JPWO2016140327A1 - Micro chamber array plate - Google Patents

Abstract

A small number of target cells (specific cells) mixed in a huge number of cells existing in a test sample can be detected with high sensitivity and high coverage. A microchamber array plate in which a plurality of microchambers, which are concave portions capable of accommodating various cells, are regularly arranged and formed on the main surface thereof in the same shape. The main surface and the microchamber have a hydrophilic surface, and the microchamber is arranged and formed so that the center of gravity of the opening shape of the recess coincides with the virtual lattice point on the main surface. Moreover, it has an opening shape that remains in 30% or less of the line segments connecting adjacent virtual lattice points.

Description

  The present invention relates to a microchamber array plate, and more particularly to a microchamber array plate that can detect a small number of mixed target cells with high sensitivity and high coverage.

  In a wide range of research fields, it is required to identify specific cells or small groups of cells containing them from samples containing multiple cells of various types. In particular, in the research and development of biotechnology related to medicine and pharmacy, and in the clinical field, a specific cell (for example, a cell having or producing a specific substance, a cell infected with a pathogen, a cancer cell, etc.) ) Is important for the progress of experiments and diagnosis of diseases. However, it is difficult to identify this specific cell or a small group of cells containing the cell when the proportion of the specific cell is low.

  For example, a malaria parasite infection as one of infections caused by infection of red blood cells with plasmodium is detected and diagnosed as follows.

  In the method of observing the presence and extent of infection with malaria parasites under a microscope, blood cells can be smeared on a slide glass, dried, stained with Giemsa, then stained, and observed with a microscope. . However, detection and diagnosis require time and advanced observation techniques, and the detection sensitivity is very low. For example, the detection sensitivity (detection limit) is such that it can barely be detected when at least about 0.01% of all blood cells are infected in a required time of about 40 minutes.

  In addition, in the method using the immunochromatography method, a test kit is also sold so that the test can be easily performed, and the presence / absence and degree of infection can be observed with a simple operation. For example, in the required time of about 20 minutes, the detection sensitivity is comparable to that observed under the microscope described above. On the other hand, combined use with other methods is recommended because false positives are likely to occur.

  Furthermore, in the method using the PCR method, a relatively high detection sensitivity can be obtained unlike the observation under the microscope described above. On the other hand, it takes about 5 hours to obtain the result, and the operation is complicated and an advanced observation technique is required. In addition, depending on the state of the sample and the primers, it may be difficult to set reaction conditions and cannot be detected. Therefore, the PCR method alone is not suitable for infection diagnosis.

  In order to compensate for the above-described drawbacks, a new method for detecting an infectious disease that is quick, simple, and high in detection sensitivity is required, and a method using a microarray chip has been proposed.

  Patent Document 1 discloses a system that can integrate a micro chamber and connect it to a heater or the like to perform a biochemical reaction of a biological sample with high throughput. However, the sensitivity is not so high as to be detected at the cell level, and it is difficult to detect a small number of target cells mixed in a huge number of cells with high sensitivity.

  Patent Document 2 discloses a technique for observing the action of a substance on cells by patterning a polymer on a substrate, fixing a predetermined substance on the substrate, and causing cells to act on the fixed substance. ing. However, it is difficult to fix a fixed number of cells in each pattern, and it is difficult to detect a small number of target cells mixed in a huge number of cells with high sensitivity.

  Patent Document 3 discloses an array chip in which a large number of microwells designed to store one lymphocyte are integrated. However, since only one cell can be introduced into the microwell, it is difficult to screen more than the number of microwells integrated on the array chip. Therefore, it is difficult to detect a small number of target cells mixed in a huge number (1 to 10 million) of cells with high sensitivity. Further, it is extremely difficult to further culture and PCR the introduced cells.

  Furthermore, a new detection means that can be easily used at the bedside or the like is also demanded.

  Patent Document 4 discloses a microchip in which the size of the microchamber (particularly the aspect ratio) and the water contact angle on the chip surface have values in a specific range. According to this, a small number of target cells (specific cells) mixed in a huge number of cells can be detected with high sensitivity.

Specifically, a sample containing a large number of cells is developed on the chip, and the cells are stored in each microchamber. Excess cells are removed (washed) from the microarray chip, and the cells are stored almost uniformly as a monolayer in each microchamber. By having a specific size micro-chamber and a specific water contact angle, even if a measurement sample is given and cells are not stored as a single layer in each micro-chamber, it can be washed into each micro-chamber. Cells can be stored almost uniformly as a single layer. Advantages include (i) that a specific cell that is a detection target is located under another cell by cells that overlap in the microchamber, thereby preventing detection from being difficult. (Ii) a microscope (Iii) It is possible to easily adjust the focus when observing with a confocal laser microscope, and the background can be suppressed with high sensitivity. (Iii) Another type of cell on a cell stored in a monolayer In addition, it is possible to easily and efficiently study the interaction of each cell. The chip disclosed in Patent Document 4 also discloses that an extra cell adsorbed on a portion other than the microchamber can be removed (washed) using an instrument such as a cell scraper.

JP-T-2004-502929 JP 2004-125781 A JP 2004-187676 A International Publication No. 2010/027003

  The chip described in Patent Document 4 can easily wash (remove) cells other than cells stored in a single layer in the microchamber, and as a result, can store cells in the single layer in the microchamber. On the other hand, washing (removing) cells also means not subjecting the cells in the test sample to a test. This is not a major problem when the target cell is present in the test sample with a relatively high probability, but it is detected especially when the target cell is present in the test sample with a very low probability. That is, it can be a risk factor that lowers the comprehensiveness of detection.

  The present invention has been made in view of the situation as described above, and the object of the present invention is to provide a small number of target cells (specific cells) mixed in a huge number of cells present in a test sample. It is an object of the present invention to provide a microchamber array plate that enables detection with high sensitivity and high coverage.

The present invention is a microchamber array plate in which microchambers, which are recesses capable of accommodating various cells, are arranged and formed on virtual lattice points having the same shape and regularly dispersed on the principal surface thereof, The surface and the inner surface of the microchamber have a hydrophilic surface, and the microchamber is arranged and formed so that the center of gravity of the opening shape of the recess coincides with the virtual lattice point on the main surface, and adjacent It has the opening shape which leaves the main surface in 30% or less of the line segment which connects between the centroids of the micro chamber.

According to this invention, after the test reagent containing cells is applied to the microchamber array plate, the remaining of the cells on the upper surface of the microchamber array plate can be suppressed without washing it. In addition, cells can be efficiently stored as a single layer in the microchamber. Therefore, a small number of target cells (specific cells) mixed in a vast number of cells can be reliably captured in the microchamber and detected with high sensitivity, resulting in detection with high coverage. It can be done.

  In the above-described invention, the ratio of the total area occupied by the opening shape may be 50% or more in a region defined by a closed line connecting the center of gravity of the opening shape with a straight line. Furthermore, the hydrophilic surface may have a water contact angle of 40 ° or less. According to this invention, the remaining of the cells on the upper surface of the microchamber array plate can be further suppressed, a small number of target cells can be reliably captured in the microchamber, and this can be detected with high sensitivity. Detection with completeness can be realized.

  In the above-described invention, the virtual lattice points may be formed by sequentially arranging the virtual lattice points on concentric circles centered on one virtual lattice point. Furthermore, the virtual lattice points may have a repeating regular arrangement in which three adjacent points are closest to each other at an equal distance. According to this invention, a specific cell or a small group of cells containing the specific cell can be quantified or semi-quantified in a microchamber more easily.

  In the above-described invention, the opening shape may be a circle or a regular polygon inscribed in the circle. The opening shape may be a regular hexagon, and the edge lines may be arranged in parallel with each other between the adjacent micro chambers. According to this invention, a specific cell or a small group of cells containing the specific cell can be quantified or semi-quantified in the microchamber more easily and more accurately.

  In the above-mentioned invention, the diameter of the circumscribed circle of the regular hexagon may be in the range of 20 to 500 μm. The microchamber may have a tapered side surface that decreases in cross-sectional area toward the bottom. Further, the microchamber may have a depth of 20 μm or more. The microchamber may have a ratio of the depth to the diameter of the circumscribed circle in a range of 0.35 to 1. According to this invention, a specific cell or a small group of cells containing the specific cell can be quantified or semi-quantified in the microchamber more easily and more accurately.

It is a front view of a micro chamber array plate. 1A is an enlarged view of a region 7 in FIG. 1, B is a partially enlarged perspective view of a micro chamber (inverted regular hexagonal trapezoid), and C is a cross-sectional view of the micro chamber (inverted truncated pyramid). It is the elements on larger scale of the area | region 7 of FIG. A: arrangement of microchambers, B: detection of one infected blood cell per microchamber. It is a flowchart which shows the identification method of a cell. It is a microscope picture of a micro chamber array plate. FIG. 7 is a photomicrograph of the microchamber array plate of FIG. 6 in which cells (red blood cells) are held in the microchamber. It is a microscope picture of the microchamber array plate which made water contact angle A: 80 degree | times, B: 25 degree | times, and C: 10 degree | times. It is a microscope picture of the microchamber array plate which stored the erythrocyte in the microchamber from which the major axis and depth of an opening differ. It is a microscope picture of the micro chamber array plate which hold | maintained the cell (white blood cell) in the micro chamber. It is a microscope picture of the microchamber array plate which stored erythrocytes in the microchamber. It is a microscope picture of the microchamber array plate which stored the human leukemia cell (CEM) in the microchamber. It is a microscope picture of the microchamber array plate which stored A: red blood cells and B: human leukemia cells (CEM) in the microchamber. Microchamber spacing and the number of stored cells. It is a microscope picture of the microchamber array plate which stored the human leukemia cell (CEM) in the microchamber. These are the distance between the microchambers and the number of cells remaining on the upper surface.

  As a result of intensive studies by the present inventors, a microchamber array is provided after application of cells by providing a plurality of microchambers in a specific arrangement while providing the characteristics of the microchip disclosed in Patent Document 4 described above. Without washing the plate, it is possible to suppress the remaining of cells on the top surface of the microchamber array plate, and the cells can be efficiently stored as a single layer in the microchamber. It has been found that detection of a small number of target cells (specific cells) mixed in cells with high sensitivity and high completeness can be realized.

Specifically, the microchamber array plate here has a plurality of microchambers that are concave portions capable of accommodating various cells in the same shape and regularly distributed and formed on the main surface. The main surface of the plate and the microchamber have a hydrophilic surface. And in the region A (see FIG. 1) defined by the closed line 4 connecting the centroids of the openings of the microchambers existing at the outermost part of the plurality of microchambers at any point on this upper surface, The plurality of microchambers are arranged so that at least a part of the peripheral edge of the opening of any one of the microchambers overlaps within a circle having a specific diameter centered on the point. And / or the area of the region A defined by the closed line 4 connecting the centroids of the microchamber openings existing at the outermost part of the plurality of microchambers with a straight line is 100%, and the openings of the plurality of microchambers in the region A are When the ratio of the total area occupied is the porosity, the porosity is less than 100% and 50% or more.

That is, a microchamber array plate comprising a plurality of microchambers recessed from the upper surface and capable of storing a large number of cells in the plurality of microchambers,
(I) The microchamber has the following conditions (i) and (ii):
(I) A ratio of (long diameter of microchamber opening: depth) within a range of (1: 0.35 to 1) in each of the plurality of microchambers [where the long diameter of the microchamber opening is Is defined as the length of the longest line segment that passes through the center of gravity of the opening of the microchamber and is defined by the periphery of the opening of the microchamber, and the depth of the microchamber is from the top surface to the microchamber. Defined as the length to the deepest part;
(Ii) The water contact angle of all inner surfaces of the plurality of microchambers and the upper surface of the microchamber array plate is 40 ° or less.
(II) The major axis of all the bottoms of the plurality of microchambers [where the major axis of the microchamber bottom is a line segment that passes through the center of gravity of the microchamber bottom and is defined by the periphery of the microchamber bottom. Defined as the length of the longest line segment] within the range of 20 to 500 μm, and (III) at least one condition in which the microchamber is selected from the group consisting of (iii) and (iv) below With:
(Iii) In the region A defined by the closed line 4 in which the centroids of the microchamber openings existing on the outermost sides of the plurality of microchambers are connected by a straight line, the point is determined at any point on the upper surface. The plurality of microchambers are arranged so that at least a part of the opening periphery of any one of the microchambers overlaps within a circle having a diameter x as a center; and the diameter x is 35 μm;
(Iv) The area of the region A defined by the closed line 4 connecting the centroids of the microchamber openings existing outside the plurality of microchambers by a straight line is defined as 100%, and the openings of the plurality of microchambers in the region A The void ratio is less than 100% and 50% or more when the ratio of the total area occupied by [the outermost microchamber includes all of the plurality of microchambers] A microchamber that is in contact with the closed line 5 in a region a defined by the closed line 5 that has the shortest possible perimeter and is in contact with the periphery of as many microchambers as possible. It can be an embodiment. In addition, all the openings of the microchamber have a major axis in the range of 20 to 500 μm and a depth of 20 μm or more.

A method for identifying a specific cell contained in a test sample or a small group of cells containing the specific cell from a test sample containing a large number of cells,
(A) expanding the cells in the test sample on the micro-chamber array plate, and storing the cells in the test sample in the micro-chamber; and (b) a specific cell is present in each micro-chamber. Determining whether or not,
(C) identifying a cell in a microchamber determined to contain a specific cell as a specific cell or a small group of cells containing the specific cell;
One aspect of the identification method includes:

.
Here, the specific cell may be a mammal-derived cell, an avian-derived cell, or a yeast.

Furthermore, the cell concentration of the test sample containing a large number of cells may be in the range of 1 × 10 ⁶ to 1 × 10 ⁸ cells / ml. Moreover, the number of cells in the test sample containing the large number of cells may be in the range of 1 × 10 ⁵ to 1 × 10 ⁸ .

And between the said process (a) and (b), the process of removing the cell which remain | survives on the upper surface of a micro chamber array plate can also be not included.

  According to such a microchamber array plate, the cells contained in the test sample can be stored in the microchamber without being wasted. In addition, according to the above-described identification method, compared to the conventional identification method, detection of a small number of target cells (specific cells) mixed in a huge number of cells with high sensitivity and high completeness is realized. It can be done.

  Details will be described below.

1. Microchamber Array Plate A microchamber array plate (hereinafter referred to as “MCAP”) as one embodiment of the present invention will be described with reference to FIGS.

  As shown in FIG. 1, the MCAP 1 includes a plurality of micro chambers (hereinafter referred to as “MC”) 2 as recesses recessed from the upper surface, and a large number of cells can be stored in the plurality of MCs 2.

As shown in FIG. 3, all of the plurality of MCs 2 have a ratio in the range of 1: 0.35 to 1 at the major axis (circumferential circle diameter): depth 2 b of the opening 2 c of the MC 2. Moreover, the water contact angles of all the inner surfaces of the plurality of MC2 and the upper surface of the MCAP1 are 40 ° or less.

  Here, the shape of the MCAP 1 is preferably a plate-like shape based on a thick rectangle, and the shape when the MCAP 1 is viewed in plan (that is, when the MCAP 1 is viewed from directly above the upper surface). In addition to the rectangle, at least one or more corners of the rectangle are rounded or at least one is chamfered. At this time, the length of the long side (the widest part in the interval from one short side to the other short side) is about 70 to 150 mm, the short side (in the interval from one long side to the other long side) The length of the widest portion is preferably about 25 to 90 mm. More preferably, (long side) × (short side) = (73 to 77 mm) × (24 to 27 mm) is preferable. The thickness of the MCAP 1 is about 0.5 to 22 mm, preferably about 0.8 to 1.9 mm. The thickness may be uniform, but a part or all of the upper outer edge 3 may be recessed. .

  Furthermore, referring also to FIG. 4, MCAP1 holds MCAP1 by hand in the range of about 10 to 15 mm inward from the short side of upper outer edge 3, and also fills in information such as the test sample. A frost portion may be provided. The MC2 column may be numbered.

  Referring to FIG. 1 again, it is the upper surface of the MCAP 1 and has the shortest circumference that can contain all of the plurality of MCs 2, and is 35 μm, preferably 35 μm from the closed line 5 that touches the periphery of as many MCs as possible. The area defined by the closed line 8 surrounding the outside of 30 μm, more preferably 20 μm is defined as “the upper surface of MCAP1”. The closed line 8 may overlap at least part of the upper outer edge 3 of the MCAP 1, but when the MCAP 1 has a frost portion, the upper surface of the MCAP 1 does not overlap the frost portion. The area defined by the closed line 6 that surrounds the outside of the closed line 8 by 0.001 to 2.5 mm, preferably 0.05 to 2.0 mm, more preferably 1.0 to 1.5 mm is the upper outer edge 3 of the MCAP 1. Preferably, two orthogonal sides of the upper outer edge 3 of the MCAP 1 are in contact with each other (if the upper outer edge 3 has a shape based on a rectangle, the extended lines of the long side and the short side are orthogonal) More preferably, the two long sides of the upper outer edge 3 of the MCAP 1 and the region defined by the closed line 8 are in contact with each other, more preferably the two long sides of the upper outer edge 3 of the MCAP 1 and the short side perpendicular thereto (the upper outer edge). When the shape 3 is based on a rectangle, the upper side of the MCAP 1 may be arranged so that the region defined by the closed line 6 is in contact with the extended line of the long side and the short side. Also in this case, the region defined by the closed line 6 does not overlap with the frost portion.

  The size of the upper surface of the MCAP1 is a range that can be operated by a human, and a range that can be operated by an adult human with one hand is preferable. When the rectangle is based on a long side (from one short side to the other short side) The length of the widest portion is preferably about 70 to 150 mm, and the length of the short side (the widest portion of the distance from one long side to the other long side) is preferably about 25 to 90 mm. More preferably, it is a slide glass size, and when there is no frost part (long side) × (short side) = (73 to 77 mm) × (18 to 27 mm), or when there is a frost part (long) Side) × (short side) = (58 to 62 mm) × (18 to 27 mm) is preferable. When the upper surface of the MCAP is based on a square, the length of one side (the widest part from one side to the other side facing each other) is about 18 to 150 mm, preferably 20 to 75 mm.

  Moreover, it is preferable that the thickness of MCAP1 corresponding to the upper surface of MCAP1 is uniform.

  Examples of the shape of the space in the MC2 when the upper portion of the space in the MC2 is an extended surface from the upper surface of the MCAP1 include a cylinder, a regular polygonal column, an inverted truncated cone, and an inverted regular polygonal frustum. Preferably, a cylinder, a regular octagonal column, a regular hexagonal column, a regular pentagonal column, a regular quadrangular column, a reverse frustum base, a reverse regular octagonal pyramid, a reverse regular hexagonal pyramid, a reverse regular pentagonal pyramid, a reverse regular square pyramid, A cylinder, a regular hexagonal column, a regular quadrangular column, a reverse frustum base, a reverse regular hexagonal frustum, a reverse regular square frustum, and the like are preferable.

  As shown in FIG. 2, when the shape of the space in MC2 is an inverted frustum trapezoid, when a perpendicular line n passing through the periphery of the opening 2c and perpendicular to the upper surface of MCAP1 is drawn in the cross section of MC2 (see FIG. 2C) The angle P (also referred to as a taper) formed between the perpendicular n and the inner surface of MC2 is greater than 0 ° and not greater than 30 °, preferably greater than 5 ° and not greater than 20 °. The shapes of the bottom 2a and the opening 2c of MC2 depend on the shape of the space in MC2.

  The bottom 2a of MC2 can be a flat surface or a curved surface (eg, convex surface, concave surface). However, in order to arrange cells in a single layer on the bottom 2a of MC2, it is preferably as flat as possible. Moreover, it is preferable that the shape of the space in MC2 is the same shape in one MCAP1.

  Further, the arrangement of MC2 is distributed on a lattice or diagonal lattice (virtual lattice points) in which the centers of gravity of the openings 2c of MC2 are regularly arranged on the main surface of MCAP1 when the upper surface of MCAP1 is viewed in plan view. It is preferable. Here, the lattice shape means that a line segment passing through the center of gravity of the openings 2c of two adjacent MC2s is parallel to the long side or the short side of MCAP1. Further, the diagonal lattice means that the position of the even-numbered column of MC2 is shifted from the position of the odd-numbered column of MC2 by half the position of MC2, or the position of the odd-numbered column of MC2 is the position of the even-numbered column of MC2. MC2 is arranged with a half shift. MC2 is arranged so that the centers of gravity of the openings 2c of MC2 are equally spaced, that is, virtual lattice points are sequentially arranged on a concentric circle centered on one virtual lattice point, and the virtual lattice points are arranged at the virtual lattice points. It is preferable that MC2s having the same center of gravity are arranged.

Here, as shown in FIG. 2A, line segments l1 having virtual lattice points at a constant interval d and line segments l2 obtained by moving the line segment l1 along the longitudinal direction by d / 2 are alternately arranged. With an arrangement of virtual grid points. The interval between the line segment l1 and the line segment l2 is such that three adjacent virtual lattice points can be connected so as to form the equilateral triangle 10, and the virtual lattice point is an equal distance between the three adjacent points. It becomes the repeating rule arrangement which makes it adjoin most. MC2 is arranged and formed so that the center of gravity of the shape of the opening 2c coincides with the grid on the main surface, and less than half of the line segment (one side of the equilateral triangle 10) connecting the adjacent virtual grid points. It has an opening shape in which the interval between the remaining openings is narrowed. For example, in the case of cultured cells up to about 50 μm at most, the opening interval is preferably 50 μm, preferably 40 μm, more preferably 30 μm or less.

  Further, as shown in FIG. 2A, the major axis of the opening 2c of the MC2 is defined as the length of the longest line segment among the line segments passing through the center of gravity of the opening 2c of the MC2 and defined by the periphery of the opening 2c of the MC2. The That is, it matches the diameter of the circumscribed circle P in the shape of the opening 2c. 2B, the depth of MC2 is defined as the length from the upper surface to the deepest part of MC2.

  The upper limit value of the ratio of the major axis of the opening 2c to the depth 2b in MC2 of MCAP1 is 1, but is preferably 0.85, and more preferably 0.8. The lower limit of this ratio is 0.35, preferably 0.4, and more preferably 0.45.

  The lower limit value of the major axis of the opening 2c of MC2 is 20 μm, preferably 30 μm, more preferably 50 μm. Alternatively, based on the ratio of the depth described later and the above (long diameter of the opening 2c of MC2: depth), the lower limit value of the long diameter of the opening 2c of MC2 is any of 20, 23.5, 25, 44, 50, and 57.1 μm. You can choose from. Moreover, although the upper limit of the major axis of the opening 2c of MC2 is 500 μm, it can be set appropriately from any one of 425, 400, 225, 200, and 175 μm according to the major axis of the opening 2c of MC2. The range of the major axis of the opening 2c of MC2 can be set by appropriately combining these upper limit value and lower limit value.

  The short diameter of the opening 2c of the MC2 is defined as the length of the shortest line segment among the line segments that pass through the center of gravity of the opening 2c of the MC2 and are defined at the periphery of the opening 2c of the MC2. The short diameter of the opening 2c of MC2 is not particularly limited as long as cells can be stored in MC2. Moreover, you may set from the shape of the opening 2c, and the long diameter of the opening 2c of MC2. However, in order to store cells in MC2, at least the lower limit of the minor axis is preferably 20 μm or more, preferably 50 μm or more, more preferably 80 μm or more. The upper limit value of the minor axis can be set according to the major axis of the opening 2c of MC2. In addition, when the shape of the opening 2c is circular, the major axis and the minor axis are equal.

  The depth of MC2 can be determined to be a preferable value if the value of the long diameter of the opening 2c of MC2 is determined by the ratio of (the long diameter of the opening 2c of MC2: depth) described above. However, since MC2 stores cells, it is preferable to have a depth of 20 μm or more. That is, it is particularly preferable that the long diameter and the depth of the opening 2c of the MC 2 have the above-described ratio, the long diameter has the above value, and the depth is 20 μm or more.

  The water contact angle of all inner surfaces of MC2 and the upper surface of MCAP1 is 40 ° or less, preferably 10 ° or less, more preferably 8 ° or less, and further preferably 5 ° or less.

  The water contact angle is measured as follows. The water contact angle is measured using a known method θ / 2. The θ / 2 method is a method for obtaining the contact angle from the angle of the straight line connecting the left and right end points and the vertex of the droplet with respect to the solid surface. For example, when obtaining the water contact angle on the surface of MCAP1, distilled water is dropped on a plurality of different locations on the surface, and the contact angles of the dropped liquid droplets are obtained. The value obtained by this can be called a water contact angle. For example, according to the above method, using a contact angle meter (manufactured by Kyowa Interface Science Co., Ltd.), 1 to 2 μl of distilled water is dropped at a plurality of different points (more than 5 points) of MCAP1 to measure the water contact angle. it can.

Furthermore, the major axis of all the bottom portions 2a of the plurality of MC2 is in the range of 20 to 500 μm. Here, the major axis of the bottom 2a of the MC2 is defined as the length of the longest line segment among the line segments that pass through the center of gravity of the bottom 2a of the MC2 and are defined by the periphery of the bottom 2a of the MC2.

  The major axis of the bottom 2a can be appropriately set according to the major axis of the opening 2c of the MC2, the depth 2b of the MC2, and the taper angle P.

  Specifically, as shown in FIG. 2C, the distance from the intersection of the perpendicular line n and the horizontal line m drawn horizontally so as to contact the outer edge of the bottom of MC2 to the inner side surface of MC2 is represented by Δw.

  The length of Δw is a value obtained by multiplying the depth 2b of MC2 by tanP. For example, if the depth of MC2 is 20 μm and the taper angle P is 20 °, the length of Δw is about 7.3 μm. The major axis of the bottom 2a of MC2 having the inverted frustum-shaped space is a value obtained by subtracting the value obtained by multiplying Δw by 2 from the major axis of MC2.

  The minor axis of the bottom 2a of the MC2 is defined as the length of the shortest line segment among the line segments that pass through the center of gravity of the bottom 2a of the MC2 and are defined at the periphery of the bottom 2a of the MC2. The short diameter of the bottom 2a of MC2 is not particularly limited as long as cells can be stored in MC2. Moreover, you may set from the shape of the bottom part 2a, and the long diameter of the bottom part 2a of MC2. However, in order to store cells in MC2, at least the lower limit of the minor axis is preferably 5 μm or more, preferably 10 μm or more, more preferably 20 μm or more. The upper limit value of the minor axis can be set according to the major axis of the bottom 2a of MC2. In addition, when the shape of the bottom part 2a is circular, a major axis and a minor axis become equal.

Here, as shown in FIG. 1, in a region A defined by a closed line 4 in which the centers of gravity of the openings 2c of MC2 existing outside the plurality of MC2 are connected by a straight line, at any point on the upper surface. A plurality of MC2s are arranged so that at least a part of the periphery of the opening 2c of any MC2 overlaps within a circle 9 (see FIG. 3) having a diameter x centered on the point, and the diameter x is 35 μm.

  Further, as shown in FIG. 1, the area of the region A defined by the closed line 4 that connects the centroids of the openings 2c of the MC2 existing outside the plurality of MC2 by a straight line is defined as 100%, When the ratio of the total area occupied by the openings 2c of MC2 is defined as the porosity, the porosity is less than 100% and 50% or more.

  Here, the outermost MC2 has the shortest circumference that can include all of the plurality of MC2 and is closed within a region a defined by the closed line 5 that touches the periphery of as many MC2 as possible. 5 is defined as MC2 in contact with 5.

  The lower limit of the diameter x is not particularly limited as long as it is larger than 0 μm, but is preferably 5 μm, more preferably 10 μm, still more preferably 20 μm, and still more preferably 30 μm. When the diameter x is 40 μm or more, the ratio of cells remaining between MC2 and MC2 increases when the test sample is developed, which is not preferable, and the upper limit of the diameter x is preferably 35 μm or less.

  The upper limit of the porosity is not particularly limited as long as it is smaller than 100%, but is preferably 90%, more preferably 80%, and further preferably 70%. When the porosity is 50% or less, the ratio of remaining cells between MC2 and MC2 becomes high when developed, and the lower limit of the porosity is preferably 50% or more.

  MCAP1 can store cells uniformly in a single layer due to the shape of MC2 described above, and can efficiently store cells in MC2 when a test sample containing cells is expanded on MCAP1 by the arrangement of MC2. it can. In other words, by narrowing the interval between MC2 and MC2, the proportion of cells remaining on the interval can be lowered, and a small number of target cells (specific cells) mixed in a huge number of cells are highly sensitive and have high coverage. Can be detected.

  Further, in the area A defined by the closed line 4 in which the centroids of the openings 2c of the MC2 existing on the outermost part of the plurality of MC2 are connected by a straight line, any point on the upper surface is specified with the point as the center. A plurality of MC2s are arranged so that at least a part of the periphery of the opening 2c of any MC2 overlaps within a circle having a diameter of and / or the opening 2c of the MC2 existing at the outermost part of the plurality of MC2 When the area of the region A defined by the closed line 4 in which the centers of gravity are connected by a straight line is 100% and the ratio of the total area occupied by the openings 2c of the plurality of MC2 in the region A is the porosity, the porosity is 100. By making it less than 50% and more than 50%, it is possible to suppress the remaining of cells on the upper surface of MCAP1 without washing MCAP1 after application of the cells, and to efficiently make the cells into a monolayer in MC2. In storage That. This makes it possible to quantify or semi-quantify specific cells or a small group of cells containing the specific cells contained in a test sample that can quickly estimate the number of cells analyzed in one MCAP1. It becomes. Here, semi-quantitative means obtaining an approximate content or content of specific cells contained in the test sample.

  Here, it is preferable that one MCAP1 can hold as many cells as possible. The cells are retained in MCAP1 means that cells are stored in MC2 provided in MCAP1, and MCAP1 can retain more cells as the area of area A is larger and the porosity is higher. .

  More specifically, the number of cells stored in one MCAP1 is specifically at least 10,000 or more when the number of cells stored in all the chambers existing in one MCAP1 is added, preferably More than 100,000 cells, more preferably more than 1 million cells, still more preferably more than 10 million cells.

  For example, when identifying target cells that can be present at a ratio of 1 to 1 million cells, it is preferable that one MCAP1 can hold 1 million or more cells. More preferably, it can carry 2 million to 10 million cells, more preferably 3 million to 10 million cells. In this case, it is particularly preferable to provide 10,000 or more chambers in one MCAP1.

  In addition, it is preferable that at least 10 cells can be stored in one MC2, more preferably 50 to 500 cells, and even more preferably 50 to 300 cells can be stored.

  For example, when 100,000 MC2s are provided in one MCAP1, and about 10 cells are stored in each MC2, about 1 million cells are held per MCAP.

  Materials for MCAP1 are polystyrene, polyethylene, polypropylene, polyamide, polycarbonate, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), cyclic olefin copolymer (COC) and other polymers, silicon and other metals, glass, quartz glass, Examples include a combination of a plurality of materials such as a polymer and glass or metal laminated together (for example, PDMS and glass fiber). Preferred are polystyrene, PMMA, glass, silicon and the like.

  MCAP1 may or may not have translucency, but preferably has translucency, and when MCAP1 is observed with an inverted microscope, at least the bottom of MC2 is It preferably has translucency.

2. Method for Producing Microchamber Array Plate The method for producing MCAP1 is not particularly limited, and can be produced according to a known method.

  MCAP1 can also be produced by a method of directly processing MC2 on a plate-like substrate of a predetermined material, a method of attaching a film having micro through holes or MC2 formed on these substrates, or plastic molding. Preferably, plastic molding is used. Specifically, MC2 is a microfabrication technique in the field of semiconductor research, such as a lithography method (optical lithography, electron beam lithography, etc.), a drilling technique for drilling holes using a microdrill, laser processing, etc. It can be produced by a technique for producing a hole.

  The method for producing MCAP1 by plastic molding is not particularly limited, but a method in which a mold for MCAP1 is produced in advance and molded by injection molding or the like is preferable. The method for producing the mold is not particularly limited, but a method of producing a plastic master by lithography and etching and then producing a mold for MCAP1 by electroforming to the master is exemplified. More specifically, production of a plastic master by X-ray lithography and etching, which is one of electron beam lithography methods, electroforming (plating) of nickel or the like on the master, and injection molding method using the mold According to the LIGA (Lithographie Galvanoformung Abformung) process for producing a plastic structure by combining the above, MCAP1 made of the above polymer (plastic) can be produced.

  In order to make the bottom of MC2 flatter, a method of molding MCAP1 by injection molding after forming a mold by a LIGA process or the like is preferable.

  It is preferable that the water contact angles of all inner surfaces of MC2 in MCAP1 and the upper surface of MCAP1 are 40 ° or less. In other words, it is preferable to make all the inner surfaces of MC2 and the upper surface of MCAP1 hydrophilic. When highly hydrophobic polystyrene, polyethylene, polypropylene, polyamide, polycarbonate, PDMS, PMMA, COC, etc. are used as the MCAP1 substrate, a hydrophilic treatment is performed as necessary to achieve the hydrophilic surface. Can do. Examples of the hydrophilic treatment method include plasma treatment and corona discharge treatment. Preferable is plasma treatment, and oxygen plasma treatment is exemplified. For example, when the substrate of MCAP1 is a hydrophobic material (polymer such as polystyrene or PMMA), it is preferable to perform hydrophilic treatment such as oxygen plasma treatment. In addition, when the MCAP1 substrate is made of a material having relatively high hydrophilicity (such as silicon or glass) and the water contact angle is 40 ° or less, such treatment is not necessary. In order to enhance, a hydrophilization treatment of a known technique such as polishing may be applied.

3. Method for identifying a specific cell or a small group of cells containing the specific cell The method for identifying a specific cell or a small group of cells containing the specific cell comprises: Is a method for identifying a specific cell or a small group of cells containing the specific cell.

  Here, examples of the test sample include a specimen collected from an individual, a diluted solution of the specimen, a suspension of cells collected from the specimen, or a suspension of cultured cells or cultured cells. For ease of development of the test sample on the upper surface of MCAP1, the test sample is preferably prepared as a cell suspension after diluting the specimen collected from the individual or collecting the cells from the specimen. Examples of specimens collected from individuals include peripheral blood, bone marrow, lymph, cerebrospinal fluid, joint fluid, amniotic fluid, ascites, effusion fluid such as ascites, pleural effusion, biopsy material, surgically removed tissue, and the like. Biopsy material and surgically removed tissue can be obtained by digesting the extracellular matrix using an enzyme such as trypsin or collagenase according to a known method, if necessary, and then performing filter filtration to obtain a cell suspension. it can. Cells contained in the test sample (hereinafter also referred to as “cell group”) may be unicellular organisms such as bacteria, rickettsia, yeast, and protozoa, as well as animal-derived cells or plant-derived cells. For example, mammal-derived cells, avian-derived cells, insect-derived cells, plant cultured cells and the like can be mentioned. The cell group may be composed of the same cell or a plurality of types of cells.

  The target specific cell is, for example, a cell expressing a specific gene or a cell in which biological substances such as nucleic acids, proteins, lipids, and sugars are excessive or insufficient than usual. Can be detected from various cell groups. Such specific cells may be cells that exist in nature or may be cells that have been subjected to artificial treatment. Specific cells include, for example, pathogenic cells, lesion cells, cells infected with pathogenic bacteria or pathogenic organisms, mutated cells, unknown cells having specific properties, and the like.

  Here, examples of the artificial treatment include physical treatment (eg, electromagnetic wave irradiation), chemical treatment (eg: drug treatment), genetic engineering treatment (eg: gene recombination treatment), and the like. Among such artificial treatments, a treatment that has a known effect on cells can be applied to the cell group, and a cell that does not show the effect or a cell that shows the effect more strongly can be identified as a specific cell. For example, cells exhibiting resistance or high sensitivity to drug treatment can be detected as specific cells.

  Here, the small group of cells containing specific cells refers to a group of cells containing specific cells and stored per MC.

  Furthermore, according to this identification method, a specific cell contained in the test sample or a small group of cells containing the specific cell can be identified from a test sample containing a large number of cells.

In the present identification method, the test sample containing a large number of cells means, for example, that the total number of cells stored in MC2 on one MCAP1 is within a range of 0.5 × 10 ^{4 to} 0.5 × 10 ¹⁰ , The test sample is preferably in the range of 0.5 × 10 ^{6 to} 0.5 × 10 ⁸ , more preferably 0.5 × 10 ^{7 to} 2.5 × 10 ⁷ . In this identification method, the cell concentration in the test sample may vary depending on the cell type, and examples thereof include 1 × 10 ⁴ to 1 × 10 ¹⁰ cells / ml. Preferably it is 1 * 10 < ⁶ > -1 * 10 < ⁸ > cells / ml, More preferably, it is 5 * 10 < ⁶ > -5 * 10 < ⁷ > cells / ml.

  When diluting a specimen or preparing a suspension of cultured cells or cultured cells, physiological saline, PBS, α-MEM medium, Dulbecco MEM medium, RPMI 1640 medium, LB broth, YPAD medium, SC medium, etc. It can select suitably according to the cell contained in this test sample. The mammalian cell culture medium may contain 1 to 20% fetal calf serum or the like. Moreover, when it has the property to which cells adhere | attach, EDTA can also be added as needed. The suspension of cultured cells and cultured cells can be prepared according to a known method.

  The identification method includes the following steps S1 to S3 as shown in FIG.

Step S1 includes a step of expanding the cells in the test sample to MCAP1 described in the section “1. Microchamber array plate” and storing the cells in the test sample in MC2. The test sample is adjusted to the cell concentration as necessary, and then developed on the upper surface of MCAP1. The amount of the test sample developed on the upper surface of the MCAP1 is not particularly limited as long as the region A on the single MCAP1 is covered. However, in order to prevent cell loss, the amount of the test sample is 0.3 to ³ per 1 mm ² of the area A. It is preferable to develop so as to be about 0.5 μl. As a method for developing the test sample, it is preferable that the test sample is dropped from above the MCAP 1 onto the upper surface using a micropipette or the like so that the test sample is developed on the region A.

  In addition, when MCAP1 is used after a long time (eg, one day) since its manufacture, a hydrophilic recovery treatment (eg, ultrasonic cleaning) of MCAP1 is performed before the test sample is dropped. It is preferable. When performing ultrasonic cleaning, select appropriately according to the cells contained in the test sample such as physiological saline, PBS, α-MEM medium, Dulbecco MEM medium, RPMI 1640 medium, LB broth, YPAD medium, SC medium, etc. Can do. More preferably, the same liquid as that used to prepare the test sample is used.

  After the test sample is spread on the upper surface of MCAP1, the MCAP is allowed to stand for a certain period of time, preferably 5 to 30 minutes, preferably 10 to 15 minutes. Can be arranged in a single layer.

  Here, it is possible to suppress the remaining of cells on the upper surface of the MCAP1 without washing, and the cells can be efficiently stored in a single layer in the MC2, so that the necessity is small. If desired, step S1 'may be performed after step S1 to remove (wash) excess cells from MCAP1.

  The method for removing excess cells from MCAP1 is not particularly limited as long as it is a washing method that does not agitate the cells in MC2. For example, a method of gently pouring the above-described PBS, cell culture medium, or the like with a micropipette or the like and washing the upper surface of MCAP1 can be mentioned.

  Step S2 includes a step of determining whether or not a specific cell exists in each MC2. For example, when the target specific cell is a cell that undergoes a morphological change, the cell that caused the morphological change can be identified by observing the morphology of the cell under a microscope. The MC in which the specific cell is present can be determined to be the MC in which the specific cell is present. In addition, when there is a substance that specifically binds to the specific cell, the cell bound to the substance is detected by detecting the binding of the substance after acting on the cell group in the test sample. The MC in which the specific cell exists can be determined as the MC in which the specific cell exists. Examples of such substances include cell imaging reagents, staining reagents, antibodies and aptamers. Alternatively, when there is a substrate that specifically reacts with an enzyme or the like contained in the specific cell, the reaction to the substrate can be detected, and the cell in which the reaction is recognized can be determined to be a specific cell, The MC in which the specific cell exists can also be determined as the MC in which the specific cell exists.

  As a method for detecting the presence of a cell imaging reagent, staining reagent, antibody or aptamer binding, or reaction with a specific substrate, it is preferable to use a fluorescent dye. For example, staining a cell group with a fluorescent cell imaging reagent or staining reagent; contacting a specific substrate that emits fluorescence by reacting with a specific enzyme, or specific to an antigen in a specific cell By applying a fluorescent label to an antibody or aptamer that binds to the antibody, and then bringing the antibody or aptamer into contact with a cell group, only specific cells in the test sample can be labeled with a fluorescent dye. The labeled fluorescent dye can be observed with a microscope or a fluorescent scanner. Then, a cell labeled with a fluorescent dye can be determined to be a specific cell, and an MC in which the specific cell is present can be determined to be an MC in which the specific cell is present.

  The step of labeling specific cells with a fluorescent dye may be performed before the test sample is developed on MCAP1, or may be performed after the cells are stored in MC2.

  For example, when blood cells infected with pathogenic microorganisms are detected from blood cells, the nuclei of pathogenic microorganisms in infected blood cells are fluorescently stained before deploying the blood cells to MCAP1, and fluorescence microscopes or array scanners are used. Or the like, the cell in which the fluorescence is detected can be determined to be a specific cell, and the MC in which the specific cell is present can be determined to be the MC in which the specific cell is present. Alternatively, after developing blood cells on MC2, the nucleus of pathogenic microorganisms in the blood cells can be fluorescently stained, and the cells in which the fluorescence is detected can be determined to be specific cells, and the specific cells are present. The MC to be determined may be an MC in which a specific cell exists. For example, when trying to detect infected blood cells on the bedside etc., it is better to fluorescently stain the nuclei of pathogenic microorganisms in the infected blood cells before spreading the blood cells on the microarray chip. Since it becomes simple, infected blood cells can be detected more easily. In addition, for example, it is not limited to the nucleus of pathogenic microorganisms in blood cells. For example, a specific protein (specific amino acid sequence) is fluorescently labeled by using a fluorescently labeled antibody, and a fluorescently labeled probe is used. Specific gene sequences may be fluorescently labeled.

  In addition, when it takes time to detect fluorescence when performing step S2, the upper surface of MCAP1 may be covered with a cover glass or the like in order to prevent the inside of MC2 from drying.

  Step S3 includes a step of identifying a cell in MC2 that is determined to have a specific cell as a specific cell or a small group of cells containing the specific cell.

  This identification method may include a step of measuring the fluorescence intensity or the number of fluorescently labeled cells. By performing this measurement, when the fluorescence intensity within one specific cell is strong, or when there are many fluorescently labeled cells, the expression level of the protein, lipid or nucleic acid in the specific cell is large, or the cell It can be tested that the degree of denaturation or pathogen infection is high. For example, as shown in FIG. 4, it is possible to arrange 50 MCs on the MCAP so as to be 50 vertically and 200 horizontally, and store 100 blood cells in each MC2. When one infected blood cell is detected per one MC, the infection rate can be determined as 1%.

  Further, in this method, for example, even if a microarray chip holds a total of 1 million blood cells and one of the blood cells is infected, this infected blood cell can be detected. In this case, according to the present identification method, it can be said that detection is possible even if the infected blood cells are present only in 0.0001% in the test sample.

  Furthermore, this identification method may further include a step of observing specific cells labeled with a fluorescent dye with a fluorescence microscope or the like. By performing the observation, it is possible to determine the pathogenic bacteria, the type of cells, and the like.

  Furthermore, for example, by using MCAP1, cells that have a specific property by the treatment can be selected from a group of cells that have been artificially treated.

  In addition, by using MCAP1, cells having the specific property can be selected particularly strongly. More specifically, for example, cells that produce the substance can be detected particularly efficiently from a plurality of cells transformed to produce a particular substance by genetic recombination treatment. Alternatively, for example, cells that have lost a specific function due to electromagnetic wave irradiation treatment or drug treatment can be detected. In addition, cells having resistance or high sensitivity to these treatments can be detected.

  Moreover, this detection method can be suitably used not only in a laboratory but also in a clinical site, for example.

  Moreover, according to this identification method, a cell is stored in the chamber provided in MCAP1. For this reason, in the detection method of the present application, at the time of detection, it is easy to understand in which part of the MCAP1 the target specific cell exists. From this, after the detection, the specific cells can be easily collected and further analyzed by the PCR method.

  Further, in this detection method, it is possible to store cells uniformly and efficiently in each MC2 provided in MCAP1. That is, in the detection method of the present application, when the MCs included in the MCAP 1 have the same shape, approximately the same number of cells can be stored.

  By this identification method, it is possible to preferably detect infected blood cells particularly in infectious diseases. Examples of infectious diseases include protozoan infections represented by malaria parasites, viral infections represented by human immunodeficiency virus (HIV), bacterial infections represented by Mycobacterium tuberculosis, and the like.

  Among these infectious diseases, for example, in infections caused by malaria parasites, malaria parasites infect red blood cells. In this case, the specific cells to be detected in this detection method are blood cells infected with malaria parasites, that is, red blood cells infected with malaria parasites. The malaria parasites are exemplified by four types of Plasmodium falciparum, Plasmodium falciparum, Plasmodium falciparum and oval malaria parasites.

  For example, in HIV infection, HIV infects certain lymphocytes (T cells). In this case, the specific cells to be detected in this detection method are blood cells infected with HIV, that is, certain lymphocytes (T cells) infected with HIV. Examples of HIV include two types, HIV-1 and HIV-2.

  For example, in tuberculosis, certain types of white blood cells (monocytes) are infected with M. tuberculosis. In this case, the specific cells to be detected in this detection method are blood cells infected with M. tuberculosis, that is, certain white blood cells (monocytes) infected with M. tuberculosis.

  This identification method can also detect infected blood cells in other infectious diseases.

  Examples of blood cells are cells contained in blood, and examples of blood cells include red blood cells, platelets, and white blood cells (lymphocytes, monocytes, neutrophils, eosinophils, basophils). For example, a test sample containing a plurality of these blood cells can be used as a test sample containing a plurality of cells for use in the present detection method. For example, blood or body fluid containing a plurality of blood cells can be used as the test sample.

  This identification method can also detect specific cells in cultured cells. The cultured cells are preferably exemplified by cell lines derived from mammals or insects (for example, HEK293 cells, Hela cells, 3T3 cells, COS-7 cells, CHO cells, Jurkat cells, Sf9 cells, etc.) and yeast.

  In this identification method, even if the target specific cell is present in a very small amount in the test sample (for example, even if the test sample is present in the cell in a proportion of 0.001 to 0.00001%), the identification It is particularly advantageous when using a test sample in which the presence ratio of specific cells serving as detection targets is extremely small. As such an example, for example, cancer cells circulating in the blood include circulating tumor cells (CTC). Moreover, for example, fetal nucleated red blood cells present in the blood of pregnant women can be mentioned. Moreover, for example, cancer stem cells present in a cancer cell group can be mentioned. Cancer stem cells have the ability to self-replicate and proliferate, are present in very few malignant tumors (cancers), and express the same markers (eg, CD34, CD133, CD117, Sca-1 etc.) as stem cells According to this identification method, it can be preferably detected.

  A cell expressing a specific gene or protein can also be an application target of this identification method.

4). Identification kit The identification kit is a kit for detecting specific cells from a test sample containing a plurality of cells, includes MCAP1, and the following are exemplified.

  The identification kit is preferably a buffer solution, a culture solution, and the like that can be used when contacting cells with MCAP1, and further a buffer solution, a culture solution, a surfactant, and the like that can be used when expanding cells into MCAP1. Further, a buffer solution, a culture solution, a surfactant, a washing solution for enzymes, etc. that can be used when removing (washing) excess cells from MCAP1 can be included. In addition, a micropipette or the like that can be used when contacting and expanding cells with MCAP1 and further removing cells from MCAP and the like can be included.

  Although the necessity is small because the remaining of cells on the upper surface of MCAP1 can be suppressed, if necessary, the identification kit can be further used to remove and wash excess cells adsorbed on portions other than MC2. Instruments such as a cell scraper may be included. In addition, a device for detecting a specific cell as a detection target can be included, and examples of the device include a fluorescence microscope and a microarray scanner. In addition, for example, when a specific cell as a detection target is finally detected by fluorescent staining, a fluorescent staining agent (for example, a fluorescent dye, a fluorescently labeled antibody, etc.) for staining the specific cell is included. It may be.

  Further, a manual for using the identification kit may be included.

  By using such an identification kit, even if the proportion of specific cells that are detection targets is low in the test sample (for example, only 0.0001% is present), the specific cells can be quickly and easily It is possible to detect cells.

5. Drug candidate substance screening method The above-mentioned MCAP1 can be preferably used for drug candidate substance screening. Since almost the same number of cells are stored in each MC2, the state of the cells in each MC2 becomes uniform, and when different drug candidate substances are added to each MC2, what influence each drug candidate substance has on the cells It is easy to compare and evaluate.

  In such drug candidate substance screening methods, the influence of various drug candidate substances on cells can be measured simply and rapidly, and substances exhibiting desired activity can be efficiently selected.

  First, a drug candidate substance is added to MC2 holding cells. The same drug candidate substance may be added to all MCs 2 provided in MCAP1, or the drug candidate substance to be added may be changed by MC2. Further, it is preferable that some MC2 is not added with a drug candidate substance, and cells retained in these MC2 are used as a control.

  After the drug candidate substance is added, the influence of the drug candidate substance on the cells retained in MC2 is measured. The method for measuring the influence may be appropriately set according to the drug candidate substance to be used, the cell to be used, and the desired activity.

  For example, if you want to screen for a substance that acts as an anticancer agent, keep cancer cells in MC2, add a drug candidate substance, and measure the rate and time at which the cancer cells die. Good. Based on the measurement result, a substance exhibiting a desired activity may be selected from drug candidate substances subjected to the experiment. For example, in the example of screening a substance that acts as the above-described anticancer agent, a substance that has an effect of killing cancer cells with high efficiency may be selected. A large number of drug candidate substances can be screened easily and rapidly.

  Moreover, it is preferable that MCAP1 used for the screening method of a drug candidate substance is MCAP1 mentioned above. With this MCAP1, cells can be stored in MC2 as a single layer more uniformly and efficiently.

  Further, according to this MCAP1, cells can be cultured in a chamber for at least several days. For this reason, if a drug candidate substance screening method is performed using MCAP1, after the drug candidate substance is added, the cells are cultured and maintained for several days or more, and then the influence of the substance on the cells can be measured. .

  Furthermore, in MCAP1, since almost the same number of cells are held in each MC2, the state of the cells in each MC2 can be made uniform. Therefore, if a drug candidate substance screening method using MCAP1 is carried out, it is easy to compare and evaluate what influence each drug candidate substance has on cells when different drug candidate substances are added to each MC2. .

As mentioned above, although the Example by this invention and the modification based on this were demonstrated, this invention is not necessarily limited to this, A person skilled in the art will deviate from the main point of this invention, or the attached claim. Various alternative embodiments and modifications could be found without doing so.

<Storage of cells (red blood cells) in MC2>
Using MCAP1 as shown in FIG. 6 and the following, the state of storage of cells (red blood cells) in MC2 was observed.

Substrate: Polystyrene surface treatment: Oxygen plasma treatment Water contact angle: 10 °
MC2 formation: LIGA process and injection molding MC2 space shape: inverted regular hexagonal frustum (taper angle 20 °)
Major diameter of opening 2c = 100 μm
Depth = 50μm
Ratio of major axis of the opening 2c: depth = 1: about 0.58
The major axis of the bottom 2a = about 63.6 μm
x = 113.4 μm
Distance between MC2: Distance between centers of opening 2c (regular hexagon) 200 μm
(Equally spaced)

  Here, MCAP1 was produced by the LIGA process and injection molding. First, PMMA was patterned by X-ray lithography, and unnecessary PMMA was removed by etching to produce a PMMA master. Thereafter, nickel was electroformed (plated) on the master to produce a mold. A polystyrene MCAP was produced by injection molding using the produced mold. The surface of the chip substrate was hydrophilized by subjecting this surface to an oxygen plasma treatment with a reactive ion etching (RIE) apparatus (manufactured by Samco Corporation) with an output of 200 W and a treatment time of about 20 seconds. In addition, in order to prevent the cell suspension from spreading and prevent air from entering the MC2, the hydrated MCAP1 was sonicated in advance. The sample was immersed in a beaker containing a medium (RPMI1640) and sonicated for about 5 minutes using a commercially available ultrasonic cleaning apparatus.

  The water contact angle of the hydrophilic surface was measured by the θ / 2 method. That is, 1 to 2 μl of distilled water is dropped onto a plurality of different surface positions (5 points or more), and the water contact angle is measured at room temperature using a contact angle meter (manufactured by Kyowa Interface Science Co., Ltd.), and the average value is obtained. From this, the water contact angle was determined.

  Observation of red blood cells (cell size: about 8 to 10 μm at a long place) was performed as follows.

First, erythrocytes obtained by collecting blood from a human and centrifuging (4 ° C., 1500 g, 30 min) are suspended in a medium (RPMI1640), and a erythrocyte suspension derived from human (red blood cell concentration in suspension: 1 × 10 ⁸ cells). / Ml). 200 μl of this erythrocyte suspension was applied onto MCAP1 using a micropipette, the erythrocytes were contacted and developed, and allowed to stand for 15 minutes. Further, the upper surface of MCAP1 was washed with 1 ml of a culture solution (RPMI1640) using a micropipette, and then the erythrocytes stored in MC2 were observed with an inverted microscope.

An example is shown in FIG. Since cells (red blood cells) can be observed in a circle with the same diameter at the bottom 2a of MC2, cells are uniformly stored in this bottom 2a side by side (single layer). In this observation, since the microscope is focused on the bottom of MC2, the microscope aperture is not focused on MC opening 2c. For this reason, in FIG. 7 (FIG. 6 is also the same), the periphery of the opening 2c of MC2 is shown as a black shadow. The same applies to the following observations.

<Water contact angle and cell deployment efficiency>
By changing the oxygen plasma treatment conditions, three types of MCAP (water contact angles 80 °, 25 °, 10 °) with different water contact angles were prepared and red blood cells were observed. Compared.

MCAP1 used for the observation of red blood cells described above is manufactured with the following modifications.
MC2 space shape: inverted frustoconical shape (taper angle 20 °)
Major diameter of opening 2c = 100 μm
Depth = 100μm
Ratio of major axis of the opening 2c: depth = 1: 1
Bottom major axis = about 27.2 μm
Oxygen plasma treatment: RIE equipment (manufactured by Samco Corporation)
Water contact angle 80 °: Output 200W, 3 seconds Water contact angle 25 °: Output 200W, 5 seconds Water contact angle 10 °: Output 200W, 20 seconds

  An example is shown in FIG. Here, the cell deployment efficiency is the ratio of the number of MC2 in which cells are stored to the total number of MC2 in MCAP1.

  As shown in FIG. 8A, when the water contact angle was 80 °, almost no cells entered MC2. As shown in FIG. 8B, about 40% of cells could be stored when the water contact angle was 25 °. Further, as shown in FIG. 8C, when the water contact angle was 10 °, 90% or more of cells could be stored. In addition, it became possible to store many cells at a water contact angle of 40 °, and cells could be stored well (90% or more) when the water contact angle was 5 °.

  From the above, the water contact angle is 40 ° or less, preferably 10 ° or less, more preferably 8 ° or less, and further preferably 5 ° or less.

<The major axis and depth of the opening 2c of MC2 and the retention ability of red blood cells>
Next, when the major axis and depth of the opening 2c of the MC2 of the MCAP1 were changed, the difference in the storage of the cells in the MC2 was examined.

  Three types of plates A to C were manufactured using MCAP1 having an inverted frustoconical MC2 (taper angle of 20 °) with the major axis and depth of the opening 2c as shown in Table 1 below. Each of the plates A to C is the same as the above-described MCAP1 except for the major axis and depth and the distance between each MC2.

  As described above, 200 μl of the red blood cell suspension was added to the plates A to C, and the red blood cells were left to stand for 15 minutes after contact and development. Thereafter, washing was performed, and MC2 storing red blood cells was detected using an inverted microscope.

  As shown in FIGS. 9A and 9C, in the plates A and C, since the cells were observed in a circular shape with the same diameter in the bottom 2a of the MC2, it can be seen that the cells could be stored in the MC2 without overlapping. When the number of cells was counted, almost the same number of cells were stored in each MC2. The erythrocytes are uniformly held in a monolayer on the bottom 2a of each MC2. On the other hand, as shown in FIG. 9B, MC2 in which red blood cells were not adhered to the bottom surface 2a could be confirmed in the plate B having a large major axis. This is considered to be because the red blood cells adhering to the bottom 2a of MC2 were washed away in the washing step. That is, with MC2 having a major axis as large as about 500 μm, it was considered difficult to keep the cells uniformly in a single layer in the chamber when the depth was 150 μm or less. Further, in the plates A and C, since the red blood cells are uniformly held in a single layer on the bottom of each MC2, it can be said that the opening 2c major axis: depth ratio of the MC2 is preferably about 1: 0.35 to 1. .

<Observation of leukemia cells>
Next, the test sample containing leukemia cells was observed using the same MCAP1 as described above.

Human cultured leukemia cells (CCRF-CEM) were adjusted to about 1.0 × 10 ⁷ cells / ml with RPMI 1640 medium and developed on MCAP1 using a micropipette or the like. After standing for about 15 minutes, the chip was further washed with a micropipette using RPMI 1640 medium, and the cells in MC2 were observed with an inverted microscope.

As shown in FIG. 10, cells (leukemia cells) were observed in a circular shape with the same diameter at the bottom 2a of MC2, and it was confirmed that the cells were uniformly arranged in a single layer (single layer). That is, nucleated cell leukemia cells can be observed in the same manner as erythrocytes. Since cancer cells contained in the leukocyte fraction in whole blood can also be detected, in particular, circulating cancer cells (CTC) in the blood can also be detected.

<Influence of porosity>
Next, the ratio of the opening 2c of MC2 to the main surface of MCAP1, that is, the influence of the porosity was observed.

Three types of MCAP1 (plate D, plate E, and plate F) having an inverted frustoconical MC2 (taper angle 20 °) as shown in Table 2 below were manufactured.

  Here, in the plates D, E, and F where the value of x (see FIG. 3) is 40, 30, and 20 μm, the centroids of the openings 2c of the MC2 are equally spaced, and these centroids are placed on the diagonal lattice points. It arranges so that it may arrange. The size of MCAP1 is about 76 mm × about 25 mm in long side × short side and about 1.0 mm in thickness. The width of the frost portion is about 10 mm. The long side of region A is about 53 mm and the short side is about 20 mm.

The red blood cell suspension is prepared in the same manner as described above, and the number of cells is 5 × 10 ⁷ cells / ml. On the other hand, the human cultured leukemia cell (CCRF-CEM) suspension was adjusted to 1 × 10 ⁷ cells / ml with PBS (RPMI1640) after culturing the cells.

400 to 500 μl (0.36 to 0.4 μl per 1 mm ^{2 of} region A) of erythrocyte suspension or human culture (CEM) cell suspension was developed on the upper surface of plates D to F. In addition, washing | cleaning operation was not performed, but in order to store a cell in a chamber, it left still for 15 minutes after expansion | deployment, and observed the state of the cell in a chamber using the inverted microscope.

  As shown in FIG. 11A, when the erythrocyte suspension is used, in the plate E (X = 30 μm), cells are observed in a circular shape with the same diameter on the bottom 2a of MC2 and stored without gaps. On the other hand, as shown in FIG. 11B, in the plate D (X = 40 μm), there was a gap between the stored cells (see the arrow). Therefore, when the interval between the openings 2c of the MC2 is set to 30 μm, the cells can be more uniformly stored as a single layer in the bottom 2a in the MC2.

As shown in FIG. 12, the same was true when leukemia cell (CEM) suspension was used. That is, in the plate E (X = 30 μm), cells are observed in a circular shape with the same diameter on the bottom 2a of the MC 2 and stored without gaps. On the other hand, in the plate D (X = 40 μm), there is a gap between the stored cells.

Further, as shown in FIG. 13, in the plate F (X = 20 μm), both the erythrocyte suspension and the leukemia cell (CEM) suspension are observed in the bottom 2a of the MC2 in a circular shape with the same diameter and stored without gaps. It was. In other words, the cells were uniformly stored as a single layer.

As shown in FIG. 14, both the erythrocyte suspension and leukemia cell (CEM) suspension stored almost the same number of cells regardless of the value of X in plates E and F. The number was decreasing. Here, assuming that the radius of the circumscribed circle of the regular hexagon of the opening 2c is r and the distance between the openings 2c is L, a geometrical relationship is established with the value of X, and X is smaller than 30 μm. In other words, 30% or less of L has an opening 2c that leaves the upper surface of MCAP1.

  Furthermore, as shown in FIG. 15, when the focus of the microscope is aligned with the upper surface of MCAP1, it can be seen that cells remain on the upper surface (see the arrows).

As shown in FIG. 16, the number of remaining cells is 23 / 10,000 μm ² in plate D, and 20 and 13 / 10,000 μm ² in plates E and F, respectively. When washing is not performed, the number of cells remaining on the upper surface of MCAP1 is smaller when the interval between MC2 is about 30 μm or less, and more cells can be efficiently stored in MC2 as a single layer.

  As described above, it was considered that detection of a small number of target cells (specific cells) mixed in an enormous number of cells with high sensitivity and high completeness could be realized.

1 Microchamber array plate (MCAP)
2 Microchamber (MC)
2a Bottom 2c Opening

Claims (11)

A microchamber array plate formed on virtual lattice points in which microchambers, which are concave portions capable of accommodating various cells, have the same shape and are regularly dispersed on the principal surface,
The main surface and the inner surface of the microchamber have a hydrophilic surface,
The microchamber is arranged and formed so that the center of gravity of the opening shape of the recess coincides with the virtual lattice point on the main surface, and among the line segments connecting the centers of gravity of the adjacent microchambers A microchamber array plate having the opening shape that leaves the main surface at 30% or less.   2. The microchamber array according to claim 1, wherein a ratio of a total area occupied by the opening shape in a region defined by a closed line obtained by connecting the center of gravity of the opening shape with a straight line is 50% or more. plate. The microchamber array plate according to claim 2, wherein the hydrophilic surface has a water contact angle of 40 ° or less.   The micro-chamber array plate according to claim 1, wherein the virtual lattice points are formed by sequentially arranging the virtual lattice points on a concentric circle centered on one virtual lattice point.   5. The microchamber array plate according to claim 4, wherein the virtual lattice points have a repeating regular arrangement in which three adjacent points are closest to each other at an equal distance. 6. The microchamber array plate according to claim 5, wherein the opening shape is a circle or a regular polygon inscribed in the circle.   The microchamber array plate according to claim 6, wherein the opening shape is a regular hexagon, and the edge lines of the adjacent microchambers are arranged in parallel to each other.   The micro chamber array plate according to claim 7, wherein the diameter of the circumscribed circle of the regular hexagon is in the range of 20 to 500 µm.   9. The micro chamber array plate according to claim 8, wherein the micro chamber has a tapered side surface with a cross-sectional area decreasing toward the bottom.   The micro chamber array plate according to claim 9, wherein the micro chamber has a depth of 20 μm or more. The micro chamber array plate according to claim 10, wherein the micro chamber has a ratio of the depth to the diameter of the circumscribed circle within a range of 0.35 to 1.

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