer frame body 20 to the bottom plate portion 2, accurate positioning of the cell placement portion 6, the lid portion 14, the peripheral wall portion 22, and the two suction ports 16 can be achieved, making assembly easy.

[0099] In the device 1, the screening cells C, the detection particles B, and the detection reagent D are supplied to the wells 50 from above the cell mounting surface 4 of the cell mounting membrane 6. The liquid in the wells 50 can be discharged from the through-holes 52. Discharge can be performed more efficiently by suction from the suction holes 16A. Suction from the suction holes 16A may be performed by suctioning the liquid from the suction holes 16A using a micropipette or the like. Alternatively, a suction pump may be connected to the suction port 16, and the suction from the suction holes 16A may be performed using the suction pump.

[0100] <Carrier particles> The carrier particles are the same as the carrier particles B1 described above in the section (screening method for secretion-producing cells). The carrier particles may be carrier particles (detection particles B in the first embodiment) to which a substance (binding substance) capable of binding to the target secretion is immobilized. The substance capable of binding to the target secretion is the same as the binding substance B2 described above in the section (screening method for secretion-producing cells).

[0101] The carrier particles may not have a binding substance immobilized thereon. In this case, the carrier particles are capable of immobilizing a binding substance. The carrier particles may be surface-modified so as to be capable of immobilizing a binding substance, or may be formed of a material capable of immobilizing a binding substance. 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 member of the binding pair, such as a carrier particle coated with streptavidin. When the binding substance is immobilized using a passive adsorption method, the carrier particles may be made of a material that has the property of adsorbing the binding substance. When the binding substance is a peptide or a protein, the carrier particles may be polystyrene particles, gold nanoparticles, or the like. If no binding substance is immobilized on the carrier particles, the user can immobilize any binding substance on the carrier particles.

[0102] <Other configurations> The screening kit of this embodiment may include other components in addition to the above-mentioned device and carrier particles, such as a detection reagent for the target secretion product, a medium or buffer solution, a labeling reagent for the binding substance, and instructions for use.

[0103] The detection reagent for the target secretion product is the same as detection reagent D described above in the section (Screening method for secretion-producing cells). The medium or buffer solution may be the same as those exemplified in the section (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 immobilized on the carrier particle. For example, when the carrier particle is coated with streptavidin, the labeling reagent may be a biotin labeling reagent.

[0104] <How to use> The screening kit of this embodiment can be used to carry out the screening method according to the first aspect, and can be used as described above in the section (Screening method for secretory cells). EXAMPLES

[0105] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0106] <Cell preparation> A gene encoding mouse anti-mouse RANKL monoclonal antibody (hereinafter referred to as "mouse monoclonal antibody") was introduced into 293FT cells. The gene-introduced cells were cultured in MEMα medium supplemented with fetal bovine serum (FBS) for 48 hours and used as cells for secretion assay.

[0107] <Preparation of detection particles> 5 μL of goat anti-mouse antibody (Jackson ImmunoResearch Inc. Code: 115-005-003) was added to 500 μL of Polybead Polystyrene Microspheres (particle size 6.0 μm; 07312, Polysciences; hereinafter referred to as "beads") washed with phosphate buffer (PBS), and reacted overnight at 4° C. to bind the goat anti-mouse antibody to the beads. The beads were washed with PBS to remove unreacted antibody. Next, 1% BSA / PBS was added to the beads, and the beads were reacted at 4° C. for 1 hour, and then the beads were washed with PBS. These were used as detection particles.

[0108] Secretion Assay [Process A] A secretion assay was performed using a device equipped with multiple wells with through-holes of 2 μm diameter 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), and the detection particles were spread over the inside of the wells. Next, the cell culture medium prepared in the above <Cell Preparation> was seeded into the wells, and the cells were trapped in the wells.

[0109] [Process B] Subsequently, Hoechst 33342 (H342, Dojindo Laboratories) and Alexa488-labeled goat anti-mouse IgG antibody (type number "A11001", Life Technologies) were diluted 500-fold with MEMα medium supplemented with fetal bovine serum (FBS) and added to the wells. With the detection particles, cells, and Hoechst 33342- and Alexa488-labeled goat anti-mouse IgG antibodies in the wells, the device was left standing in a CO2 incubator at 5% CO2 concentration and 37°C for 3 hours. This caused the cells to secrete mouse monoclonal antibodies. The secreted mouse monoclonal antibodies were captured by the goat anti-mouse antibodies of the detection particles, and further bound to the Alexa488-labeled goat anti-mouse IgG antibodies. This resulted in the formation of an antigen-antibody complex consisting of the goat anti-mouse antibody-mouse monoclonal antibody-Alexa488-labeled goat anti-mouse IgG antibody.

[0110] [Process C(1)] After leaving the device in a CO2 incubator for 3 hours, the device was observed under a fluorescence microscope (Keyence, model number BZ-9000). Figure 14(A) is a representative fluorescence microscope photograph taken from the bottom side of the well. As a result, the cell nuclei were detected by the blue fluorescence of Hoechst 33342 (Hoechst; arrow). In addition, the mouse monoclonal antibody bound to the detection particle was detected by the green fluorescence of Alexa488 (Alexa; arrow).

[0111] [Process D(1)] From the fluorescent signal detected in step C(1), it was possible to identify the wells in which secretion-producing cells secreting mouse monoclonal antibodies were captured (Merge; arrow).

[0112] [Process E (cleaning process)] The solution in the wells was discharged from the through-holes, and PBS was added to the wells to wash the detection particles, thereby removing unreacted Alexa488-labeled goat anti-mouse IgG antibody. [Process C(2)] The washed device was observed under a fluorescence microscope. Figure 14(B) is a representative fluorescence micrograph taken from the bottom side of the well. As a result, the cell nuclei were detected by the blue fluorescence of Hoechst 33342 (Hoechst; arrow). In addition, the mouse monoclonal antibody bound to the detection particle was detected by the green fluorescence of Alexa488 (Alexa; arrow). In addition, the washing process removed the unreacted Alexa488-labeled goat anti-mouse IgG antibody, reducing the background. As a result, the fluorescent signal of the mouse monoclonal antibody captured by the detection particle became clear.

[0113] [Process D(2)] From the fluorescent signal detected in step C(2), it was possible to identify the wells in which secretion-producing cells secreting mouse monoclonal antibodies were captured (Merge; arrow).

[0114] From the above results, it was confirmed that the screening method of the present invention can screen secretion-producing cells that produce a target secretion product. Furthermore, by performing a washing step, the background was reduced, and the signal of the target secretion product secreted from the secretion-producing cells could be made clearer. [Explanation of symbols]

[0115] 1 Device 2 Bottom plate part 4 Cell placement surface 6 Cell mounting membrane (cell mounting part) 8 Liquid inlet / outlet 10 Flow Path 12 Flow path end 14 Lid (fluid storage section) 16 Suction port 16A Suction hole 17 Foam discharge surface 18 Engagement wall 20 Outer frame 22 Peripheral wall section 24 Engagement groove 26 Protrusion 28 Partition 30 Ribs 32 Engagement groove 34 Step part 36 Frame 36A Wall section 38 Slope 40 Engagement ridge 42 Engagement ridge 44 Engagement groove 46 Spacer 47 Notch 50 wells 50a bottom 52 Through hole A, A-1, A-2, A-3 Secretions C, C-1, C-2, C-3 Screening cells B Beads B1 Carrier particles B2 binding substance B2' Cell membrane protein D Detection Reagent D1 binding substance D2 labeled substance

Claims

1. A method for screening a secretion-producing cell, which screens a cell that produces a secretion of interest from a plurality of cells, comprising the steps of: A step A of capturing the cells and detection particles capable of capturing the target secretion product and having a size that does not allow the cells to pass through a plurality of wells having through-holes at the bottom thereof; A step B of allowing the cells captured in the multiple wells to produce secretions; A step C of detecting the target secretion captured by the detection particles; and step D of identifying a well in which a cell producing the target secretion product is captured from the plurality of wells using the detection result as an index, The well has a size capable of capturing the cells on a cell-by-cell basis while capturing one or more of the detection particles. A method for screening secretion-producing cells.

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

3. 3. The method for screening secretion-producing cells according to claim 1, wherein the detection particles are carrier particles to which a substance capable of binding to the target secretion is immobilized.

4. 3. The method for screening secretory cells according to claim 1 or 2, wherein the detection particle is a cell containing a cell membrane protein having binding ability to the target secretory product.

5. A screening kit for secretory cells for screening cells that produce a secretory product of interest from a plurality of cells, comprising: A device including a plurality of wells and carrier particles, the well has a size that allows the cell to be captured on a single cell basis while capturing one or more of the carrier particles, and has a through-hole at the bottom that is large enough that the cell cannot pass through; The carrier particles have a size that does not pass through the through holes, and have a substance having a binding ability to the target secretion immobilized thereon. Screening kit for secretory cells.

6. The device, The secretion-producing cell screening kit according to claim 5 , further comprising a flow path connected to the through-hole and disposed below the through-hole.

7. The secretion-producing cell screening kit according to claim 6 , wherein the device further comprises a suction hole communicating with the flow path.

8. The screening kit for secretory cell according to any one of claims 5 to 7, wherein the secretory product is an antibody.

Images