WO2021132480A1

WO2021132480A1 - Culture substrate and culture dish

Info

Publication number: WO2021132480A1
Application number: PCT/JP2020/048493
Authority: WO
Inventors: 千佳土屋
Original assignee: Ａｇｃ株式会社
Priority date: 2019-12-27
Filing date: 2020-12-24
Publication date: 2021-07-01
Also published as: JPWO2021132480A1

Abstract

The purpose of the present invention is to provide a culture substrate which has excellent adhesion between a culture substrate and a frame body and allows easy observation of cells or the like, and a culture dish including the culture substrate.　Provided is a plate-shaped culture substrate 10 in which at least a part of one surface 10a is a culture region 12, wherein the culture region 12 has a plurality of microwells, and the total amount of warpage of both surfaces is 400 μm or less. Also provided is a culture dish including a culture substrate 10 and a frame body.

Description

Culture medium and culture vessel

The present invention relates to a culture medium and a culture container.

Spheroid culture is well known in which cells derived from humans and animals are artificially cultured in a culture vessel or the like and aggregated three-dimensionally. It is known that in spheroid culture, cell populations form a three-dimensional structure and cells interact with each other, so that they are closer to the three-dimensional structure in vivo and exhibit superior characteristics compared to planar adhesive culture. ing. In fact, spheroid culture is often used for anti-cancer drug screening using cancer cells and for proliferation and differentiation of pluripotent stem cells.

In Patent Document 1, the opening area expands as it approaches the opening end, a recess for accommodating cells and a culture solution is provided at the bottom of the container body, and a plurality of microwells for assembling cells by gravity are provided in the recess. The culture vessel provided on the bottom surface of the above is disclosed. If the culture vessel is not flat, it will be difficult to focus the cells, and it will be easily affected by external light, making it difficult to observe the cells. Therefore, it is desired that the culture vessel has excellent flatness.

In Patent Document 2, in a cell culture plate in which a sheet having microwells is attached to the back surface of a well plate having through holes to form the bottom surface of the wells, injection-molded pingates are arranged at specific positions. It is disclosed to improve the flatness of the.

Japanese Patent No. 5921437 Japanese Unexamined Patent Publication No. 2015-195768

However, as in Patent Document 2, in a culture vessel in which a base material having a plurality of microwells is attached to a frame body, even if the frame body is highly flat, the adhesiveness between the base material and the frame body is lowered, or cells are formed. May be difficult to observe.

An object of the present invention is to provide a culture medium having excellent adhesion between the culture medium and the frame and easy observation of cells and the like, and a culture container provided with the culture medium.

The present invention has the following aspects.
[1] A plate-shaped culture substrate in which at least a part of one surface is a culture region,
The culture area has a plurality of microwells and has a plurality of microwells.
A culture substrate having a total warpage of 400 μm or less on both surfaces.
[2] The culture medium according to [1], wherein the average diameter of the openings of the microwells is 20 μm or more and 1500 μm or less.
[3] The culture medium according to [1] or [2], wherein the average depth of the microwells is 10 μm or more and 1500 μm or less.
[4] The culture medium according to any one of [1] to [3], wherein the plurality of microwells are formed by unevenness formed of a curved surface.
[5] The culture medium according to any one of [1] to [4], wherein the average number of microwells per unit area is 10 pieces / cm ^{2 or more.}
[6] The culture medium according to any one of [1] to [5], wherein the material of the culture medium is synthetic resin or glass.
[7] The culture medium according to any one of [1] to [6], wherein the average thickness of the culture medium is 50 μm or more and 2000 μm or less.
[8] The culture medium according to any one of [1] to [7], wherein the total area of one of the surfaces is 5 cm ² or more and 700 cm ^{2 or less.}
[9] The culture substrate according to any one of [1] to [8], wherein the ratio of the total area of the culture region to the total area of the one surface is 10% or more and 100% or less.
[10] A culture container comprising the culture substrate according to any one of [1] to [9] and a frame.
[11] The culture vessel according to [10], wherein the container shape is any of a flask shape, a petri dish shape, a microplate shape, and a multilayer flask shape.
[12] The culture vessel according to [10] or [11], wherein the culture substrate is attached to the bottom of the frame.
[13] The method for producing a culture medium according to any one of [1] to [9], which comprises performing an annealing treatment in which the culture medium is sandwiched between a pair of flattening plates and heated. A method for producing a culture medium.
[14] The method for producing a culture medium according to [13], wherein the load condition in the annealing treatment is 100 g or more and 2000 g or less.
[15] The method for producing a culture medium according to [13] or [14], wherein the heating conditions in the annealing treatment are 80 ° C. or higher and 100 ° C. or lower.

According to the present invention, it is possible to provide a culture medium having excellent adhesion between the culture medium and the frame and observing cells easily, and a culture container provided with the culture medium.

It is a perspective view which showed an example of the culture medium of this invention. It is sectional drawing which showed an example of the culture container of this invention. It is a top view which showed an example of the culture container of this invention. FIG. 3 is a cross-sectional view taken along the line AA of the culture vessel of FIG. It is sectional drawing which showed an example of the plurality of microwells of a culture area.

The following terms have the following meanings.
The “culture region” means a region on the surface of the culture substrate that constitutes the bottom surface of the portion in which the cells of the culture vessel are cultured and has a plurality of microwells.
A "microwell" is a depression having an opening diameter of 2000 μm or less and a depth of 2000 μm or less.
A "continuous curved surface" is a surface that does not include a flat surface.
"Microwell opening diameter" is the diameter of the microwell opening in the reference plane. When the opening shape of the microwell on the reference plane in a plan view is not a perfect circle, the diameter of the circumscribed circle of the opening shape is defined as the diameter of the opening of the microwell.
The "microwell depth" is the distance between the deepest part of the microwell and the reference plane.
The “reference surface of the culture medium” is a surface including the outer peripheral edge of the surface including the culture region of the culture medium.
For example, when a plurality of microwells are formed on the surface of the culture substrate by laser irradiation, the surface of the culture substrate before laser irradiation coincides with the reference plane. When the culture region is partially provided on a part of the surface of the culture substrate, the surface of the region other than the culture region among the surfaces including the culture region of the culture substrate coincides with the reference plane.
The "frame body" is a member that constitutes a culture container together with a culture base material, and is a member that includes a side wall portion that surrounds the culture area with the culture base material attached to the bottom.

[Culture medium]
The culture substrate of the present invention is a plate-shaped substrate, and is a culture region in which at least a part of one surface has a plurality of microwells. In a state where the culture substrate of the present invention is attached to the bottom of the frame constituting the culture vessel, the region constituting the bottom surface of the portion of the culture medium in which the cells are cultured is formed on the surface facing upward of the culture substrate. It becomes a culture area. The culture region may be partially formed on the surface of the culture substrate or may be formed on the entire surface of the culture substrate.

For example, as shown in FIGS. 1 and 2, in the culture container 100 in which the culture base 10 is attached to the bottom surface 112a of the petri dish-shaped frame 110, the entire surface 10a facing upward of the culture base 10 is cultured. It becomes the area 12. As shown in FIGS. 3 and 4, in the microplate-shaped culture container 200 in which the culture base 10 is attached to the bottom of the frame 210 having a plurality of through holes 212, the surface of the culture base 10 facing upward. Of 10a, the portion that closes the lower end side of each through hole 212 is the culture region 12.
It should be noted that the dimensions and the like of the figures exemplified in the description in the specification are examples, and the present invention is not necessarily limited thereto, and the present invention can be appropriately modified without changing the gist thereof. Is. For example, in the frame 110 of FIG. 1, the culture substrate may be attached to the lower side of the frame without the bottom surface 112a.

The amount of warpage of both surfaces of the culture substrate is 400 μm or less. That is, both the amount of warpage of the surface of the culture substrate including the culture region and the amount of warpage of the surface opposite to the surface including the culture region are 400 μm or less. When the amount of warpage of the entire surface of the culture base material is not more than the above upper limit value, the adhesiveness between the culture base material and the frame is excellent. In addition, the flatness of the culture vessel provided with the culture substrate becomes excellent, the cells can be easily focused, and the cells are less likely to be affected by external light, so that the cells can be easily observed. The amount of warpage of both surfaces of the culture substrate is preferably 380 μm or less, more preferably 350 μm or less.
The amount of warpage of the entire surface of the culture substrate is the difference between the maximum value and the minimum value of the amount of warpage measured by a non-contact shape measurement system using a laser displacement meter.

The ratio of the total area of the culture area to the total area of the surface including the culture area of the culture substrate is preferably 10% or more and 100% or less, and more preferably 30% or more and 90% or less. When the ratio is equal to or higher than the lower limit of the above range, spheroids having a desired size are likely to be formed. When the ratio is not more than the upper limit of the above range, the culture medium is less likely to be distorted, and the adhesiveness to the frame is improved.

The total area of the surface including the culture region of the culture substrate ^{is preferably 5 cm 2} or more and 700 cm ² or less. When the culture substrate is used in the form of a microplate, it is 80 cm ² or more and 120 cm ² or less, when it is used in the dish shape, it is 9 cm ² or more and 150 cm ² or less, and when it is used in the flask shape, it is 25 cm ² or more and 700 cm ² or less, large. When used in the form of a multi-layer flask, 250 cm ² or more and 700 cm ² or less per stage is more preferable. When the total area of the surface including the culture region is equal to or more than the lower limit of the above range, the entire bottom surface of the frame can be covered, and the adhesiveness with the frame is improved. When the total area of the surface including the culture region is not more than the upper limit of the above range, the culture substrate is less likely to be distorted and the adhesiveness to the frame is improved.
When a plurality of culture regions are provided on the surface of the culture substrate, the total area of the culture regions is the total area of the plurality of culture regions.

The plan-view shape of the culture substrate can be appropriately set according to the shape of the frame to be attached, and examples thereof include a round shape and a rectangular shape.

The average thickness of the culture medium is preferably 50 μm or more and 2000 μm or less. When the culture substrate is glass, the average thickness is more preferably 100 μm or more and 250 μm or less, and further preferably 130 μm or more and 200 μm or less. When the culture substrate is a resin, it is more preferably 300 μm or more and 1800 μm or less, further preferably 500 μm or more and 1500 μm or less, and even more preferably 810 μm or more and 1000 μm or less. When the average thickness of the culture base material is within the above range, the deformation of the culture base material due to the formation of microwells can be reduced.

The culture area of the culture medium has a plurality of microwells. By having a plurality of microwells in the culture region, during cell culture, cells fall into the microwells and spheroids are formed in the wells.

For example, as shown in FIG. 5, the plurality of microwells 14 included in the culture region 12 of the culture substrate 10 are formed by irregularities formed of curved surfaces. As described above, it is preferable that the plurality of microwells are composed of irregularities composed of continuous curved surfaces. If the inside of the microwell is a curved surface, a beautiful spherical spheroid is likely to be formed. When the boundary surface between the microwells is a curved surface, cells are less likely to remain on the boundary surface and easily fall into the microwells. The boundary surface between the microwells may be flat. In FIG. 5, the dotted line indicates the reference plane of the culture medium 10. This reference plane is a plane including the outer peripheral edge of the surface including the culture region 12 of the culture substrate 10.
A plurality of microwells composed of irregularities formed of curved surfaces can be formed by, for example, laser irradiation.

The opening shape of the microwell in a plan view can be appropriately selected according to the type of cells to be cultured and the size of the spheroid to be formed, and examples thereof include a round shape, an elliptical shape, a polygonal shape, and a donut shape. ..

The formation pattern of a plurality of microwells can be appropriately selected according to the type of cells to be cultured and the shape of the frame to be attached, and examples thereof include a lattice shape, a polygonal shape, a honeycomb shape, a round shape, and a donut shape. When the culture area is surrounded by the side wall portion of the frame, the microwell formation pattern may be adjusted according to the opening shape of the side wall portion in a plan view. For example, in the case of a microplate-shaped culture vessel in which the opening shape of the side wall portion of the frame body is round in the plan view, the formation pattern can be made round.

The average diameter d (FIG. 5) of the opening of the microwell can be appropriately adjusted according to the desired size of the spheroid, and is preferably 20 μm or more and 1500 μm or less, more preferably 100 μm or more and 1000 μm or less, and further preferably 200 μm or more and 800 μm or less. When the average diameter d of the opening of the microwell is equal to or more than the lower limit of the above range, a spheroid having a desired size is likely to be formed. When the average diameter d of the opening of the microwell is equal to or less than the upper limit of the above range, it is easy to prevent cells from spilling from the microwell, and it is easy to form a spheroid having a uniform size.
The diameter of the opening of the microwell is measured by a laser microscope (manufactured by KEYENCE CORPORATION) or the like.

The average depth D (FIG. 5) of the microwell is preferably 10 μm or more and 1500 μm or less, more preferably 50 μm or more and 1000 μm or less, and further preferably 100 μm or more and 600 μm or less. When the average depth D of the microwells is equal to or greater than the lower limit of the above range, spheroids having a desired size are likely to be formed. When the average depth D of the microwells is equal to or less than the upper limit of the above range, it is easy to prevent cells from spilling from the microwells, and it is easy to form spheroids having a uniform size.
The depth of the microwell is measured by a laser microscope (manufactured by KEYENCE CORPORATION) or the like.

The opening area of the microwell is preferably 0.15 mm ² or more 0.50 mm ² or less, more preferably 0.20 mm ² or more 0.35 mm ² or less, more preferably 0.24 mm ² or more 0.30 mm ² or less. When the opening area of the microwell is equal to or larger than the lower limit of the above range, spheroids having a desired size are likely to be formed. When the opening area of the microwell is equal to or less than the upper limit of the above range, it is easy to prevent cells from spilling from the microwell, and it is easy to form a spheroid having a uniform size.
The opening area of the microwell is measured by a laser microscope (manufactured by KEYENCE CORPORATION) or the like.

The average number of microwells per unit area in the culture region ^{is preferably 10 pieces / cm 2} or more, more preferably 15 pieces / cm ² or more, and even more preferably 20 pieces / cm ² or more. The average number of microwells per unit area in the culture region ^{is preferably 10,000 pcs / cm 2} or less, more preferably 5,000 pcs / cm ² or less, and even more preferably 1000 pcs / cm ² or less. When the average number of microwells per unit area is equal to or greater than the lower limit, spheroids having a desired size are likely to be formed.

As the material of the culture substrate, synthetic resin or glass is preferable.
Examples of the synthetic resin constituting the culture substrate include acrylic resin, polystyrene resin, polyester resin, polycarbonate resin, polypropylene resin, silicone resin, polyethylene terephthalate (PET) resin, vinyl chloride, high-density polyethylene, polyether sulfan, and the like. Examples thereof include PET copolymers, polypropylene resins, Permanox ™, cycloolefin polymer resins, Cytop ™, and the like. When a silicone resin is used as the culture substrate, it is easy to prevent cells from adhering to the surface of the culture substrate. The synthetic resin constituting the culture substrate may be one kind or two or more kinds.
Synthetic resins include substances that suppress cell adhesion (eg, phospholipid polymers (eg 2-methacryloyloxyethyl phosphorylcholine), polyhydroxyethyl methacrylate, fluorine-containing compounds, polyethylene glycol, etc.) and colorants (eg, oxidation). Titanium, carbon, etc.) may be blended.

Examples of the glass constituting the culture medium include quartz glass, borosilicate glass, phosphate glass, and chemically strengthened glass. The glass constituting the culture substrate may be one kind or two or more kinds.

A cell adhesion inhibitor (protein adhesion inhibitor) may be applied to the culture region on the surface of the culture substrate to form a film. By forming the coating film on the surface of the culture region including the inner surface of the microwell, it is possible to prevent cells from adhering to the surface of the culture region. Therefore, it becomes easy for cells to aggregate in the microwell to form a spheroid, and it becomes easy to take out the spheroid from the microwell.

Examples of the cell adhesion inhibitor include phospholipid polymers (2-methacryloyloxyethyl phosphorylcholine, etc.), polyhydroxyethyl methacrylate, 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.

The method for producing the culture medium is not particularly limited. For example, a method of molding using a mold having a cavity surface having a shape complementary to the surface shape of a culture region having a plurality of microwells, and a process of forming a plurality of microwells on the surface of a molded base material. The method of applying can be exemplified.

Examples of the method for forming a plurality of microwells after molding include laser irradiation (CO ₂ laser, YAG laser, excimer laser, etc.), nanoimprint, press, and the like. In particular, when a resin base material _{is irradiated with a CO 2} laser to form microwells, the resin dissolves and vaporizes to smooth the surface of the culture region, so that beautiful spherical spheroids are likely to be formed. Become.

After forming the microwells, it is preferable to perform an annealing treatment in which the culture substrate is sandwiched (loaded) between a pair of flattening plates and heated. When a microstructure such as a microwell is formed, the culture medium itself is easily distorted, but by performing the annealing treatment, it becomes easy to obtain a culture medium having a warp amount of 400 μm or less on both surfaces.
Examples of the material of the flattening plate include stainless steel (SUS) and quartz glass.

The load condition (weight stone condition) for the annealing treatment is preferably 100 g or more and 2000 g or less, and more preferably 500 g or more and 1200 g or less. When the weight stone condition is within the above range, the warp of the culture medium can be uniformly reduced. As the weight stone, it is preferable to use a weight stone having high flatness (warp amount <5 μm) so that the load is evenly applied.
The heating conditions for the annealing treatment are preferably 80 ° C. or higher and 100 ° C. or lower, and more preferably 80 ° C. or higher and 90 ° C. or lower. When the heating conditions are equal to or higher than the lower limit of the above range, the stress in the culture medium can be relaxed and the warpage can be reduced. When the heating conditions are not more than the upper limit of the above range, deformation due to melting of the culture substrate can be suppressed.

[Culture container]
The culture vessel of the present invention includes the culture medium of the present invention and a frame.
In the culture vessel of the present invention, the culture base material of the present invention is attached to the bottom of the frame, so that the culture region on the surface of the culture base material becomes the bottom surface of the portion for culturing cells.

Examples of the container shape of the culture vessel of the present invention include a flask shape, a petri dish shape, a microplate shape, and a multilayer flask shape. The shape of the culture vessel is preferably one of a flask shape, a petri dish shape, a microplate shape, and a multilayer flask shape from the viewpoint that a beautiful spherical spheroid is easily formed.

The shape of the culture vessel can be adjusted by the shape of the frame.
Examples of the petri dish-shaped culture container include the culture container 100 illustrated in FIG. In the culture container 100, the culture base material 10 is attached to the bottom surface 112a of the petri dish-shaped frame 110 so that the culture region 12 faces upward.

The frame body 110 includes a bottom portion 112 and a side wall portion 114 rising from the peripheral edge portion of the bottom portion 112. In the culture container 100, the entire surface 10a of the culture base material 10 is the culture region 12, and the culture region 12 is surrounded by the side wall portion 114 of the frame body 110.

Examples of the microplate-shaped culture container include the culture container 200 illustrated in FIGS. 3 and 4. In the culture container 200, the culture base material 10 is attached to the bottom of the frame body 210 having a plurality of through holes 212.

The frame body 210 includes a flat plate portion 214 having a rectangular shape in a plan view, and a plurality of tubular portions 216 hanging from the lower surface of the flat plate portion 214. The inside of the tubular portion 216 is a through hole 212 leading to the upper surface of the flat plate portion 214. In the culture vessel 200, the portion of the surface 10a of the culture substrate 10 surrounded by each tubular portion 216 is the culture region 12. As described above, in the culture vessel 200, each culture region 12 is surrounded by the side wall portion 216a of the tubular portion 216, and a plurality of recesses 218 for culturing cells are formed.

The arrangement pattern of the recesses 218 in the culture vessel 200, that is, the arrangement pattern of the through holes 212 in the frame body 210 is not particularly limited, and examples thereof include a matrix shape and a staggered shape.
The opening shape of the recess 218 in a plan view is not particularly limited, and examples thereof include a round shape and a rectangular shape.

The average diameter of the opening of the recess 218, that is, the average diameter of the opening of the through hole 212 is preferably 1.5 mm or more and 40 mm or less, and more preferably 1.7 mm or more and 35 mm or less. When the average diameter of the opening of the recess 218 is equal to or more than the lower limit of the above range, it is preferable because it is suitable for drug discovery screening of the formed spheroid. When the average diameter of the opening of the recess 218 is equal to or less than the upper limit of the above range, the shaking of the medium is suppressed and the spheroids can be prevented from popping out.

The average depth of the recess 218 is preferably 4.0 mm or more and 20 mm or less, and more preferably 5.0 mm or more and 18 mm or less. When the average depth of the recesses 218 is equal to or greater than the lower limit of the above range, a sufficient amount of the minimum amount of medium necessary for culturing can be sufficiently added. When the average depth of the recess 218 is equal to or less than the upper limit of the above range, the spheroid can be easily taken out in the recess.
The number of recesses 218 in the culture vessel 200 is not particularly limited, and can be, for example, 1 or more and 1536 or less.

The area of each culture region 12 in the culture container 200 of the microplate shape is preferably 7.0 cm ² or more 80 cm ² or less, more preferably 7.5 cm ² or more 75 cm ² or less. When the area of the culture region 12 is equal to or greater than the lower limit of the above range, the minimum number of cells required for culture can be dropped. When the area of the culture region 12 is equal to or less than the upper limit of the above range, the movement of spheroids due to the shaking of the medium can be reduced.
The area of each culture region 12 can be adjusted by adjusting the diameter of the through hole 212 of the frame body 210.

As the flask-shaped culture container, for example, a culture container in which the culture base material 10 is attached so that the culture region 12 faces upward can be exemplified on the bottom surface of the flask-shaped frame.

The method of attaching the culture substrate to the bottom of the frame is not particularly limited, and examples thereof include a method of adhering the culture substrate to the bottom of the frame by pressure bonding, welding, and an adhesive.
The adhesive used for adhering the culture substrate is, for example, a liquid or tape-based adhesive using a silicone-based, instantaneous-based, epoxy-based, ultraviolet-curable, polypropylene-based, acrylic-based, rubber-based, or polyethylene-based adhesive. Can be exemplified.

As described above, in the present invention, the culture vessel is composed of two members, the frame and the culture base, and the amount of warpage of the entire surface of both the culture bases is controlled to 400 μm or less. As a result, the adhesion between the culture substrate and the frame is excellent, and the culture area in the culture vessel is flattened, so that the cells can be easily focused, are less affected by external light, and the cells can be easily observed. Become.

The technical scope of the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the spirit of the present invention.
For example, in the case of the culture base material 10 applied to the microplate-shaped culture container 200, a plurality of microwells may be formed on the entire surface 10a of the culture base material 10, and a frame body forming each recess 218. It may be partially formed only in the region surrounded by the side wall portion 216a of 210.

In the

culture containers

100 and 200 described above, one culture substrate was attached to one frame, but the culture container of the present invention is not limited to such a mode. For example, a mode in which a plurality of culture substrates are attached to one frame may be used. The embodiment in which a plurality of culture substrates are attached to one frame is advantageous in that it can be easily applied to mass culture in a large culture vessel such as a large flask shape or a petri dish shape. Further, since the culture medium of the present invention is excellent in flatness, even in a mode in which a plurality of culture substrates are attached to one frame, the adhesion is excellent and cells can be easily observed.

In addition, it is possible to replace the constituent elements in the above-described embodiment with well-known constituent elements as appropriate without departing from the spirit of the present invention, and the above-mentioned modified examples 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.
[Measurement of warpage amount]
Device name: Using Dyvoce (manufactured by Kozu Seiki Co., Ltd.), the amount of warpage of the entire surface of the culture substrate was measured by a non-contact shape measurement system using a laser displacement meter. In the measurement, the culture medium of the sample was supported at three points, and the measurement points were continuously moved in one direction at a pitch of 1 mm and a moving speed of 50,000 μm / sec in each of the X and Y directions. The amount of warpage was measured on both the surface including the culture region (plane A) and the surface opposite to the surface containing the culture region (plane B). The amount of warpage of the entire surface of the culture substrate was defined as the difference between the maximum value and the minimum value of the measured amount of warpage.

[Example 1]
A rectangular plate-shaped base material (average thickness: 1000 μm) of 150 mm × 105 mm was molded using polystyrene resin. Next, the entire surface of one surface of the plate-shaped substrate _{is irradiated with a CO 2} laser to form a plurality of microwells composed of irregularities having continuous curved surfaces as illustrated in FIG. did.
After forming the microwells, the base material was sandwiched between a pair of quartz glass flattening plates and subjected to annealing treatment under the conditions of a load of 585 g and a temperature of 80 ° C. for 3 hours to obtain a culture base material.

[Example 2]
A culture substrate was produced in the same manner as in Example 1 except that the annealing treatment was not performed.

[Evaluation of cell observation]
The culture substrates of Examples 1 and 2 were adhered to the bottom of the frame having the same shape as the frame 210 illustrated in FIGS. 3 and 4 with a synthetic rubber adhesive to prepare a microplate-shaped culture container. .. HepG2 cells were seeded in the culture area of the culture vessel, and qualitative evaluation was performed by microscopic observation on the 5th day. In microscopic observation, those in which it was confirmed that spheroid formation was formed were judged to have good cell observability (〇), and those in which spheroid formation could not be confirmed were judged to have poor cell observability (×).

[Evaluation of adhesiveness]
Adhesion was evaluated based on the presence or absence of medium leakage during cell culture in the evaluation of cell observability. In the visual judgment, the medium with no leakage was judged to have good adhesiveness (◯), and the medium with leakage was judged to have poor adhesion (×).

Table 1 shows the evaluation of each example.

As shown in Table 1, the culture medium of Example 1 in which the amount of warpage of both surfaces is 400 μm or less is superior to the culture medium of Example 2 in which the amount of warpage exceeds 400 μm, and has excellent adhesiveness to the frame. Also, cell observation was easy.

The entire contents of the specification, claims, drawings and abstract of Japanese Patent Application No. 2019-238095 filed on December 27, 2019 are cited here as disclosure of the specification of the present invention. It is something to incorporate.

10 ... Culture substrate, 10a ... Surface, 12 ... Culture area, 14 ... Microwell, 100 ... Culture container, 110 ... Frame, 112 ... Bottom, 112a ... Bottom, 114 ... Side wall, 200 ... Culture container, 212 ... Through hole, 210 ... frame body, 214 ... flat plate portion, 216 ... tubular portion, 216a ... side wall portion, 218 ... recessed portion.

Claims

A plate-shaped culture substrate in which at least a part of one surface is a culture region,
The culture area has a plurality of microwells and has a plurality of microwells.
A culture substrate having a total warpage of 400 μm or less on both surfaces.
The culture medium according to claim 1, wherein the average diameter of the openings of the microwells is 20 μm or more and 1500 μm or less.
The culture medium according to claim 1 or 2, wherein the average depth of the microwells is 10 μm or more and 1500 μm or less.
The culture medium according to any one of claims 1 to 3, wherein the plurality of microwells are formed by unevenness formed of a curved surface.
The culture medium according to any one of claims 1 to 4, wherein the average number of microwells per unit area is 10 pieces / cm 2 or more.
The culture medium according to any one of claims 1 to 5, wherein the material of the culture medium is synthetic resin or glass.
The culture medium according to any one of claims 1 to 6, wherein the average thickness of the culture medium is 50 μm or more and 2000 μm or less.
The culture medium according to any one of claims 1 to 7, wherein the total area of one of the surfaces is 5 cm 2 or more and 700 cm 2 or less.
The culture substrate according to any one of claims 1 to 8, wherein the ratio of the total area of the culture area to the total area of the one surface is 10% or more and 100% or less.
A culture container comprising the culture medium and the frame according to any one of claims 1 to 9.
The culture vessel according to claim 10, wherein the container shape is any of a flask shape, a petri dish shape, a microplate shape, and a multilayer flask shape.
The culture vessel according to claim 10 or 11, wherein the culture substrate is attached to the bottom of the frame.
The method for producing a culture medium according to any one of claims 1 to 9, wherein the culture medium is annealed by sandwiching the culture medium between a pair of flattening plates and heating the culture medium. Method.
The method for producing a culture medium according to claim 13, wherein the load condition in the annealing treatment is 100 g or more and 2000 g or less.
The method for producing a culture medium according to claim 13 or 14, wherein the heating conditions in the annealing treatment are 80 ° C. or higher and 100 ° C. or lower.