Microplates

Abstract

To provide a microplate in which cells are easily aggregated in wells, and a cell mass is easily formed.SOLUTION: A microplate 1 having a plurality of wells 14, where a bottom face 16 of wells 14 is a concave surface recessed downward, a low adhesive coating film made of cell adhesion inhibitor is formed on the bottom face 16, and when the radius of the bottom face 16 of the wells 14 is R(mm) in a plan view of the wells 14, a surface roughness Ra of the surface of the low adhesive coating film within a circular region of a radius r (note that r=0.4×R) having the center of the bottom face 16 of wells 14 as a center is 0.1 μm or less.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a microplate. [Background technology]

[0002] Cultured cells are widely used in basic research to elucidate life phenomena, drug discovery research, and the like. In particular, three-dimensional cell masses (spheroids) formed by the aggregation of cells obtained by three-dimensional culture have a three-dimensional structure similar to that in vivo, and are therefore expected to improve test accuracy compared to cells obtained by two-dimensional culture. A microplate with multiple wells arranged vertically and horizontally in a matrix is ​​known as a culture vessel used for three-dimensional culture (Patent Document 1).

[0003] When a culture medium containing suspended cells is dispensed into each well and cultured, the cells aggregate in each well to form a cell mass. If the bottom of the well is concave downward, the cells in each well are likely to aggregate. In addition, in order to inhibit cells from adhering to the bottom of the well, a cell adhesion inhibitor is applied to the bottom of the well to form a low-adhesion coating film. In addition, the well shape, such as the opening angle and curvature radius of the well concave surface, affects the formability of spheroids (for example, Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2019-22527 A Summary of the Invention [Problem to be solved by the invention]

[0005] However, even if the recess of the well has a shape suitable for spheroid formation, if the surface of the well is rough, the cells may get caught in the middle of the well and form incomplete spheroids, or the rate of cell aggregation may be slowed down, resulting in reduced ability to form cell clusters.

[0006] An object of the present invention is to provide a microplate in which cells are likely to associate and form cell clusters within the wells. [Means for solving the problem]

[0007] The present invention has the following aspects. [1] A microplate having a plurality of wells, the bottom surface of the well is a concave surface that is recessed downward; A low adhesion coating film made of a cell adhesion inhibitor is formed on the bottom surface, A microplate in which, when viewed in a plane of the well, the surface roughness Ra of the surface of the low adhesion coating film within a circular area of ​​radius r (where r = 0.4 x R) centered on the center of the bottom surface of the well is 0.1 μm or less, where R (mm) is the radius of the bottom surface of the well. [2] The microplate according to [1], wherein the surface roughness Ra of the bottom part of the well is maximum within the circular region. [3] The microplate according to [2], wherein the surface roughness Ra within the circular region increases toward the bottom. [4] The bottom surface of the well is a concave surface that is hemispherically recessed downward, The microplate according to any one of [1] to [3], wherein the radius of curvature of the arc of the bottom surface of the well in a cross section cut in the height direction through the bottommost part of the bottom surface of the well is 0.5 mm or more and 5 mm or less. [5] The bottom surface of the well is a concave surface that is conically recessed downward; The microplate according to any one of [1] to [3], wherein in a cross section obtained by cutting the well in the height direction so as to pass through the bottommost part of the bottom surface of the well, the inclination angle of the bottom surface of the well with respect to the height direction of the well is 20° or more and 70° or less. [6] The microplate according to any one of [1] to [5], wherein the cell adhesion inhibitor is 2-methacryloyloxyethyl phosphorylcholine or a fluorine-containing polymer. Effect of the Invention

[0008] According to the present invention, a microplate can be provided in which cells can rapidly migrate to the bottommost part of the concave bottom surface within the wells, whereby they can rapidly aggregate and form cell clusters. [Brief description of the drawings]

[0009] [Figure 1] FIG. 2 is a cross-sectional view of a microplate according to an embodiment. [Diagram 2] FIG. 2 is an enlarged cross-sectional view of a well of the microplate in FIG. 1. [Diagram 3] FIG. 11 is a cross-sectional view of a microplate according to another embodiment. [Figure 4] FIG. 3 is an enlarged cross-sectional view of a well of the microplate in FIG. 2. [Diagram 5] FIG. 1 shows the results of observing the bottom surface of the wells 2 hours, 6 hours, and 48 hours after the start of culture in Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The meanings and definitions of terms used in this specification are as follows. A numerical range expressed by "to" means that the numerical values ​​before and after the "to" are the lower and upper limits of the numerical range. When the shape of the bottom surface of the well in plan view is not a perfect circle, the "center of the bottom surface of the well" refers to the center of an inscribed circle relative to the shape of the bottom surface of the well in plan view.

[0011] [Microplate] An example of an embodiment of the microplate of the present invention will be described below with reference to the drawings. Note that the dimensions of the drawings shown in the following description are merely examples, and the present invention is not necessarily limited thereto, and can be appropriately modified and implemented within the scope of the present invention.

[0012] As shown in Figures 1 and 2, the microplate 1 of this embodiment comprises an upper surface portion 10, a peripheral wall portion 12 hanging vertically from the outer edge of the upper surface portion 10, and a plurality of bottomed cylindrical wells 14 each having a circular opening formed on the upper surface 10a of the upper surface portion 10.

[0013] In the microplate 1, the bottom surface 16 of each well 14 serves as the culture surface. In this example, the well 14 has a bottom surface 16 that is concave and recessed downward in a hemispherical shape. This allows cells to move more easily toward the bottom 18 within the well 14, and the cells are more likely to meet together to form cell clusters. Note that the bottom surface 16 of the well 14 is not limited to a hemispherical concave surface, and may be, for example, a concave surface that is recessed downward in a conical shape.

[0014] The opening shape of the wells 14 in 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 may be, for example, 4 to 1536. For example, there may be 96 wells 14 arranged in a rectangular matrix of 8×12 in a vertical and horizontal view, or there may be 384 wells 14 in a 16×24 matrix, or there may be 1536 wells 14 in a 32×48 matrix.

[0015] A low adhesion coating film made of a cell adhesion inhibitor that inhibits cell adhesion is formed on the bottom surface 16 of the well 14. The formation of the low adhesion coating film inhibits cells from adhering to the well surface, and the cells can associate at the bottom of the well to form spheroids. The low adhesion coating film can be formed, for example, by applying a cell adhesion inhibitor. Examples of cell adhesion inhibitors include phospholipid polymers (such as 2-methacryloyloxyethyl phosphorylcholine), polyhydroxyethyl methacrylate, fluorine-containing compounds, and polyethylene glycol. Among these, 2-methacryloyloxyethyl phosphorylcholine or a fluorine-containing polymer is preferred as the cell adhesion inhibitor because of its high affinity with cells. As the cell adhesion inhibitor, one type may be used alone, or two or more types may be used in combination.

[0016] In a plan view of the well 14, the radius of the bottom surface 16 of the well 14 is R (mm), and r = 0.4 × R. In this case, in a plan view of the microplate 1, the surface roughness Ra of the surface of the low adhesion coating film in a circular region (hereinafter also referred to as "circular region A") of radius r centered on the center of the bottom surface 16 of the well 14 is 0.1 μm or less. Note that the surface roughness Ra of the surface of the low adhesion coating film in the circular region A being 0.1 μm or less means that the surface roughness Ra is 0.1 μm or less at any position in the circular region A.

[0017] If the surface roughness Ra within the circular region A of the bottom surface 16 is 0.1 μm or less, the cells will be more likely to migrate toward the bottommost portion 18 within the well 14, and the cells will be more likely to associate with each other and form cell clusters. The surface roughness Ra within the circular region A of the bottom surface 16 is preferably 0.07 μm or less, and more preferably 0.05 μm or less. The smaller the surface roughness Ra within the circular region A, the better, and the substantial lower limit is about 0.02 μm. The surface roughness Ra is an arithmetic mean roughness, and is a value calculated by the following formula 1 when the roughness curve is expressed as y=f(x) (where the unit is micrometers (μm) and L is the measurement length). The surface roughness Ra is measured by the method described in JIS B0601:2001 (ISO4287:1997).

[0018]

number

[0019] Within the circular region A of the bottom surface 16, the surface roughness Ra may be uniform or non-uniform as long as it is 0.1 μm or less. Within the circular region A, it is preferable that the surface roughness Ra of the bottommost part 18 of the bottom surface 16 of the well 14 is maximum. Furthermore, within the circular region A, it is more preferable that the surface roughness Ra increases toward the bottommost part 18 of the bottom surface 16 of the well 14. This makes it relatively difficult for cells to move at the bottommost part 18 in the well 14, and cells tend to gather at the bottommost part 18 in the well 14, making it even easier to form cell clusters.

[0020] The radius R of the bottom surface 16 of the well 14 is preferably 0.2 mm or more and 20 mm or less, more preferably 0.5 mm or more and 10 mm or less, and even more preferably 1 mm or more and 5 mm or less. If the radius R of the bottom surface 16 is equal to or more than the lower limit of the above range, the operability when seeding cells and the observation of spheroids are excellent. If the radius R of the bottom surface 16 is equal to or less than the upper limit of the above range, a large number of spheroids can be formed on one microplate. When the shape of the bottom surface 16 of the well 14 in plan view is not a perfect circle, the radius R is the radius of an inscribed circle relative to the shape of the bottom surface 16 in plan view.

[0021] In a cross section of well 14 cut in the height direction so as to pass through bottommost part 18 of bottom surface 16 of well 14, the radius of curvature of the arc of bottom surface 16 is preferably 0.5 mm to 5 mm, more preferably 1 mm to 4 mm, and even more preferably 1.5 mm to 3.5 mm. If the radius of curvature is equal to or less than the upper limit of the range, cells in well 14 tend to migrate toward bottommost part 18, and cells tend to associate with each other to form cell clusters. If the radius of curvature is equal to or more than the lower limit of the range, spheroids can be formed efficiently and observation is excellent.

[0022] The material of the microplate 1 is preferably resin or glass. Examples of the resin include polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, acrylic resin, polycarbonate resin, and silicone resin. The resin is preferably one selected from polystyrene resin, polyester resin, polyethylene resin, polyethylene terephthalate (PET), polyvinyl chloride, high density polyethylene, polyethersulfane, PET copolymer, Permanox (Thermo Fisher Scientific trademark), cycloolefin polymer resin, Cytop (AGC trademark), acrylic resin, polycarbonate resin, and silicone resin, and polystyrene resin is particularly preferable because of its high transparency and low drug adsorption. The resin constituting the microplate 1 may be one type or two or more types.

[0023] The glass is not particularly limited, and examples thereof include quartz glass, borosilicate glass, phosphate glass, and aluminosilicate glass. The glass may be chemically strengthened to prevent cracking. The glass constituting the microplate 1 may be one type or two or more types.

[0024] The peripheral wall 12 of the microplate 1 may be transparent or opaque, and is preferably opaque from the viewpoint of observation. When the peripheral wall 12 is opaque, the color tone is more preferably black. The method for making the peripheral wall 12 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.

[0025] The manufacturing method of the microplate 1 is not particularly limited, and the microplate 1 can be molded by, for example, injection molding or compression molding. Among them, injection molding is preferred because it is easy to manufacture the microplate 1. The surface roughness Ra of the surface of the low adhesion coating film in the circular area A of the bottom surface 16 of the well 14 can be adjusted by the selection of the molding material, the mold finishing process such as polishing, and the molding method, and further by the low adhesion coating method.

[0026] As described above, in the microplate 1, the bottom surface 16 of the well 14 is concave, and the surface roughness Ra of the surface of the low adhesion coating film within the circular area A of the bottom surface 16 is controlled to 0.1 μm or less. Therefore, even if a low adhesion coating film is formed on the bottom surface 16 of the well 14, the cells tend to migrate toward the bottommost part 18 within the well 14 during cell culture, and the cells tend to associate with each other to form cell clusters.

[0027] The microplate of the present invention is not limited to the above-mentioned embodiment. For example, the microplate of the present invention may be the microplate 2 exemplified in Figures 3 and 4. The microplate 2 has the same configuration as the microplate 1 except that the wells 14 have a bottom surface 16A instead of the bottom surface 16. In Figures 3 and 4, the same parts as those in Figures 1 and 2 are denoted by the same reference numerals and will not be described.

[0028] The bottom surface 16A of the well 14 of the microplate 2 is a concave surface that is recessed downward in a conical shape. In the microplate 2, the surface roughness Ra of the surface of the low adhesion coating film in the circular area A of the bottom surface 16A is controlled to 0.1 μm or less, so that cells tend to migrate toward the bottommost part 18 even if a low adhesion coating film is formed, and the cells tend to associate with each other to form cell clusters.

[0029] In a cross section of well 14 cut in the height direction so as to pass through bottommost part 18 of bottom surface 16A of well 14, the inclination angle θ (FIG. 4) of bottom surface 16A with respect to the height direction of well 14 is preferably 20° or more and 70° or less, more preferably 25° or more and 60° or less, and even more preferably 35° or more and 55° or less. If the inclination angle θ is equal to or less than the upper limit of the range, cells in well 14 tend to migrate toward bottommost part 18, so that cells tend to associate with each other to form cell clusters. If the inclination angle θ is equal to or more than the lower limit of the range, spheroids can be easily observed under a microscope.

[0030] In addition, within the scope of the invention, the components in the above-described embodiments may be replaced with well-known components, and the above-described modified examples may be combined as appropriate. EXAMPLES

[0031] The present invention will be described in detail below 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.

[0032] [Surface roughness Ra] The surface roughness Ra is the arithmetic mean roughness measured by the method described in JIS B0601:2001 (ISO4287:1997).

[0033] [Example 1] A 96-well (8×12) microplate was prepared as shown in FIG. 1 and FIG. 2, in which the bottom surface of each well is a concave surface recessed downward in a hemispherical shape, and the radius R of each well is 3.2 mm. A fluoropolymer was applied to the bottom surface of each well as a cell adhesion inhibitor to form a low adhesion coating film. r=0.4×R. In plan view, the surface roughness Ra of the surface of the low adhesion coating film within a circular region A of radius r centered on the center of the bottom surface 16 of the well 14 increases toward the bottom, and the surface roughness Ra of the bottom was 0.041 μm. Ra was measured using a white light interference three-dimensional shape measuring instrument (Zygo, Newview6200).

[0034] [Example 2] A microplate similar to that of Example 1 was produced, except that the surface roughness Ra of the surface of the low adhesion coating film in the circular region A increases toward the bottom, and the surface roughness Ra at the bottom was 0.218 μm.

[0035] [Culture test] Using the microplates obtained in each example, HepG2 cells were seeded in each well so that the number of cells per well was 2500, and cultured in an incubator at 37°C, 5% CO2, and saturated with water vapor. Using a microscope (ZEISS, Axio Observer7), the bottom of the wells was observed 2 hours, 6 hours, and 48 hours after the start of culture. The observation results of Example 1 are shown in Figure 5.

[0036] As shown in Figure 5, in Example 1, where the surface roughness Ra in the circular region A on the bottom of the well was 0.1 µm or less, the cells tended to migrate toward the bottom in the well, and the cells met with each other to form cell clusters. On the other hand, in Example 2, where the surface roughness Ra in the circular region A on the bottom of the well was more than 0.1 µm, the formation of cell clusters was insufficient. [Explanation of symbols]

[0037] 1,2...microplate, 10...upper surface portion, 12...peripheral wall portion, 14...well, 16,16A...bottom surface, 18...bottom portion.

Claims

1. A microplate having a plurality of wells, wherein the bottom surface of each well is a concave surface recessed downward, a low-adhesion coating film made of a cell adhesion inhibitor is formed on the bottom surface, and when the radius of the bottom surface of each well is R (mm) in a planar view of the well, the surface roughness Ra of the surface of the low-adhesion coating film within a circular region of radius r (where r = 0.4 x R) centered on the center of the bottom surface of the well is 0.1 μm or less, and the surface roughness Ra of the bottommost part of each well is maximum within the circular region.

2. A microplate having a plurality of wells, the bottom surface of each well being a concave surface recessed downward, a low-adhesion coating film made of a cell adhesion inhibitor being formed on the bottom surface, the surface roughness Ra of the surface of the low-adhesion coating film within a circular region of radius r (where r = 0.4 x R) centered on the center of the bottom surface of the well in a planar view of the well being, being 0.1 μm or less when the radius of the bottom surface of the well is R (mm), and the surface roughness Ra within the circular region increases toward the bottommost portion.

3. 2. The microplate of claim 1, wherein the surface roughness Ra within the circular region increases toward the bottom.

4. 4. The microplate according to claim 1, wherein the bottom surface of the well is a concave surface that is hemispherically recessed downward, and the radius of curvature of the arc of the bottom surface in a cross section obtained by cutting the well in the height direction so as to pass through the bottommost part of the bottom surface of the well is 0.5 mm or more and 5 mm or less.

5. The bottom surface of the well is a concave surface that is conically concave downward, The microplate according to any one of claims 1 to 3, wherein an inclination angle of the bottom surface of the well with respect to the height direction of the well in a cross section obtained by cutting the well in the height direction so as to pass through the bottommost part of the bottom surface of the well is 20° or more and 70° or less.

6. 6. The microplate according to claim 1, wherein the cell adhesion inhibitor is 2-methacryloyloxyethyl phosphorylcholine or a fluorine-containing polymer.

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