JP2020114191A - Culture container, culture method and culture device - Google Patents

Abstract

To provide a culture container comprising a culture part on which a plurality of recesses for storing a culture object such as a sphere are formed, capable of preventing movement of the sphere or the like between the recesses even when a culture medium is fed at a flow rate of 1 mL/minute or greater.SOLUTION: There is provided a culture container comprising a culture part on which a plurality of recesses for storing a sphere, are formed, the culture container has at least one port, a depth of the recess is 50-500 μm, at least a part of a side wall of the recess is almost vertical, and a length in a vertical direction of the almost vertical part of the side wall is longer than a maximum diameter of the sphere.SELECTED DRAWING: Figure 3

Description

TECHNICAL FIELD The present invention relates to a cell culture technique, and more particularly to a technique for efficiently producing spheres.

In recent years, in the case of culturing a large amount of adherent cells such as stem cells such as iPS cells and ES cells, not only the cells are allowed to adhere to a culture container to proliferate, but also a material having low adhesiveness to cells is applied. A method of culturing cells in a three-dimensional state closer to that of a living body by forming spheres (spheroids, aggregates) and organoids using a culture vessel equipped with a microwell plate and the like is used. ..

In the culture of spheres using such a culture container, there is a problem that the spheres jump out of a well (recess) and move to other wells, and it is difficult to obtain a sphere having a desired size and shape.
That is, in order to exchange the medium, when the medium is fed into a culture bag (a bag-shaped culture container) provided with a large number of wells in the culture section, the sphere easily jumps out of the well and moves to another well ( Hereinafter, this is referred to as movement of spheres), so that the flow rate of the medium needs to be extremely slowed, and rapid medium exchange was extremely difficult. Therefore, such a culture vessel is not suitable for long-term culture that requires frequent medium replacement.

Japanese Patent No. 2716646

The migration of spheres frequently occurs when using a culture bag with shallow wells, but it is preferable to use a culture bag with shallow wells in order to properly perform medium exchange.
That is, in the case of using a culture bag having a shallow well having a well depth of less than 500 μm, the movement of the sphere generally occurs unless the velocity (flow rate) of feeding the medium is 0.1 mL/min or less. Therefore, there is a problem that it takes a long time to completely discharge the medium from the culture bag. For example, when the amount of medium in the culture bag is 10 mL, it takes 100 minutes to discharge at a flow rate of 0.1 mL/min, which may cause damage to cells.

It is also conceivable to use culture bags with deep wells to prevent migration of the spheres. However, such a culture bag has a problem that the medium cannot be exchanged sufficiently.
For example, using a culture bag having 1000 wells having an opening width of 2 mm and a depth of 700 μm, 2 mL of a medium in which lactic acid was dissolved was filled, and then 10 mL of a medium without lactic acid was added at a flow rate of 1 mL/min. Then, the solution was transferred into the culture bag. Then, the amount of medium remaining in the well was calculated by measuring the lactic acid concentration of the medium in the well. As a result, it was found that about 20% of the original medium remained in the wells. In this case, it was expected that the 200 μm spheres would be entirely buried in the remaining original medium, which would hinder the culture.

On the other hand, it is also possible to invert the culture bag in a state where a predetermined treatment is applied so that the spheres do not jump out from the well, and the medium is discharged from the well. However, even with this method, about 7% of the medium remained in the wells due to surface tension. Further, when the culture bag is inverted, the sphere may be damaged, such as sticking to the upper surface of the culture bag. Therefore, it is desirable to exchange the culture medium by feeding the culture medium to the culture bag.
On the other hand, it is not unthinkable to carry out the culture by not replacing all the medium but replacing only half of the medium. However, when actually carried out, it was not possible to appropriately create spheres of iPS cells by this method.

Currently, spheres for research are being prepared by using a culture container such as a rigid microwell plate. However, with such a conventional method, it is difficult to obtain a large culture area and it is not suitable for mass production of spheres.
In order to create a large amount of spheres, it is preferable to use a culture bag having a shallow well with a size of about A4 (about 600 cm ² ), for example. When culture is performed using such a culture bag with a liquid thickness of the medium of 2 mm to 10 mm, the culture bag is filled with 120 ml to 600 ml of the medium. In this case, even if the flow rate of the medium is set to 1 mL/min to 10 mL/min, it is possible to discharge the medium without causing movement of spheres. However, when culturing with a liquid thickness of the medium of 1 mm or less, movement of the spheres occurs unless the flow rate of the medium is 0.1 mL/min or less. Therefore, for example, when the amount of the medium is 60 mL, the medium is It took 10 hours to discharge, and it was difficult to put into practical use.

Therefore, the inventors of the present invention have conducted diligent research to make at least a part of the side wall of the well substantially vertical even in a culture container having a shallow well having a depth of about 50 to 500 μm, and to determine the vertical direction of the substantially vertical portion. The present invention has been completed by finding that by making the length of the sphere larger than half the maximum diameter of the sphere, it is possible to prevent the movement of the sphere even when the medium is fed at a flow rate of 1 mL/min or more in the medium exchange.

Here, Patent Document 1 can be cited as a prior document that describes a culture container including a well in which at least a part of the side wall is substantially vertical.
Patent Document 1 discloses a culture vessel having a funnel-shaped bottom surface with an angle of 120 degrees or less, a hemispherical shape with a radius of curvature of 10 mm or less, or a shape in which the central portion of the bottom surface is a flat surface. However, these culture vessels are intended to form spheres in a rigid culture vessel, and cannot solve the above-mentioned problems in the case of feeding the medium to the culture bag.

That is, for example, when a culture bag made of a soft packaging material is used, it is necessary to greatly change the flow rate of the culture medium according to the thickness between the upper surface portion and the lower surface portion thereof, so that the movement of the spheres is prevented and Control has been an important issue.
For example, as described above, if the liquid thickness of the medium is 2 mm or more, it is possible to discharge the medium without causing the movement of the spheres even if the flow rate of the medium is 1 mL/min. If it is 1 mm or less, the movement of the sphere will occur unless the flow rate of the medium is slowed down to about 0.1 mL/min. Further, when the liquid thickness of the medium was 0.5 mm or less, the flow rate of the medium had to be 0.05 mL/min or less.

The present invention has been made in view of the above circumstances, and in a culture container having a culture part in which a plurality of recesses for accommodating a culture object such as a sphere is formed, the medium is supplied at a flow rate of 1 mL/min or more. An object of the present invention is to provide a culture container, a culture method, and a culture device capable of preventing the movement of spheres or the like between recesses even when liquid is fed.

In order to achieve the above object, the culture container of the present invention is a culture container having a culture part in which a plurality of recesses for accommodating spheres are formed, and at least one port is provided, and the depth of the recesses is provided. Is 50 to 500 μm, at least a part of the side wall of the recess is substantially vertical, and the vertical length of the substantially vertical portion of the side wall is longer than half the maximum diameter of the sphere.

Further, the culture method of the present invention is a method of controlling the flow rate of the medium by using the above-mentioned culture container, injecting the medium from one port and discharging the medium from the other port.

Further, the culture apparatus of the present invention is provided with a pressing plate in contact with the upper surface of the culture container, the culture container is pressed by the pressing plate, the pressing plate, the injection or discharge of the medium to the culture container With this, it is movable in the vertical direction.

According to the present invention, in a culture container having a culture part having a plurality of recesses for accommodating an object to be cultured such as spheres, even if the medium is fed at a flow rate of 1 mL/min or more, It is possible to provide a culture container, a culture method, and a culture device capable of preventing the movement of spheres and the like.

It is a schematic diagram which shows the structure of the culture container which concerns on embodiment of this invention. It is a schematic diagram which shows the structure of the modification of the culture container which concerns on embodiment of this invention. It is a schematic diagram which shows the culture|cultivation part in the culture container which concerns on embodiment of this invention. It is a figure which shows the photograph of the culture container which concerns on embodiment of this invention. It is a schematic diagram which shows the structure of the culture apparatus which concerns on embodiment of this invention. It is a schematic diagram which shows the state which has arrange|positioned the modification of the culture container in the culture apparatus which concerns on embodiment of this invention. FIG. 3 is a view showing a micrograph of a culture part of the culture container prepared in Example 1. FIG. 3 is a view showing a micrograph showing a state in which resin beads are placed in the culture container prepared in Example 1 to feed a medium. FIG. 3 is a view showing a micrograph showing a state where iPS cell spheres are housed in the culture container prepared in Example 1. FIG. 5 is a view showing a micrograph showing a state in which T cells are accommodated in the culture container created in Example 2.

Hereinafter, embodiments of a culture container, a culture method, and a culture apparatus of the present invention will be described in detail with reference to the drawings. However, the present invention is not limited to the specific contents of the following embodiments and examples.

As shown in FIG. 1, the culture container 10 of the present embodiment is a culture container having a culture part 11 in which a plurality of recesses (wells) for accommodating an object to be cultured such as a sphere is formed. Two ports 13 are provided. A tube 14 is connected to the port 13 so that the medium 1 is injected into the culture container 10 and the medium 1 is discharged from the culture container 10 by a pump or the like (not shown) arranged in the tube 14. Has become.

Further, in FIG. 1, the culture container 10 is made of a soft packaging material, is formed in a bag shape, and has a top plate portion 12. The distance (liquid thickness L) between the culture section 11 and the top plate section 12 is changed according to the amount of the medium 1 injected into the culture container 10.
Further, a modified example of the culture container 10 of the present embodiment is shown in FIG. In this modification, the liquid thickness of the medium 1 in the culture container 10a can be made more uniform, and all the recesses can be brought into contact with the installation surface in parallel. This makes it possible to reduce the difference in the proliferative property of the culture object and the variation in the number of cells at the time of cell seeding. Other configurations are the same as those of the culture container 10 shown in FIG.

As shown in FIG. 3, at least a part of the side wall of the plurality of recesses in the culture section 11 is formed in a substantially vertical shape. The culture object 2 is accommodated in these recesses, and the vertical length (b) of the substantially vertical portion of the side wall is longer than half the maximum diameter (c) of the culture object 2.
The shape of the region where the side wall of the recess is substantially vertical can be, for example, a columnar shape, a quadrangular prism shape, or the like. Further, the arrangement of the plurality of recesses in the culture section 11 may be, for example, a staggered pattern or a lattice pattern.
With such a configuration of the recess in the culture container 10, when the medium 1 is fed from the port 13 to the culture container 10, the culture object 2 pops out of the recess even if the flow rate is 1 mL/min or more. Therefore, it is possible to prevent it from moving to another recess.

When the culture object 2 is a sphere, the depth (a) of the recess is preferably 50 to 500 μm. This is because if the depth (a) of the recess is larger than 500 μm, it may be difficult to sufficiently replace the medium in the recess even when the medium 1 is fed to the culture container 10. Further, it is because the culture container having the recesses deeper than 500 μm has a high difficulty in processing.

Here, one sphere can be formed by aggregating about 200 to several thousand single cells (single cells). The size of the sphere thus formed is small, about 50 μm to 100 μm, and large, about 200 μm to 300 μm.
Therefore, the depth (a) of the recess is preferably 75 to 500 μm, and more preferably 100 to 500 μm. In addition, the depth (a) of the recess is preferably 100 to 400 μm, and more preferably 100 to 250 μm.

The shape of the bottom portion in the recess is not particularly limited, but when the culture object 2 is a sphere, in order to facilitate aggregation of single cells and favorably form a sphere, a cone shape, a hemisphere shape Or, it is preferable that the shape is rounded.

When the culture object 2 is a single cell, the depth (a) of the recess is preferably 5 to 50 μm. This is because the size of the single cell is about 6 μm to 15 μm, and often about 10 μm. Therefore, in this case, the depth (a) of the recess is preferably 6 to 50 μm, and more preferably 10 to 50 μm. In addition, the depth (a) of the recess is preferably 10 to 40 μm, and more preferably 10 to 25 μm.

The shape of the opening of the recess is not particularly limited, and may be circular or rectangular such as square. Further, the width of the opening of the recess can be appropriately set according to the size of the culture object 2.
For example, when the culture object 2 is a sphere, the lower limit of the diameter of the circle or the inscribed circle of the opening of the recess is 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, 100 μm or more, 110 μm or more, 120 μm or more, 150 μm or more. The above may be applied. The upper limit of the diameter of the circle or the inscribed circle of the opening of the recess may be 1 mm or less, 900 μm or less, 800 μm or less, 700 μm or less, 500 μm or less.
When the culture object 2 is a single cell, the lower limit of the diameter of the circle or the inscribed circle of the opening of the recess may be 5 μm or more, 6 μm or more, 8 μm or more. The upper limit of the diameter of the circle or the inscribed circle of the opening of the recess may be 50 μm or less, 40 μm or less, 30 μm or less.

In addition, the culture container 10 of the present embodiment preferably has two ports 13, and these ports 13 are provided in the vertical direction (parallel to the bottom surface) with respect to the substantially vertical portion of the side wall of the recess, and both ends of the culture container are provided. It is preferable that they are provided facing each other.
In addition, it is preferable to use such a culture vessel 10 to inject the medium 1 from one port 13 and discharge the medium 1 from the other port 13 to control the flow rate of the medium.
At this time, the flow rate of the medium 1 is preferably controlled to 1 mL/min or more, more preferably 3 mL/min or more, further preferably 5 mL/min or more, and more preferably 10 mL/min or more. It is even more preferred to control.

A photograph of such a culture container of the present embodiment is shown in FIG. Further, FIG. 7 shows a micrograph of a part of the culture part of the culture container. This culture container is provided with about 50,000 recesses in the culture section, the depth of the recesses is 250 μm, the vertical length of the substantially vertical portion of the side wall of the recesses is 170 μm, and the opening of the recesses is The shape is square, the diameter of the inscribed circle is 260 μm, the bottom of the recess is pyramidal, and the spacing between the openings of the recess is 40 μm.

Further, two ports are provided so as to face each other at both ends of the culture container in a direction perpendicular to a substantially vertical portion of the side wall of the recess.
As will be described later, a sphere having a diameter of 200 μm is put in the recess of the culture container, the medium is injected from one port of the culture container, the medium is discharged from the other port, and the flow rate of the medium is 3 mL/min or the like. Controlled to. As a result, it was not observed that the resin beads jumped out of the recess and moved to other recesses.

Here, the flow rate of the medium in the present embodiment can be calculated from the amount of medium to be injected and discharged, but the actual flow rate of the medium in the culture container 10 varies depending on the location in the culture container 10 and the change in the liquid thickness. .. That is, since the flow velocity in the vicinity of the port 13 is the highest in the culture container 10, it is important that the culture object 2 does not move from the concave portion in this region.
Further, as the liquid thickness is reduced by pressing the top plate portion 12, the flow velocity becomes faster. Therefore, even in such a case, it is important that the culture object 2 does not move from the recess.

As a material for the culture vessel 10 of the present embodiment, a polyolefin resin such as polyethylene or polypropylene can be preferably used. Examples thereof include polyethylene, a copolymer of ethylene and α-olefin, a copolymer of ethylene and vinyl acetate, an ethylene and acrylic acid or methacrylic acid copolymer, and an ionomer using a metal ion. Further, polyolefin, styrene-based elastomer, polyester-based thermoplastic elastomer, silicone-based thermoplastic elastomer, silicone resin and the like can also be used. Furthermore, silicone rubber, soft vinyl chloride resin, polybutadiene resin, ethylene-vinyl acetate copolymer, chlorinated polyethylene resin, polyurethane thermoplastic elastomer, polyester thermoplastic elastomer, silicone thermoplastic elastomer, styrene elastomer, for example, SBS (styrene/butadiene/styrene), SIS (styrene/isoprene/styrene), SEBS (styrene/ethylene/butylene/styrene), SEPS (styrene/ethylene/propylene/styrene), polyolefin resin, fluorine resin, etc. Good.

Moreover, when the culture object 2 is a sphere, it is preferable to perform a low-adhesion surface treatment coating on the culture part 11 in the culture container 10 of the present embodiment so that the spheres and single cells do not adhere to each other. .. Specifically, it is preferable to apply a cell adhesion inhibitor (cell low adhesion treatment agent) in advance.
As the cell adhesion inhibitor, phospholipid polymer, polyvinyl alcohol derivative, phospholipid/polymer complex, polyhydroxyethyl methacrylate, polyvinyl alcohol, agarose, chitosan, polyethylene glycol, albumin and the like can be used. Also, these may be used in combination.

When the culture object 2 is a sphere, the culture container 10 of the present embodiment is used for a step of forming a sphere, a step of culturing and proliferating while maintaining a sphere state, a step of inducing differentiation in a sphere state, and the like. You can
In addition, a method of forming spheres using differentiated cells obtained from iPS cells, ES cells, and other stem cells that have already undergone a differentiation-inducing step, or a method in which differentiation-induced cells are cryopreserved and thawed again to form spheres It can also be used in methods and the like.

In addition, in the top plate portion 12, a top plate protrusion is provided inside the culture container 10 so that the width of the top protrusion is smaller than the width of the opening of the recess and the height of the top protrusion is reduced. The diameter is made smaller than the minimum diameter of the culture object 2 and a gap is formed between the top surface 111 and the top plate portion 12 when the top plate protrusion contacts a part of the top surface 111 of the culture portion 11. Is also preferable.

When the culture container 10 of the present embodiment has such a configuration, due to the shape of the recess of the culture section 11, it is possible to prevent the movement of the sphere between the recesses even when the medium is fed at a flow rate of 1 mL/min or more. In addition, for example, when the medium needs to be exchanged more quickly, the top plate protrusion is brought into contact with a part of the upper end surface 111 to form a gap smaller than the size of the culture object 2 as the upper end surface 111. It can be in a state of being formed on the top plate portion 12. This makes it possible to prevent the movement of the culture object such as the sphere while allowing the liquid matter such as the medium to pass through the gap.

Further, as shown in FIG. 5, the culture device of the present embodiment includes a culture container 10, a loading table 20, guide pins 21, and a pressing plate 30. Further, FIG. 6 shows an example in which the culture container 10a shown in FIG. 2 is arranged in this culture device.
The culture container 10 is arranged on the loading table 20, and the pressing plate 30 is arranged in contact with the upper surface thereof. Guide pins 21 are provided upright at four corners of the loading table 20, and the pressing plate 30 is supported by these guide pins 21 so as to be vertically movable.

That is, the culture container 10 can be fixed so as to be pressed with a constant pressure by the weight of the pressing plate 30. In addition, a spring material can be installed on the guide pin 21 and the pressing plate 20 can be appropriately pressed against the culture container 10. As a result, the pressing plate 30 can be moved in the vertical direction as the culture medium is poured into or discharged from the culture container 10.
According to the culture apparatus of the present embodiment, with such a configuration, the flow rate of the medium in the culture container 10 can be controlled more appropriately.

As described above, according to the culture container, the culture method, and the culture device of the present embodiment, in the culture container having the culture part having a plurality of recesses for accommodating the culture object such as the sphere, the medium It is possible to prevent the movement of spheres or the like between the concave portions even when the solution is sent at a flow rate of 1 mL/min or more.

Hereinafter, a test performed for confirming the effect of the culture container according to the embodiment of the present invention will be described.

(Example 1)
As the culture container of Example 1, the depth of the recess was 250 μm, the vertical length of the substantially vertical portion of the side wall of the recess was 170 μm, the shape of the opening of the recess was square, and the inscribed circle was The diameter was 260 μm, the bottom of the recess was pyramid-shaped, the spacing between the openings of the recess was 40 μm, and the culture section was equipped with 50,000 recesses. A micrograph of the prepared culture container is shown in FIG. The photograph on the left side of the figure shows a part of the culture vessel, and the photograph on the right is an enlarged part thereof.
In addition, a simulated sphere and a real sphere were prepared as culture objects. The pseudo sphere is a resin bead having a uniform diameter of 200 μm, and the actual sphere is an iPS sphere having a diameter of about 80 μm.

StemFit AK02N (product number RCAK02N, Ajinomoto Co., Inc.) is used as the medium, and after filling the inside of the culture container, the artificial spheres and the real spheres are separately injected into the culture container and dispersed throughout the whole, and the inside of the recess Housed in. FIG. 8 shows a micrograph of the state in which the resin beads are placed and the medium is being sent. Further, FIG. 9 shows a micrograph of the culture container in a state in which the iPS cell spheres are contained.

Then, the pump attached to the port was driven to feed the medium at three flow rates of 0.2 mL/min, 1 mL/min, and 3 mL/min, and a microscope was used to check whether the spheres had moved. It was confirmed by visual observation.
As a result, in the culture container of Example 1, spheres did not move at all three flow rates of both the artificial sphere and the real sphere.

(Reference example 1)
As the culture vessel of Reference Example 1, the depth of the recess was 500 μm, the side wall of the recess was rounded, the shape of the opening of the recess was circular, the diameter was 1 mm, and the distance between the openings of the recess was Was 50 μm, and a culture part having 4000 recesses was prepared.
Further, as the culture objects, similar to Example 1, a pseudo sphere and a real sphere were prepared.

After the culture medium similar to that used in Example 1 was filled in the culture container, the artificial spheres and the real spheres were separately injected into the culture container, dispersed throughout, and housed in the recess.
Then, the pump attached to the port was driven to feed the medium at three flow rates of 0.2 mL/min, 1 mL/min, and 3 mL/min, and a microscope was used to check whether the spheres had moved. It was confirmed by visual observation.
As a result, in the culture container of Reference Example 1, spheres did not move at all three flow rates of both the artificial sphere and the real sphere.

(Comparative Example 1)
As the culture vessel of Comparative Example 1, the depth of the recess was 250 μm, the sidewall of the recess was rounded, the shape of the opening of the recess was circular, the diameter was 1 mm, and the distance between the openings of the recess was Was 50 μm, and a culture part having 4000 recesses was prepared.
Further, as the culture objects, similar to Example 1, a pseudo sphere and a real sphere were prepared.

After the culture medium similar to that used in Example 1 was filled in the culture container, the artificial spheres and the real spheres were separately injected into the culture container, dispersed throughout, and housed in the recess.
Then, the pump attached to the port was driven to feed the medium at three flow rates of 0.2 mL/min, 1 mL/min, and 3 mL/min, and a microscope was used to check whether the spheres had moved. It was confirmed by visual observation.
As a result, in the culture container of Comparative Example 1, movement of spheres occurred at all three flow rates of both the simulated sphere and the real sphere.

(Example 2)
As the culture container of Example 2, the depth of the recess was 30 μm, the vertical length of the substantially vertical portion of the side wall of the recess was 20 μm, the shape of the opening of the recess was square, and the inscribed circle was The diameter was 20 μm, the bottom of the recess was pyramid-shaped, the spacing between the openings of the recess was 10 μm, and the culture part was provided with 100,000 recesses.
In addition, a single cell (human T cell) was prepared as a culture object.

After filling the culture medium with the same medium as in Example 1, T cells were injected into the culture vessel, dispersed throughout, and housed in the recesses. FIG. 10 shows a micrograph of a culture container containing T cells.
Then, the pump attached to the port was driven to feed the medium at three flow rates of 0.2 mL/min, 1 mL/min, and 3 mL/min, and a microscope was used to check whether single cell migration occurred. It was confirmed by the visual observation used.
As a result, in the culture container of Example 2, single cell migration did not occur at all three flow rates.

Needless to say, the present invention is not limited to the above embodiments and examples, and various modifications can be made within the scope of the present invention. For example, the cell type and the size of the culture vessel are not limited to those shown in the examples, and may be of a size capable of forming, for example, 500,000 to 1 million spheres or tens of millions of single cells. It can be appropriately changed, for example, by making it culturable.

INDUSTRIAL APPLICABILITY The present invention can be suitably used for efficiently producing a large number of uniformly-sized spheres arranged at equal intervals.

10 culture container 11 culture part 12 top plate part 13 port 14 tube 20 loading platform 21 guide pin 30 pressing plate

Claims (9)

A culture container having a culture part having a plurality of recesses for accommodating spheres, comprising at least one port, the depth of the recesses being 50 to 500 μm, and at least one side wall of the recesses. The culture vessel is characterized in that the portion is substantially vertical, and the vertical length of the substantially vertical portion of the side wall is longer than half the maximum diameter of the sphere. What is claimed is: 1. A culture container having a culture part having a plurality of recesses for accommodating single cells, comprising at least one port, the depth of the recesses being 5 to 50 µm, At least a part is substantially vertical, and the vertical length of the substantially vertical portion of the side wall is longer than half the maximum diameter of a single cell. The culture container according to claim 1 or 2, wherein the culture container is made of a soft packaging material. The culture container according to any one of claims 1 to 3, which has a bag shape. 5. The culture container according to claim 4, wherein two ports are provided so as to face each other at both ends of the culture container in a direction perpendicular to the substantially vertical portion of the side wall. The culture vessel according to any one of claims 1 to 5, wherein the bottom of the recess has a pyramidal shape, a hemispherical shape, or a rounded shape. A culture method comprising using the culture container according to claim 5 to inject a medium from one port and discharge the medium from the other port to control the flow rate of the medium. The culture method according to claim 7, wherein the flow rate of the medium is controlled to 1 mL/min or more. A pressing plate is provided in contact with the upper surface of the culture container according to any one of claims 1 to 6, the culture container is pressed by the pressing plate, and the pressing plate injects or discharges a culture medium into the culture container. A culture device which is movable in the vertical direction with