JP2022078638A - Microplate - Google Patents

Abstract

To provide a microplate suitable for an automatic culture apparatus, which makes it easy to focus on cultured cells in each well in automatic observation and has excellent observation efficiency.SOLUTION: A microplate 1 comprises: an upper surface portion 10 having a rectangular plan view shape; a peripheral wall portion 12 vertically hung from the outer edge portion of the upper surface portion 10; and a plurality of bottomed cylindrical wells 14 in which a circular opening is formed in an upper surface 10a of the upper surface portion 10. Regarding the deepest parts of two wells 14, which are farthest from each other, the distance d1 between the deepest parts of two wells 14 is 400 μm or less in the direction perpendicular to a reference plane calculated by a reference plane calculation method.SELECTED DRAWING: Figure 2

Description

The present invention relates to a microplate.

Cultured cells are often used as specimens in basic research and drug discovery research to elucidate biological phenomena, and culture vessels for obtaining a large number of specimens are widely used. Conventionally, the evaluation of cultured cells has mainly been carried out by cell metabolism or property evaluation by metabolites such as MTT assay and ELISA (Enzyme-Linked Immuno Sorbent Assay). On the other hand, the three-dimensional cell mass (spheroid) in which cells are aggregated, which is obtained by three-dimensional culture, has a three-dimensional structure as in the living body. Therefore, in recent years, the importance of morphological observation using cell masses has increased.

As a culture container used for three-dimensional culture, a microplate having a plurality of wells and having a large number of fine wells (microwells) having a pore size of 100 to 1,000 μm formed on the bottom surface (culture surface) of each well is known. (Patent Document 1). When cells are sown on the culture surface, the cells associate in each microwell to form a cell mass. The ANSI (American National Standards Institute) / SBS standard is known as a standard for microplates.

Japanese Patent No. 6400575

As a cell culture method, a method of culturing a large amount of cells at high throughput using an automatic culture device has been developed. However, with conventional microplates, even if they meet ANSI / SBS standards, if the height of the bottom of the container is different, it is difficult to accurately match the amount of cells and culture medium collected from each well, and the same plate. The uniformity of the evaluation system within may decrease.

As for cell observation, a method of observing a large number of cells with high throughput using an automatic observation device has been developed. However, with conventional microplates, it is difficult to automatically focus on cultured cells in each well even if they meet the ANSI / SBS standard, and the efficiency of automatic observation may decrease.

It is an object of the present invention to provide a microplate suitable for an automatic culture apparatus, which makes it easy to focus on cultured cells in each well in automatic observation and has excellent observation efficiency.

The present invention has the following aspects.
[1] A microplate having a plurality of wells, in which the deepest portions of the wells are farthest from each other in the direction perpendicular to the reference plane calculated by the following reference plane calculation method. Microplate with a distance of 400 μm or less.
(Reference plane calculation method)
Of all the wells, select four wells that are located at each apex of the square or rectangle in plan view and that maximize the area of the square or rectangle. Of the above four wells, xy passes through the deepest part of two wells diagonally located in a plan view (however, if the bottom surface of the well is a flat bottom, it is the center of the bottom surface of the well in a plan view). It has a plane, the midpoint of the deepest part of the two wells is the origin, and the xy coordinates of the deepest part of the four wells are (-X, Y), (X, Y), (-, respectively. A three-dimensional Cartesian coordinate system (x, y, z) that becomes (X, −Y), (X, −Y) is set. In the three-dimensional Cartesian coordinate system (x, y, z), the coordinates of the deepest part of the four wells are (-X, Y, Z1), (X, Y, Z2), (-X, -Y, When Z3) and (X, −Y, Z4) are used, the plane represented by the following equations 1 to 4 is used as the reference plane.

[2] A microplate having a plurality of wells, wherein the distance between the reference plane calculated by the reference plane calculation method and the bottom of each well is 200 μm or less.
[3] The plurality of wells are arranged in a matrix of n × m rectangles vertically and horizontally in a plan view, and among the plurality of wells, four wells located at four corners are used as the reference. The microplate according to [1] or [2], wherein the reference plane is calculated by a plane calculation method.
[4] When the bottom surface of the well is cut along a plane parallel to the depth direction passing through the center of the bottom surface in a plan view, the cross-sectional shape is a downwardly convex arc shape, a downwardly convex fold line shape, or a downwardly convex fold line shape. The microplate according to any one of [1] to [3], which is linear.
[5] A frame body and a flat plate base material are provided, and the frame body includes a plurality of tubular portions forming a peripheral wall portion of each of the wells, and the flat plate base material is provided on the lower end side of the plurality of tubular portions. The microplate according to any one of [1] to [4], wherein the microplate is attached to form the plurality of wells.
[6] The microplate according to any one of [1] to [5], wherein a plurality of fine wells are formed on the bottom surface of the well.

According to the present invention, it is possible to provide a microplate which is suitable for an automatic culture apparatus and which is easy to focus on the cultured cells of each well in automatic observation and has excellent observation efficiency.

It is a top view of the microplate of an embodiment. FIG. 1 is a cross-sectional view taken along the line II of the microplate of FIG. It is explanatory drawing explaining the reference plane calculation method. It is sectional drawing which showed the bottom surface of each well of a microplate enlarged. It is sectional drawing of the microplate of another embodiment. It is sectional drawing of the microplate of another embodiment.

The meanings and definitions of the terms used herein are as follows.
The numerical range represented by "-" means a numerical range in which the numerical values before and after "-" are the lower limit value and the upper limit value.
The “deepest part of the well” means the deepest point on the bottom surface (culture surface) of the well, and when the bottom surface of the well is a flat bottom, it is the center of the bottom surface of the well in a plan view.

Hereinafter, an example of an embodiment of the microplate of the present invention will be shown and described with reference to the drawings. It should be noted that the dimensions and the like of the figures exemplified in the following description are examples, and the present invention is not necessarily limited to them, and the present invention can be appropriately modified without changing the gist thereof. ..

[First Embodiment]
As shown in FIGS. 1 and 2, in the microplate 1 of the present embodiment, the upper surface portion 10 having a rectangular plan view shape, the peripheral wall portion 12 vertically hung from the outer edge portion of the upper surface portion 10, and the upper surface portion 10 A plurality of bottomed tubular wells 14 having a circular opening formed on the upper surface 10a of the above surface are provided.

In the microplate 1, the bottom surface 16 of each well 14 is the culture surface. The cross-sectional shape when the bottom surface 16 of the well 14 of this example is cut along a plane parallel to the depth direction passing through the center of the bottom surface 16 in a plan view is linear. That is, the well 14 of this example has a flat bottom with a flat bottom surface 16. The cross-sectional shape when the bottom surface 16 of the well 14 is cut along a plane parallel to the depth direction passing through the center of the bottom surface 16 in a plan view is not limited to a straight line, but is a downwardly convex arc shape and a downwardly. It may be a convex bent line or the like.

The arrangement pattern of the wells 14 in the microplate 1 is not particularly limited, and examples thereof include a pattern in which n × m rectangular matrices are arranged vertically and horizontally, and a staggered pattern. The opening shape of the well 14 in a plan view is not limited to a circle, and may be, for example, a rectangle. The number of wells 14 is not particularly limited, and examples thereof include 6 to 1536.
In the microplate 1 of this example, 96 wells 14 are arranged vertically and horizontally in a matrix of 8 × 12 rectangles in a plan view. It may be 16 × 24 384 wells or 32 × 48 1536 wells.

In the microplate 1, the distance d1 between the deepest portions of the two wells 14 where the deepest portions of the wells 14 are farthest from each other in the direction perpendicular to the reference plane calculated by the reference plane calculation method described later is 400 μm or less.

(Reference plane calculation method)
Of all the wells 14, four wells 14 are selected that are located at each apex of the square or rectangle in plan view and that maximize the area of the square or rectangle. In this example, as shown in FIG. 1, of the 96 wells 14 arranged in a matrix of 8 × 12 rectangles vertically and horizontally, the four wells 14a to 14d located at the four corners are the vertices of each. The rectangle P when located in is the rectangle having the largest area.

Then, in the microplate 1, a three-dimensional Cartesian coordinate system (x, y, z) satisfying the following three conditions (1) to (3) is set.
(1) Of the four wells 14a to 14d, it has an xy plane passing through the deepest part of the two wells 14b and 14c located diagonally in a plan view.
(2) The midpoint of the deepest part of the two wells 14b and 14c located diagonally is the origin.
(3) The deepest xy coordinates of the four wells 14a to 14d are (-X, Y), (X, Y), (-X, -Y), and (X, -Y), respectively.
However, when the bottom surface of the well 14 is a flat bottom as in this example, the deepest portion of the well 14 is the center of the bottom surface 16 of the well 14 in a plan view.

In this three-dimensional Cartesian coordinate system (x, y, z), the coordinates of the deepest part of the four wells 14a to 14d are set to A (-X, Y, Z1), B (X, Y, Z2), and C (, respectively. Let it be -X, -Y, Z3) and D (X, -Y, Z4). Then, of the four wells 14a to 14d, the deepest portions (points B and C) of the diagonally located wells 14b and 14c pass through, and the deepest portions (points A and points A) of the remaining two wells 14a and 14d are passed. The surface with the smallest distance from D) is set as the reference surface.

Specifically, in the three-dimensional Cartesian coordinate system (x, y, z) shown in FIG. 3, the equation representing the reference plane k is given by the following equation 1.
z = ax + by + c ・ ・ ・ Equation 1

When the surface represented by the equation 1 passing through the points B and C is moved around the axis with the balance axis m passing through the points B and C as the central axis, the distance between the points A and D becomes the smallest. The plane is set as the reference plane k.
Since the surface represented by the equation 1 passes through the points B and C, substituting the coordinates of the points B and C into the equation 1 leads to the following equations 5 and 6, respectively.
Z2 = aX + bY + c ・ ・ ・ Equation 5
Z3 = -aX-bY + c ... Equation 6

The following equation 4 is derived by taking the sum of equations 5 and 6.
Z2 + Z3 = 2c
c = (Z2 + Z3) / 2 ... Equation 4

Taking the difference between equations 5 and 6, the following equation 3 is derived.
Z2-Z3 = 2aX + 2bY
2bY = (Z2-Z3) -2aX
b = {(Z2-Z3) /2-aX}/2Y
b = 1 / Y {(Z2-Z3) / 2-aX} ... Equation 3

Substituting Equation 3 and Equation 4 into Equation 1 leads to Equation 7 below.
Z = ax + 1 / Y {(Z2-Z3) /2-aX} y + (Z2 + Z3) / 2 ... Equation 7

A point on the reference plane k whose x-coordinate and y-coordinate are the same as the point A is defined as a point A'(-X, Y, Z1'). By substituting the coordinates of the point A'in equation 7, the following equation 8 is derived.
Z1'= -aX + 1 / Y {(Z2-Z3) /2-aX} Y + (Z2 + Z3) / 2
Z1'= -2aX + Z2 ... Equation 8

Similarly, a point on the reference plane k whose x-coordinate and y-coordinate are the same as the point D is defined as a point D'(X, −Y, Z3'). By substituting the coordinates of the point D'in equation 7, the following equation 9 is derived.
Z4'= aX-1 / Y {(Z2-Z3) /2-aX} Y + (Z2 + Z3) / 2
Z4'= 2aX + Z3 ... Equation 9

When the distance between the surface represented by the equation 1 (the surface represented by the equation 7) and the points A and D is the minimum, the distance between the points A and A'in the z-axis direction and the points D and the points The sum of the distances from D'is minimized.
By taking the square of the difference between the z-coordinates of the point A and the point A'and substituting the equation 8, the following equation 10 is derived. Similarly, by taking the square of the difference between the z-coordinates of the point D and the point D'and substituting the equation 9, the following equation 11 is derived.
(Z1-Z1') ² = (Z1 + 2aX-Z2) ² ... Equation 10
(Z4-Z4') ² = (Z4-2aX-Z3) ² ... Equation 11

The sum of equations 10 and 11 leads to equation 12.
(Z1 + 2aX-Z2) ² + (Z4-2aX-Z3) ²
= 8a ² X ² + 4aX (Z1-Z2-Z4 + Z3) + (Z1-Z2) ² + (Z4-Z3) ²
= 8X ² {a ² + (1 / 2X) × (Z1-Z2-Z4 + Z3) a} + (Z1-Z2) ² + (Z4-Z3) ² ... Equation 12

When Q = (1 / 2X) × (Z1-Z2-Z4 + Z3), the formula 12 is represented by the following formula 13.
8X ² {a ² + (a / 2X) x (Z1-Z2-Z4 + Z3)} + (Z1-Z2) ² + (Z4-Z3) ²
= 8X ² {a ² + aQ} + (Z1-Z2) ² + (Z4-Z3) ²
= 8X ² {(a + Q / 2) ² -Q ^2/4 } + (Z1-Z2) ² + (Z4-Z3) ² ... Equation 13

Since equation 13 is the minimum, that is, the distance between the surface represented by equation 1 and the points A and D is the minimum when a + Q / 2 = 0, a = −Q / 2. Substituting Q = (1 / 2X) × (Z1-Z2-Z4 + Z3) into this leads to the following equation 2.
a = -1 / 2 x (1 / 2X) x (Z1-Z2-Z4 + Z3) ... Equation 2

From the above, the reference plane k is a plane represented by the equations 1 to 4.

In the microplate 1, the distance d1 between the deepest portions of the two wells 14 in the direction perpendicular to the reference plane k is 400 μm or less, preferably 200 μm or less, preferably 50 μm or less. More preferred. If the distance d1 is equal to or less than the upper limit, the height deviation of the bottom surface 16 (culture surface) of each well 14 is small. It is easy and the uniformity of the evaluation system in the same plate is improved. In addition, the height deviation of the cultured cells cultured in each well 14 becomes small, and even in high-throughput observation using an automatic observation device, it is easy to focus on each cultured cell, and the observation efficiency becomes high.

As shown in FIG. 4, it is preferable that a plurality of fine wells 19 having a uniform size are formed on the bottom surface 16 of the well 14 from the viewpoint that a cell mass having a uniform size can be easily obtained.
The opening shape of the fine well 19 is not particularly limited, and examples thereof include a circular shape, an elliptical shape, a polygonal shape, and an irregular shape. Dimensions such as the average diameter, opening area, and depth of the openings of the fine wells 19 are not particularly limited and may be appropriately set. For example, the average diameter of the opening of the fine well 19 can be set to 100 to 2500 μm, the opening area can be set to 7850 to 4906250 μm ² , and the average depth can be set to 50 to 1000 μm. The number of fine wells 19 formed on the bottom surface 16 can be set to 10 to 10,000 per unit area / cm ² .

The arrangement pattern of the fine wells 19 is not particularly limited, and may be formed in a regular pattern, irregularly formed, or a mixture of regular portions and irregular portions. As a regular arrangement pattern, for example, a pattern in which fine wells are arranged at each vertex of a square arranged without gaps, a pattern in which fine wells are arranged at each vertex of a regular hexagon arranged without gaps and a fine well in the center, and a staggered pattern are used. It can be exemplified.

As the material of the microplate 1, resin can be exemplified.
Examples of the resin include polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, acrylic resin, polycarbonate resin, and silicone resin. As the resin, one selected from polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, acrylic resin, polycarbonate resin and silicone resin is preferable, and polystyrene resin is particularly preferable from the viewpoint of high transparency and low drug adsorption. preferable. The resin constituting the microplate 1 may be one kind or two or more kinds.

The peripheral wall portion 12 of the microplate 1 may be transparent or opaque, and is preferably opaque from the viewpoint of observability, and black is more preferable as the color tone. The method for making the peripheral wall portion opaque is not particularly limited, and for example, a method of adding fine particles, a method of adding a pigment, or the like can be used.

The method for manufacturing the microplate 1 is not particularly limited, and can be molded by, for example, an injection molding method or a compression molding method. Among them, the compression molding method is preferable because it is easy to manufacture the microplate 1 having a plurality of wells 14 having a distance d1 of 400 μm or less.

As a method for forming the fine well 19, for example, laser irradiation can be exemplified. When the culture surface 18 which is the bottom surface 16 of the well 14 is irradiated with laser light, the resin constituting the culture surface 18 is dissolved and vaporized to form a fine well 19 having a very smooth surface. Around the opening of the fine well 19, the melted resin may be raised to form a bank portion.

The laser light source is not particularly limited, and a CO ₂ laser can be exemplified. The arrangement and size of the fine wells 19 can be adjusted by adjusting irradiation conditions such as the irradiation position and output of the laser beam. For example, the size of the fine well can be adjusted with a laser output of 1 to 100 W and an irradiation time of 0.1 to 100 μs.

A low adhesion coat film that suppresses cell adhesion may be formed on the bottom surface 16 of the well 14 that is the culture surface. The formation of a low-adhesion coat film facilitates the removal of cultured cells. The low adhesion coat film can be formed, for example, by applying a cell adhesion inhibitor. Examples of the cell adhesion inhibitor include phospholipid polymers (2-methacryloyloxyethyl phosphorylcholine and the like), polyhydroxyethyl methacrylates, fluorine-containing compounds, and polyethylene glycol. As the cell adhesion inhibitor, one type may be used alone, or two or more types may be used in combination.
If the microplate 1 is molded from a resin having an effect of suppressing cell adhesion such as a silicone resin, it is possible to prevent cells from adhering to the bottom surface 16 without forming a low adhesion coat film.

An easy-adhesion coat film may be formed on the bottom surface 16 of the well 14 to facilitate the adhesion of cells. Examples of the material for forming the easy-adhesive coating film include collagen and gelatin. As the material for forming the easy-adhesion coating film, one type may be used alone, or two or more types may be used in combination.

[Second Embodiment]
As an embodiment of the microplate of the second embodiment, the same microplate 1 as that of the first embodiment can be exemplified. The microplate 1 of the second embodiment has the first embodiment except that the distance d2 between the reference plane k calculated by the reference plane calculation method described above and the bottom (deepest portion) of each well 14 is 200 μm or less. Is similar to. "The distance d2 is 200 μm or less" means that all the wells 14 of the microplate 1 satisfy the condition that the distance d2 between the bottom of the well 14 and the reference plane k is 200 μm or less.

The distance d2 between the reference surface k and the bottom (deepest portion) of each well 14 is 200 μm or less, preferably 150 μm or less, and more preferably 50 μm or less. If the distance d2 is equal to or less than the upper limit, the height deviation of the bottom surface 16 (culture surface) of each well 14 is small. It is easy and the uniformity of the evaluation system in the same plate is improved. In addition, the height deviation of the cultured cells cultured in each well 14 becomes small, and even in high-throughput observation using an automatic observation device, it is easy to focus on each cultured cell, and the observation efficiency becomes high.

A preferred embodiment of the microplate 1 of the second embodiment is the same as that of the first embodiment except for the distance d2.
As a method for manufacturing the microplate 1 of the second embodiment, the same method as that of the first embodiment can be exemplified, and compression molding is possible from the viewpoint that it is easy to manufacture the microplate 1 having a plurality of wells 14 having a distance d2 of 200 μm or less. The method is preferred.

As described above, in the microplate of the present invention, the distance d1 is controlled to 400 μm or less or the distance d2 is controlled to 200 μm or less in each well. As a result, the deviation of the bottom surface 16 of each well 14 is small, and it is easy to match the amount of cells and culture solution collected from each well when applied to an automatic culture device, so that the uniformity of the evaluation system in the same plate is improved. .. Further, even with an automatic observation device, it becomes easy to automatically focus on the cultured cells cultured in each well 14. Therefore, a large amount of cultured cells can be efficiently observed with high throughput by an automatic observation device.
In the present invention, it is preferable that the distance d1 is controlled to 400 μm or less and the distance d2 is controlled to 200 μm or less.

The microplate of the present invention is not limited to the above-described embodiment.
For example, the microplate of the present invention may be the microplate 2 illustrated in FIG. The microplate 2 has the same embodiment as the microplate 1 except that the well 14 has a bottom surface 16A instead of the bottom surface 16. The same parts as those in FIG. 2 in FIG. 5 are designated by the same reference numerals, and the description thereof will be omitted.

The cross-sectional shape when the bottom surface 16A of the well 14 of the microplate 2 is cut along a plane parallel to the depth direction passing through the center of the bottom surface 16A in a plan view is a downwardly convex arcuate shape. Even in the microplate 2, by satisfying both the distance d1 controlled to 400 μm or less and the distance d2 controlled to 200 μm or less, the cultured cells cultured in each well 14 even in the automatic observation device. It becomes easy to focus automatically on.

The microplate of the present invention may be the microplate 3 exemplified in FIG. The same parts as those in FIG. 2 in FIG. 6 are designated by the same reference numerals, and the description thereof will be omitted.
The microplate 3 includes a frame body 20 and a flat plate base material 22. The frame body 20 has a plurality of tubular portions having a circular opening formed in the upper surface portion 10, a peripheral wall portion 12 vertically hung from the outer edge portion of the upper surface portion 10, and the upper surface portion 10a of the upper surface portion 10 and having an open lower end. It is equipped with 24. A plurality of wells 14 are formed by attaching the flat plate base material 22 to the lower end side of the plurality of tubular portions 24.

As described above, the microplate 3 has the same embodiment as the microplate 1 except that the flat plate base material 22 which is a separate member is attached to the lower end side of each tubular portion 24 to form each well 14. .. Also in the microplate 3, by satisfying both the distance d1 controlled to 400 μm or less and the distance d2 controlled to 200 μm or less, cells collected from each well when applied to an automatic incubator can be used. It is easy to adjust the amount of the culture solution, and it is easy to automatically focus on the cultured cells cultured in each well 14 even with an automatic observation device.

In addition, it is possible to appropriately replace the components in the embodiment with well-known components without departing from the spirit of the present invention, and the above-mentioned modifications may be appropriately combined.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to the following description. Example 1 is an example, and Example 2 is a comparative example.

[Example 1]
In the manner illustrated in FIGS. 1 and 2, 96-well (8 × 12) microplates having a well bottom surface (culture surface) with a diameter of 6.45 mm were prepared, and distances d1 and d2 were set for each well. As a result of measurement, the distance d1 was 155 μm, and the maximum value of the distance d2 was 89 μm.

[Example 2]
In the manner illustrated in FIGS. 1 and 2, 6-well (8 × 12) microplates having a well bottom surface (culture surface) with a diameter of 6.45 mm were prepared, and distances d1 and d2 were set for each well. As a result of measurement, the distance d1 was 1350 μm, and the maximum value of the distance d2 was 1320 μm.

[Accuracy of medium exchange in automatic culture equipment]
In a 96-well (8 × 12) microplate as illustrated in FIGS. 1 and 2, a medium of 100 μL to 200 μL is usually added and cultured. Normally, in culturing, the medium that supplies nutrients to cells is exchanged during culturing. In the automatic culture device, the suction nozzle is set to an arbitrary height (several μm above the culture surface), and the medium is exchanged. When a suction nozzle is installed according to the highest culture surface in the microplate and the medium in each well is sucked, if there is a difference in the height of the deepest part of each well in the microplate, the amount of medium recovered will be different and the same. There will be a difference in the culture performed under the conditions, and accurate evaluation will not be possible.
Therefore, 200 μL of medium was added to each well of the microplate of each example, and the suction nozzle of the automatic dispenser was set according to the deepest part of the well having the maximum z value in the deepest part of the microplate to prepare the medium. After suction, the amount of remaining medium in the well with the smallest z value in the deepest part of the microplate was calculated from the following formula. In addition, the volume error when 200 μL of new medium was added to each well was also calculated from the following formula. Table 1 shows the measurement results of the remaining amount of medium and the volume error of each example.
Medium remaining amount = well bottom area (0.3266 cm ² ) x d1
Volume error (%) Medium remaining amount (μL) / 200 μL × 100

[Example 3]
In a 96-well (8 × 12) microplate of the embodiment illustrated in FIGS. 1 and 2, the remaining medium amount and volume error corresponding to the value of the distance d1 shown in Table 2 were similarly calculated.

As shown in Table 1, Example 1 having a distance d1 of 400 μm or less and a distance d2 of 200 μm or less has a smaller amount of medium remaining when applied to an automatic culture apparatus than Example 2 which does not satisfy these conditions. , The capacity error was also small. Further, as shown in Table 2, when the distance d1 is 400 μm or less, the capacitance error can be reduced to 7% or less.

1 to 3 ... Microplate, 10 ... Top surface, 10a ... Top surface, 12 ... Peripheral wall, 14 ... Well, 16, 16A ... Bottom surface, 20 ... Frame, 22 ... Flat plate base material, 24 ... Cylinder.

Claims (6)

A microplate with multiple wells
A microplate in which the distance between the deepest portions of the two wells, which are the farthest from each other in the direction perpendicular to the reference plane calculated by the following reference plane calculation method, is 400 μm or less.
(Reference plane calculation method)
Of all the wells, select four wells that are located at each apex of the square or rectangle in plan view and that maximize the area of the square or rectangle. Of the above four wells, xy passes through the deepest part of two wells diagonally located in a plan view (however, if the bottom surface of the well is a flat bottom, it is the center of the bottom surface of the well in a plan view). It has a plane, the midpoint of the deepest part of the two wells is the origin, and the xy coordinates of the deepest part of the four wells are (-X, Y), (X, Y), (-, respectively. A three-dimensional Cartesian coordinate system (x, y, z) that becomes (X, −Y), (X, −Y) is set. In the three-dimensional Cartesian coordinate system (x, y, z), the coordinates of the deepest part of the four wells are (-X, Y, Z1), (X, Y, Z2), (-X, -Y, When Z3) and (X, −Y, Z4) are used, the plane represented by the following equations 1 to 4 is used as the reference plane.

A microplate with multiple wells
A microplate in which the distance between the reference plane calculated by the following reference plane calculation method and the bottom of each well is 200 μm or less.
(Reference plane calculation method)
Of all the wells, select four wells that are located at each apex of the square or rectangle in plan view and that maximize the area of the square or rectangle. Of the above four wells, xy passes through the deepest part of two wells diagonally located in a plan view (however, if the bottom surface of the well is a flat bottom, it is the center of the bottom surface of the well in a plan view). It has a plane, the midpoint of the deepest part of the two wells is 0, and the xy coordinates of the deepest part of the four wells are (-X, Y), (X, Y), ( Set the three-dimensional Cartesian coordinate system (x, y, z) to be -X, -Y), (X, -Y). In the three-dimensional Cartesian coordinate system (x, y, z), the coordinates of the deepest part of the four wells are (-X, Y, Z1), (X, Y, Z2), (-X, -Y, When Z3) and (X, −Y, Z4) are used, the plane represented by the following equations 1 to 4 is used as the reference plane.

The plurality of wells are arranged in a matrix of n × m rectangles vertically and horizontally in a plan view, and among the plurality of wells, four wells located at four corners are used to calculate the reference plane. The microplate according to claim 1 or 2, wherein the reference plane is calculated by the above method. When the bottom surface of the well is cut along a plane parallel to the depth direction passing through the center of the bottom surface in a plan view, the cross-sectional shape is a downwardly convex arc, a downwardly convex fold line, or a straight line. The microplate according to any one of claims 1 to 3. A frame body and a flat plate base material are provided,
The frame includes a plurality of tubular portions forming a peripheral wall portion of each of the wells, and the flat plate base material is attached to the lower end side of the plurality of tubular portions to form the plurality of wells. Item 6. The microplate according to any one of Items 1 to 4. The microplate according to any one of claims 1 to 5, wherein a plurality of fine wells are formed on the bottom surface of the well.

Images