JP2022044569A - Method for observing microplate and cultured cells - Google Patents

Abstract

To provide a microplate that allows cultured cells in each well to be easily captured in sequence during observation and has excellent observation efficiency when applied to automated observation.SOLUTION: There is provided a microplate 1 in which a plurality of wells 14 each having a bottom surface 16 provided as a concave surface that is recessed downward is arranged in a rectangular shape in a plan view. A square S has the centers of four wells 14 located four corners as respective vertexes. Each of the centers is the deepest part of the bottom surface 16 of the well 14. An origin O is an intersection of two lines connecting midpoints of opposite sides of the square S. In XY coordinates, the longer of the two lines is an x-axis and the line perpendicular to the x-axis passing through the origin O is a y-axis. For each of the x-coordinate and the y-coordinate, when the theoretical and measured values of the x and y coordinates of the center of each well 14 are obtained, a difference of ratios of the measured maximum and minimum values of deviation from the theoretical value shall be 2.00% or less in the x-coordinate and y-coordinate.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method for observing microplates and cultured cells.

Cultured cells are often used as specimens in basic research, drug discovery research, etc. to elucidate biological phenomena, and culture vessels for obtaining a large amount of specimens are widely used. Conventional evaluation of cultured cells has mainly been carried out by cell metabolism or metabolite-based property evaluation 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 in which a plurality of wells are arranged in a matrix in the vertical and horizontal directions is known (Patent Document 1). By dispensing the culture solution into each well and culturing in large quantities, many cell masses formed in each well can be efficiently observed. The ANSI (American National Standards Institute) / SBS standard is known as a standard for microplates. The ANSI / SBS standard defines the pitch between wells arranged on the outermost side of the plate in plan view.

Japanese Unexamined Patent Publication No. 11-127843

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 a conventional microplate, it is difficult to accurately capture the cultured cells in each well even if they meet the ANSI / SBS standard, and the efficiency of automatic observation tends to decrease.

An object of the present invention is to provide a microplate which can easily capture cultured cells in each well in order at the time of observation and has excellent observation efficiency when applied to automatic observation.

The present invention has the following aspects.
[1] A microplate in which a plurality of wells are arranged in a square shape in a plan view.
The bottom surface of each of the wells is a concave surface that is recessed downward.
The deepest part of the bottom surface of the well is set as the center of the well.
In a plan view, the longest of the two straight lines is the intersection of the two straight lines connecting the midpoints of the opposite sides in the quadrangle whose apex is the center of the four wells located at the four corners. The straight line is the x-axis, and the line perpendicular to the x-axis passing through the origin O is the y-axis.
When the theoretical values of the x-coordinate and y-coordinate of the center of each well calculated from the preset pitch of each well are obtained and compared with the measured values of the x-coordinate and y-coordinate of the center of each well,
Each well represented by (x-x ₀ ) / x ₀ x 100 (where x is the measured value of the x-coordinate of the center of the well and x ₀ is the theoretical value of the x-coordinate of the center of the well). The difference between the maximum value and the minimum value of the deviation ratio in the x-axis direction is 2.00% or less.
And it is represented by (y-y ₀ ) / y ₀ x 100 (where y is the measured value of the y-coordinate of the center of the well and y ₀ is the theoretical value of the y-coordinate of the center of the well). A microplate in which the difference between the maximum value and the minimum value of the deviation ratio in the y-axis direction of each well is 2.00% or less.
[2] A microplate in which a plurality of wells are arranged in a square shape in a plan view.
The bottom surface of each of the wells is a concave surface that is recessed downward.
The deepest part of the bottom surface of the well is set as the center of the well.
In a plan view, the longest of the two straight lines is the intersection of the two straight lines connecting the midpoints of the opposite sides in the quadrangle whose apex is the center of the four wells located at the four corners. The straight line is the x-axis, and the line perpendicular to the x-axis passing through the origin O is the y-axis.
When the theoretical values of the x-coordinate and y-coordinate of the center of each well calculated from the preset pitch of each well are obtained and compared with the measured values of the x-coordinate and y-coordinate of the center of each well,
Each well represented by (x-x ₀ ) / x ₀ x 100 (where x is the measured value of the x-coordinate of the center of the well and x ₀ is the theoretical value of the x-coordinate of the center of the well). The rate of deviation in the x-axis direction is -1.00% or more and 1.00% or less.
And it is represented by (y-y ₀ ) / y ₀ x 100 (where y is the measured value of the y-coordinate of the center of the well and y ₀ is the theoretical value of the y-coordinate of the center of the well). A microplate in which the percentage of deviation of each well in the y-axis direction is −1.00% or more and 1.00% or less.
[3] A method for observing cultured cells, which automatically observes the cultured cells cultured in each well using only the microplates of [1] or [2].

According to the present invention, it is easy to capture the cultured cells of each well in order at the time of observation, and it is possible to provide a microplate having excellent observation efficiency when applied to automatic observation.

It is a top view of the microplate of an embodiment. FIG. 3 is a cross-sectional view taken along the line II of the microplate of FIG. It is sectional drawing of the microplate of another embodiment. It is a top view explaining the center of a well.

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 "preset pitch of wells" means a value (design value) set as the pitch of each well when the microplate is manufactured. For example, the pitch described in the product drawing of the microplate can be adopted as the "preset pitch".
"Center of the well" means the deepest part of the bottom surface, which is a concave surface recessed below the well.

[Microplate]
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. ..

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

In the microplate 1, the bottom surface 16 of each well 14 is the culture surface. As shown in FIG. 2, the well 14 of this example has a concave surface having a bottom surface 16 recessed downward in a hemispherical shape. The bottom surface 16 of the well 14 is not limited to the hemispherical concave surface, and may be, for example, a concave surface recessed downward in a conical shape as shown in FIG.

The opening shape of the well 14 in a plan view is not limited to a circle, and may be, for example, a rectangle or a polygon. The number of wells 14 is not particularly limited, and examples thereof include 4 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 position of each well 14 in a plan view satisfies the condition described below.
As shown in FIG. 1, in a quadrangle S whose vertices are the centers of four wells 14a to 14d located at four corners in a plan view, the intersection of two straight lines connecting the midpoints of opposite sides is defined. Let the origin be O. Further, the longer straight line of the two straight lines is defined as the x-axis, and the line perpendicular to the x-axis passing through the origin O is defined as the y-axis. However, as shown in FIG. 4A, the deepest part of the bottom surface 16 of the well 14 is the center of the well 14.

Next, on the xy coordinates with the origin O as a reference point in a plan view, the theoretical values of the x and y coordinates of the center of each well 14 theoretically calculated from the preset pitch are obtained. Further, the x-coordinate and the y-coordinate of the center of each well 14 on the xy-coordinate are actually measured. For example, the microplate 1 is observed from above by an observation device, and the position in the x-axis direction and the position in the y-axis direction of the center of each well 14 with respect to an arbitrary point in the observation image are measured. After that, the measured values at those positions are converted into x-coordinates and y-coordinates when the origin O is used as a reference point, and are used as measured values of the x-coordinates and y-coordinates of the center of each well 14.

The ratio of the deviation of the measured value of the x-coordinate of the center of each well 14 from the theoretical value (hereinafter, also referred to as “ratio Q _x ”) is expressed by the following equation 1.
(X-x ₀ ) / x ₀ x 100 ... Equation 1
However, in the above equation 1, x is an actually measured value of the x coordinate of the center of the well 14. x ₀ is the theoretical value of the x coordinate of the center of the well 14.

The ratio of the deviation of the measured value of the y coordinate of the center of each well 14 from the theoretical value (hereinafter, also referred to as “ratio Q _y ”) is expressed by the following equation 2.
(Yy ₀ ) / y ₀ × 100 ・ ・ ・ Equation 2
However, in the above equation 2, y is an actually measured value of the y coordinate of the center of the well 14. y ₀ is the theoretical value of the y coordinate of the center of the well 14.

In the microplate 1, the position of the center of each well 14 satisfies at least one of the following conditions 1 and 2.
Condition 1: The difference between the maximum value and the minimum value of the ratio Q _x obtained for each well 14 is 2.00% or less, and the difference between the maximum value and the minimum value of the ratio Q _y obtained for each well 14 is It is less than 2.00%.
Condition 2: The ratio Q _x obtained for each well 14 is within the range of -1.00% or more and 1.00% or less, and the ratio Q _y obtained for each well 14 is -1.00% or more and 1.00%. It is within the range of% or less.
Condition 2 means that the ratio Q _x and the ratio Q _y of all the wells 14 are within the range of −1.00% or more and 1.00% or less.

The microplate 1 may satisfy only condition 1, may satisfy only condition 2, or may satisfy both condition 1 and condition 2. As a result, the cultured cells cultured in each well 14 can be easily arranged at equal intervals in the vertical direction (y-axis direction) and the horizontal direction (x-axis direction) in a plan view. Therefore, even in high-throughput observation using an automatic observation device, it becomes easy to capture each cultured cell in order, the observation efficiency is improved, and oversight of the cultured cells can be suppressed. It is preferable that the microplate 1 satisfies both the conditions 1 and the condition 2 from the viewpoint that the effect of the present invention can be easily obtained.

The difference between the maximum value and the minimum value of the ratio Q _x is preferably 2.00% or less, more preferably 1.50% or less, further preferably 1.00% or less, and particularly preferably 0.80% or less.
The ratio Q _x is preferably −1.00% or more and 1.00% or less, more preferably −0.80% or more and 0.70% or less, further preferably −0.60% or more and 0.40% or less, and − It is particularly preferable that it is 0.40% or more and 0.40% or less.

The difference between the maximum value and the minimum value of the ratio _Qy is preferably 2.00% or less, more preferably 1.50% or less, further preferably 1.00% or less, and particularly preferably 0.80% or less.
The ratio Q _y is preferably −1.00% or more and 1.00% or less, more preferably −0.80% or more and 0.70% or less, further preferably −0.60% or more and 0.40% or less, and − It is particularly preferable that it is 0.50% or more and 0.30% or less.

As the material of the microplate 1, resin or glass is preferable.
Resins include polystyrene resin, polyester resin, polyethylene resin, polypropylene, polyethylene terephthalate (PET), polyvinyl chloride, high-density polyethylene, polyether sulfan, PET copolymer, Permanox (Thermofisher Scientific trademark), One selected from cycloolefin polymer resin, Cytop (AGC trademark), 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. The resin constituting the microplate 1 may be one kind or two or more kinds.

Examples of the glass include quartz glass, borosilicate glass, phosphoric acid glass, and chemically strengthened glass. The glass constituting the microplate 1 may be one kind or two or more kinds. If only the bottom surface is made of glass, there is an advantage that it is easy to observe with a microscope.

The peripheral wall portion 12 of the microplate 1 may be transparent or opaque, and is preferably opaque from the viewpoint of observability. When the peripheral wall portion 12 is opaque, it is more preferable to use black or white as the color tone because fluorescence and light emission can be blocked. The method for making the peripheral wall portion 12 opaque is not particularly limited, and for example, a method of adding fine particles, a method of adding a coloring agent such as a pigment, or the like can be used. Carbon can be used in the case of black, titanium oxide or the like can be used in the case of white.

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 injection molding method is preferable because it is easy to manufacture the microplate 1 having a plurality of wells 14 satisfying the condition 1 or the condition 2. Further, the side wall and the bottom may be separately molded and bonded to each other.

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 a cell adhesion inhibitory effect such as a silicone resin or a synthetic resin containing the cell adhesion inhibitor, cells can be formed on the bottom surface 16 without forming a low adhesion coat film. Adhesion can be suppressed.

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.
The microplate 1 may be molded from a resin having a cell adhesion effect.
In addition to the coating agent, physical treatment such as plasma treatment and corona discharge treatment may be performed to facilitate the adhesion of cells.

As described above, in the microplate of the present invention, each well satisfies at least one of condition 1 and condition 2. As a result, the cultured cells cultured in each well 14 can be easily arranged at equal intervals in a plan view. Therefore, even in high-throughput observation using an automatic observation device, it becomes easy to capture the cultured cells of each well, the observation efficiency is improved, and the oversight of the cultured cells can be suppressed.

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. 3 are designated by the same reference numerals, and the description thereof will be omitted.

The bottom surface 16A of the well 14 of the microplate 2 is a concave surface recessed downward in a conical shape. As shown in FIG. 4B, the deepest part of the bottom surface 16A of the well 14 is the center of the well 14. By satisfying at least one of the conditions 1 and 2 in the microplate 2, it becomes easy to capture the cultured cells cultured in each well 14 even with the automatic observation device.

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

[Observation method of cultured cells]
The method for observing cultured cells of the present invention is a method for automatically observing cultured cells cultured in each well using only the microplate of the present invention. For example, using only the microplate 1 that satisfies at least one of the conditions 1 and 2, the culture solution is dispensed into each well 14 to culture the cells, and the cultured cells in each well 14 are automatically observed by an automatic observation device.

Since the microplate 1 used for automatic observation of cultured cells satisfies at least one of condition 1 and condition 2, it is easy to capture the cultured cells of each well 14 even in high-throughput observation using an automatic observation device. Observation efficiency is high.

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 and Example 2]
A 96-well (8 × 12) microplate having an embodiment as illustrated in FIGS. 1 and 2 was prepared by setting the pitch setting value of each well to 9 mm.
The theoretical values (x ₀ , y ₀ ) and the measured values (x, y) of the x-coordinate and the y-coordinate of the center of each well were obtained, and the ratios Q _x and Q _y were calculated. Table 1 shows the maximum and minimum values of the ratios Q _x and Q _y , and the difference between the maximum and minimum values.

[Example 3 and Example 4]
96-well (8 × 12) microplates of aspects as illustrated in FIGS. 1 and 3 were made. Table 2 shows the maximum and minimum values of the ratios Q _x and Q _y , and the difference between the maximum and minimum values.

[Uniformity of well position]
The microplates of each example were observed with an image measuring device (NEXIV VMZ-R4540, manufactured by Nikon Corporation), the proportions Q _x and Q _y of each well were measured, and the uniformity of the well positions was evaluated according to the following criteria. The results are shown in Table 1.
◎ (excellent): The difference between the maximum value and the minimum value of the ratios Q _x and Q _y is 1.00% or less.
〇 (Good): The difference between the maximum and minimum values of the ratios Q _x and Q _y is 2.00% or less, and at least one of them exceeds 1.00%.
× (Defective): At least one of the difference between the maximum value and the minimum value of the ratios Q _x and Q _y is more than 2.00%.

1, 2, ... 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 (3)

A microplate in which multiple wells are arranged in a square shape in a plan view.
The bottom surface of each of the wells is a concave surface that is recessed downward.
The deepest part of the bottom surface of the well is set as the center of the well.
In a plan view, the longest of the two straight lines is the intersection of the two straight lines connecting the midpoints of the opposite sides in the quadrangle whose apex is the center of the four wells located at the four corners. The straight line is the x-axis, and the line perpendicular to the x-axis passing through the origin O is the y-axis.
When the theoretical values of the x-coordinate and y-coordinate of the center of each well calculated from the preset pitch of each well are obtained and compared with the measured values of the x-coordinate and y-coordinate of the center of each well,
Each well represented by (x-x ₀ ) / x ₀ x 100 (where x is the measured value of the x-coordinate of the center of the well and x ₀ is the theoretical value of the x-coordinate of the center of the well). The difference between the maximum value and the minimum value of the deviation ratio in the x-axis direction is 2.00% or less.
And it is represented by (y-y ₀ ) / y ₀ x 100 (where y is the measured value of the y-coordinate of the center of the well and y ₀ is the theoretical value of the y-coordinate of the center of the well). A microplate in which the difference between the maximum value and the minimum value of the deviation ratio in the y-axis direction of each well is 2.00% or less. A microplate in which multiple wells are arranged in a square shape in a plan view.
The bottom surface of each of the wells is a concave surface that is recessed downward.
The deepest part of the bottom surface of the well is set as the center of the well.
In a plan view, the longest of the two straight lines is the intersection of the two straight lines connecting the midpoints of the opposite sides in the quadrangle whose apex is the center of the four wells located at the four corners. The straight line is the x-axis, and the line perpendicular to the x-axis passing through the origin O is the y-axis.
When the theoretical values of the x-coordinate and y-coordinate of the center of each well calculated from the preset pitch of each well are obtained and compared with the measured values of the x-coordinate and y-coordinate of the center of each well,
Each well represented by (x-x ₀ ) / x ₀ x 100 (where x is the measured value of the x-coordinate of the center of the well and x ₀ is the theoretical value of the x-coordinate of the center of the well). The rate of deviation in the x-axis direction is -1.00% or more and 1.00% or less.
And it is represented by (y-y ₀ ) / y ₀ x 100 (where y is the measured value of the y-coordinate of the center of the well and y ₀ is the theoretical value of the y-coordinate of the center of the well). A microplate in which the percentage of deviation of each well in the y-axis direction is −1.00% or more and 1.00% or less. A method for observing cultured cells, which automatically observes the cultured cells cultured in each well using only the microplate according to claim 1 or 2.

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