Cell culture vessel

Abstract

To provide a cell culture vessel that is suitable for an automatic culture / observation device, and facilitates focusing on cultured cells in each well in automatic observation and has excellent observation efficiency.SOLUTION: A cell culture vessel having a plurality of wells has a culture surface on a bottom face of each well, and when the cell culture vessel is mounted on a flat surface plate and a distance D from the center of the culture surface to the surface plate is measured regarding each well, a difference between the maximum value Dmax and the minimum value Dmin of the distance D is 400 μm or less, and the culture surface does not have a flaw of a length of 5.0 mm or more.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to a cell culture vessel. [Background technology]

[0002] Cultured cells are often used as specimens in basic research to elucidate life phenomena and drug discovery research, and culture vessels are widely used to obtain large amounts of specimens. Conventional evaluation of cultured cells has mainly been based on characteristics of cell metabolism or metabolites, such as MTT assay and ELISA (Enzyme-Linked Immuno Sorbent Assay). On the other hand, three-dimensional cell masses (spheroids) obtained by 3D culture, in which cells are aggregated, have a three-dimensional structure similar to that in vivo. For this reason, the importance of morphological observation using cell masses has increased in recent years.

[0003] For cell observation, a method has been developed to observe a large number of cells at high throughput using an automated observation device. However, with conventional microplates, even those that meet the ANSI / SBS standard, it takes time to automatically focus on the cultured cells in each well, which can reduce the time efficiency of the automated observation.

[0004] Patent Document 1 proposes a microplate in which the distance between the deepest points of wells, based on a reference plane calculated by a reference plane calculation method, is 400 μm or less, as a microplate that is suitable for automatic culture / observation equipment and makes it easy to focus on cultured cells in each well during automatic observation. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2022-78638 A Summary of the Invention [Problem to be solved by the invention]

[0006] However, as a result of investigations by the present inventors, it was found that even in the case of the microplate described in Patent Document 1, the reference surface is not necessarily parallel to the surface of the base plate (stage) of the observation device, and therefore in automatic observation using the observation device, tilt may occur with respect to the base plate (stage) of the observation device, making focusing difficult.

[0007] An object of the present invention is to provide a cell culture vessel which is suitable for an automatic culture / observation device, which makes it easy to focus on cultured cells in each well during automatic observation, and which is time-efficient for automatic observation. [Means for solving the problem]

[0008] The present invention includes the following configurations. [1] A cell culture vessel having a plurality of wells, Each well has a culture surface on the bottom surface thereof, The cell culture vessel is placed on a flat surface plate, and the distance D from the center of the culture surface to the surface plate is measured for each well. The maximum value D of the distance D is max and the minimum value D min The difference is 400 μm or less, And, the culture surface has no scratches with a length of 5.0 mm or more. [2] The cell culture vessel according to [1], wherein the culture surface is flat. [3] The maximum value D of the distance D max and the minimum value D min The cell culture vessel according to [1] or [2], wherein the difference in height between the surfaces is 200 μm or less, and the culture surface is free of scratches with a length of 2.0 mm or more. [4] When the thickness T of each well is measured at the center of the culture surface, the maximum value T of the thickness T is max and the minimum value T min The cell culture vessel according to any one of [1] to [3], wherein the difference between the distances is 100 μm or less. [5] The cell culture vessel according to any one of [1] to [4], wherein the culture surface is coated with an extracellular matrix. [6] The cell culture vessel according to any one of [1] to [5], wherein the culture surface is coated with a low cell adhesion coating. [7] The cell culture vessel according to any one of [1] to [6], wherein the culture surface is made of a gas-permeable material. [8] The cell culture vessel according to any one of [1] to [7], wherein a housing other than the culture surface of the cell culture vessel is colored. [9] The cell culture vessel of claim 1, wherein the cell culture vessel comprises a bioplastic. Effect of the Invention

[0009] According to the present invention, there is provided a cell culture vessel which is suitable for an automatic culture / observation device, which allows easy focusing on cultured cells in each well during automatic observation, and which is excellent in time efficiency of automatic observation. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a perspective view showing a cell culture vessel according to an embodiment. [Diagram 2] FIG. 2 is a plan view showing the cell culture vessel of FIG. [Diagram 3] 3 is a cross-sectional view of the cell culture vessel shown in FIG. 2 according to a second embodiment. [Figure 4] FIG. 2 is an enlarged cross-sectional view showing the vicinity of a well in a state where the cell culture vessel is placed on a flat surface plate. [Diagram 5] FIG. 11 is a cross-sectional view showing a cell culture vessel according to another example of the embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] 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. The "center of the culture surface" refers to the center of the culture surface in plan view from above, and coincides with the center of the bottom surface (culture surface) of the well when viewed from above. If the shape of the well in plan view is circular, the center of the circle is the center of the culture surface, and if the shape of the well in plan view is a shape other than circular, the center of the culture surface is the center of a circle inscribed in that shape.

[0012] An example of an embodiment of the cell culture vessel 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 without departing from the gist of the present invention.

[0013] Fig. 1 is a perspective view showing a cell culture vessel 1 according to an embodiment of the present invention, and Fig. 2 is a plan view of the cell culture vessel 1. The cell culture vessel 1 includes an outer frame 10 having a rectangular shape in a plan view, and a plurality of cylindrical wells 12 with bottoms arranged inside the outer frame 10.

[0014] In the example of cell culture vessel 1 shown in Figures 1 to 3, the bottom surface of each well 12 serves as culture surface 13. The cross-sectional shape of the bottom surface of well 12 in this example when cut along a plane parallel to the depth direction passing through the center of the bottom surface in a plan view is linear. That is, in this example of well 12, culture surface 13 is a flat bottom. The cross-sectional shape of the bottom surface of well 12 when cut along a plane parallel to the depth direction passing through the center of the bottom surface in a plan view is not limited to a linear shape, and may be a downwardly convex arc shape (U-bottom), a downwardly convex broken line shape (V-bottom), or the like.

[0015] The opening shape of the well 12 in a plan view is circular in this example, but is not limited thereto and may be, for example, rectangular. The number of wells 12 is six in this example, but is not limited thereto. The number of wells 12 can be, for example, 6 to 1536.

[0016] 2, the wells 12 are arranged in a rectangular matrix of 2×3 pieces vertically and horizontally. Note that the arrangement pattern of the wells 12 is not limited to this pattern, and examples include a rectangular matrix of n×m pieces vertically and horizontally (other than 2×3), and a staggered pattern.

[0017] As shown in FIG. 4, the cell culture vessel 1 is placed on a flat surface plate 100, and the distance D from the center a of the culture surface 13 to the surface plate 100 is measured for each well 12. The maximum value of the distance D is D max and the minimum value D min Difference (D max -D min ) is 400 μm or less. This makes the cell culture vessel 1 suitable for an automatic culture / observation device, and makes it easy to focus on the cultured cells cultured in each well 12 during automatic observation, resulting in excellent time efficiency in automatic observation.

[0018] D max -D min is 400 μm or less, preferably 300 μm or less, more preferably 200 μm or less, and even more preferably 100 μm or less. max -D min If is equal to or less than the upper limit value, the deviation in height of the culture surface 13 of each well 12 is small, making it easy to focus on each cultured cell even in high-throughput observation using an automated observation device, and improving the time efficiency of the automated observation.

[0019] D max -D min It becomes easier to control the thickness of the wells 12 to a smaller value, for example, by providing a reinforcing rib between adjacent wells 12 that is connected to the wells 12 . 2, a reinforcing rib 20 is provided in an area surrounded by four adjacent wells 12 so as to be located at each vertex of a square when viewed from above, the reinforcing rib 20 being connected to each of the four wells 12. The reinforcing rib 20 in this example has a cylindrical portion 21 and four plate portions 22 extending radially from the cylindrical portion 21 toward each well 12 when viewed from above. The reinforcing rib 20 in this form can apply a force uniformly to the four connected wells 12 to reinforce them, which makes it easier to prevent deviations in height of each well 12 from occurring when the wells are released from the mold.

[0020] In the example shown in Fig. 2, two wells 12 adjacent to each other along the side wall 11 of the outer frame 10 as viewed from above, and a reinforcing rib 30 connected to each of the two wells 12 and the outer frame 10 is provided in an area surrounded by the outer frame 10. The reinforcing rib 30 in this example has a cylindrical portion 31 and three plate-piece portions 32 extending radially from the cylindrical portion 31 toward each well 12 and the outer frame 10 as viewed from above. The reinforcing rib 30 in this form can apply a force evenly to the two connected wells 12 and the outer frame 10 to reinforce them, which makes it easier to suppress the occurrence of a deviation in height of each well 12 when released from the mold.

[0021] Also, D max -D min can also be reduced by, for example, using a mold release agent or the like to facilitate removal of the cell culture vessel 1 from the mold.

[0022] The culture surface 13 of each well 12 in the cell culture vessel 1 according to the embodiment has no scratches with a length of 5.0 mm or more. Note that "no scratches with a length of 5.0 mm or more" means that the culture surface 13 has no scratches with a length of 5.0 mm or more in the planar direction. Since there are no scratches longer than 5.0 mm on the culture surface 13 of each well 12, disturbances caused by scratches during focusing are significantly reduced, making it easier to focus on the cultured cells in each well 12 and further improving the time efficiency of automatic observation. In order to further increase the time efficiency of automated observation, it is preferable that the culture surface 13 of each well 12 has no scratches longer than 4.0 mm in length, more preferably no scratches longer than 2.0 mm in length, and even more preferably no scratches longer than 1.0 mm in length.

[0023] The time efficiency of automated observation is further increased by ensuring that the culture surface 13 of each well 12 does not have any gouges deeper than 0.50 mm. Preferably, there are no gouges deeper than 0.40 mm, more preferably, there are no gouges deeper than 0.20 mm, and even more preferably, there are no gouges deeper than 0.10 mm.

[0024] The culture surface 13 of each well 12 has an area of ​​3.14 mm 2 The absence of any cloudiness or scratches makes automated observations more time-efficient. 2 It is preferable that there are no cloudy scratches or scratches of 0.50 mm or more in area. 2 It is more preferable that there are no cloudy scratches or scratches with an area of ​​0.12 mm or more. 2 It is even more preferable that there are no more cloudy spots.

[0025] In the cell culture vessel 1 according to the embodiment, it is easy to focus on the cultured cells in each well, and the time efficiency of automatic observation is particularly excellent. max -D min It is preferable that the thickness of the culture surface 13 is 200 μm or less, and that the culture surface 13 is free of scratches having a length of 2.0 mm or more.

[0026] When the thickness T (FIG. 3) of each well 12 at the center a of the culture surface 13 is measured, the maximum value T max and the minimum value T min The difference (T max -T min ) is preferably 100 μm or less, more preferably 80 μm or less, even more preferably 40 μm or less, and even more preferably 20 μm or less. max -T minIf is equal to or less than the upper limit, it becomes easier to focus on the cultured cells in each well 12, and the time efficiency of automatic observation becomes higher.

[0027] A low cell-adhesion coat for suppressing cell adhesion may be applied to the culture surface 13 of each well 12. By applying the low cell-adhesion coat, it becomes easier to remove the cultured cells from each well 12. The low cell adhesion coat can be achieved, for example, by applying a cell adhesion inhibitor. Examples of cell adhesion inhibitors include phospholipid polymers (e.g., 2-methacryloyloxyethyl phosphorylcholine), polyhydroxyethyl methacrylate, fluorine-containing compounds, polyethylene glycol, etc. One type of cell adhesion inhibitor may be used alone, or two or more types may be used in combination.

[0028] The culture surface 13 of each well 12 may be coated with an extracellular matrix to facilitate the adhesion of cultured cells to form a scaffold. Examples of the extracellular matrix to be used include collagen, gelatin, fibronectin, poly-D-resin, poly-L-lysine, and polyethyleneimine. One type of extracellular matrix may be used alone, or two or more types may be used in combination.

[0029] In order to facilitate obtaining cell clusters of uniform size, a plurality of fine wells of uniform size may be formed on culture surface 13 of well 12. The shape of the opening of the microwell is not particularly limited, and may be, for example, a circle, an ellipse, a polygon, or an irregular shape. The dimensions of the opening of the microwell, such as the average diameter, opening area, and depth, are not particularly limited and may be set appropriately. For example, the average diameter of the opening is 100 to 2500 μm, and the opening area is 7850 to 4906250 μm. 2 The number of fine wells formed on the culture surface 13 is 10 to 10,000 per unit area. 2 can be set to.

[0030] When forming fine wells, the arrangement pattern is not particularly limited, and they may be formed in a regular pattern, an irregular pattern, or a mixture of regular and irregular patterns. The microwells can be formed, for example, by laser irradiation using a CO2 laser or the like. When the culture surface 13 of the wells 12 is irradiated with laser light, the resin that constitutes the culture surface 13 melts and vaporizes, forming microwells with very smooth surfaces. The conditions for laser irradiation can be, for example, a laser output of 1 to 100 W and an irradiation time of 0.1 to 100 μs.

[0031] Examples of materials constituting the cell culture vessel 1 include resins such as polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, acrylic resin, polycarbonate resin, and silicone resin. Among them, polystyrene resin is preferred because of its high transparency and low drug adsorption. As the resin, one type may be used alone, or two or more types may be used in combination. From the viewpoint of low environmental impact, the cell culture vessel 1 preferably contains a bioplastic such as a biomass plastic or a biodegradable plastic.

[0032] The portion including the culture surface 13 of each well 12 is preferably made of a gas-permeable material. Representative thicknesses and ranges of the "gas-permeable material" herein include intermediate values ​​and ranges, and may be, for example, about 25.4 μm to about 635 μm, or about 38.1 μm to about 762 μm. However, if the material has high gas permeability, there is no problem even if it deviates from the above range. For example, in the case of polydimethylsiloxane polymer, it is possible to provide sufficient gas permeability with a thickness of up to about 2.54 cm. As the gas-permeable material, one type may be used alone, or two or more types may be used in combination.

[0033] In order to facilitate observation of the cultured cells, the housing of the cell culture vessel 1 may be colored other than the culture surface 13. More specifically, the peripheral wall portion 14 surrounding the culture surface 13 in the well 12 and the outer frame 10 may be colored. When the peripheral wall 14 or outer frame 10 of the well 12 in the cell culture vessel 1 is colored, the color tone is preferably black. The method for coloring the peripheral wall 14 or outer frame 10 of the well 12 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.

[0034] The method for producing the cell culture vessel 1 is not particularly limited, and the vessel can be molded by, for example, injection molding or compression molding. The cell culture vessel of the present invention can be suitably used in a method for culturing cells in each well and, after the culturing, automatically observing the cultured cells in each well using an automatic observation device having an autofocus mechanism.

[0035] As described above, the cell culture vessel according to the embodiment is placed on a flat surface plate and the distance D from the center of the culture surface to the surface plate is measured for each well. The maximum value D of the distance D is max and the minimum value D min The difference in the distance between the wells is controlled to 400 μm or less. This reduces the deviation in the culture surface of each well, making it easy for an automated observation device to automatically focus on the cultured cells in each well. This allows for efficient high-throughput observation of large numbers of cultured cells using an automated observation device.

[0036] The cell culture vessel of the present invention is not limited to the above-mentioned embodiment. For example, the cell culture vessel of the present invention may be the cell culture vessel 2 illustrated in Fig. 5. The cell culture vessel 2 has the same configuration as the cell culture vessel 1, except that each well 12 is formed by attaching a plate-like member 16 to the bottom of a main body portion 15 having an outer frame 10 and a plurality of cylindrical peripheral wall portions 14 provided inside the outer frame 10. The same parts in Fig. 5 as those in Fig. 3 are denoted by the same reference numerals, and description thereof will be omitted.

[0037] 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

[0038] The present invention will be specifically described below with reference to examples and comparative examples, but the present invention is not limited to the following descriptions. The following Examples 1 to 4 are working examples, and Example 5 is a comparative example.

[0039] [Example 1] As a cell culture vessel, max -D min A 96-well plate (AGC Technoglass) with a flat bottom having a thickness of 400 μm and no scratches of 5.0 mm or more was prepared. TIG-3 cells were cultured at 1 × 10 5 The solution was adjusted to cells / mL, 0.2 mL of the solution was seeded into each well of the 96-well plate described above, and cultured for 24 hours in a humidified incubator at 37°C under 5% CO2 / 95% air.

[0040] [Example 2] As a cell culture vessel, max -D min TIG-3 cells were cultured in the same manner as in Example 1, except that a flat-bottomed 96-well plate (AGC Technoglass) with a microparticle size of 300 μm and no scratches of 4.0 mm or more was used.

[0041] [Example 3] As a cell culture vessel, max -D min TIG-3 cells were cultured in the same manner as in Example 1, except that a flat-bottomed 96-well plate (AGC Technoglass) with a microparticle size of 200 μm and no scratches of 2.0 mm or more was used.

[0042] [Example 4] As a cell culture vessel, max -D min TIG-3 cells were cultured in the same manner as in Example 1, except that a flat-bottomed 96-well plate (AGC Technoglass) with a microparticle size of 100 μm and no scratches of 1.0 mm or more was used.

[0043] [Example 5] As a cell culture vessel, max -D min TIG-3 cells were cultured in the same manner as in Example 1, except that a flat-bottomed 96-well plate (AGC Technoglass) with a 420 μm hole and a scratch of 5.2 mm or more was used.

[0044] [Evaluation of time efficiency of automated observation] In each case, after incubation, automatic observation was performed using a fluorescent and phase contrast motorized inverted microscope system (Carl Zeiss Axio Observer7) and the time required for measurement was measured. The results are shown in Table 1.

[0045] [Table 1]

[0046] As shown in Table 1, in Example 1, image acquisition of all 96 wells was completed in about 40 minutes, and clear images without scratches were obtained. In Example 2, image acquisition of all 96 wells was completed in about 25 minutes, and clear images without scratches were obtained. In Examples 3 and 4, image acquisition of all 96 wells was further shortened to about 20 minutes and about 15 minutes, and clear images without scratches were obtained. On the other hand, in Example 5, it took about 60 minutes to acquire images of all 96 wells using the autofocus mechanism. [Explanation of symbols]

[0047] 1 cell culture vessel 2 Cell culture vessel 10 Outer Frame 11 Side wall 12 well 13 Culture surface 14 Peripheral wall 15 Main body 16 Plate-shaped member 20 Reinforcing rib 21 Cylindrical part 22 Plate piece 30 Reinforcing rib 31 Cylinder section 32 Plate piece 100 Plate

Claims

1. A cell culture vessel having a plurality of wells, Each well has a culture surface on the bottom surface thereof, The cell culture vessel is placed on a flat surface plate, and the distance D from the center of the culture surface to the surface plate is measured for each well. The maximum value D of the distance D is ｍａｘ and the minimum value D ｍｉｎ The difference is 400 μm or less, And, the culture surface has no scratches having a length of 5.0 mm or more.

2. The cell culture vessel according to claim 1 , wherein the culture surface is flat.

3. The maximum value D of the distance D ｍａｘ and the minimum value D ｍｉｎ 2. The cell culture vessel according to claim 1, wherein the difference between the thickness and the thickness of the cell culture vessel is 200 μm or less, and the culture surface is free of scratches having a length of 2.0 mm or more.

4. When the thickness T of each well is measured at the center of the culture surface, the maximum value T of the thickness T is ｍａｘ and the minimum value T ｍｉｎ The cell culture vessel according to claim 1 , wherein the difference is 100 μm or less.

5. The cell culture vessel according to claim 1 , wherein the culture surface is coated with an extracellular matrix.

6. The cell culture vessel according to claim 1 , wherein the culture surface is coated with a low cell adhesion coating.

7. The cell culture vessel according to claim 1 , wherein the culture surface is made of a gas-permeable material.

8. The cell culture vessel according to claim 1 , wherein a housing other than the culture surface of the cell culture vessel is colored.

9. The cell culture vessel of claim 1 , wherein the cell culture vessel comprises a bioplastic.

Images