WO2017163378A1

WO2017163378A1 - Culture vessel

Info

Publication number: WO2017163378A1
Application number: PCT/JP2016/059439
Authority: WO
Inventors: 宏昭紀伊; 魚住　孝之; 泰次郎清田; 林　大輔; 春男大久保
Original assignee: 株式会社ニコン; 住友ベークライト株式会社
Priority date: 2016-03-24
Filing date: 2016-03-24
Publication date: 2017-09-28

Abstract

The purpose of the present invention is to provide a culture vessel which allows for highly precise observation without increasing costs. This culture vessel is provided with: a bottom wall part (10) having a culture surface (51); a side wall part (30) disposed along the peripheral edge of the bottom wall part and having an inner peripheral surface (31); a peripheral edge surface (55) disposed to space the outer peripheral edge of the culture surface and the inner peripheral surface apart from each other by a predetermined distance and having a contiguous part (56) which is contiguous with the inner peripheral surface; and a culture chamber for cultivation using a predetermined amount of liquid, the culture chamber being surrounded by the culture surface, the side wall part and the peripheral edge surface. The volume of the culture chamber to the contiguous part is less than the predetermined amount. The predetermined amount is an amount of liquid which is contained in the culture chamber such that the liquid surface is in contact with the inner peripheral surface at a position above the contiguous part where the inner peripheral surface is contiguous with the peripheral edge surface.

Description

Culture vessel

The present invention relates to a culture vessel. More specifically, the present invention relates to a culture vessel having a meniscus effect.

Recently, an apparatus capable of time-lapse observation and live observation of a plurality of samples (stem cells, iPS cells, various differentiated cells, etc.) while maintaining position reproducibility has been used. This type of device enables culturing of cells in a plurality of samples in a culture vessel, and automatic observation / photographing within the culture environment of the cells being cultured. There are advantages such as less damage caused by environmental exposure during microscopic observation, and automatic observation, recording and management of cells in culture, reducing the burden on the cultivator.

When a culture solution is placed in a culture vessel, a concave meniscus is generated at the interface due to the interfacial tension of the solution. In particular, when a gel is used for culturing, this tendency becomes more prominent because the viscosity of the solution before gelation is high and the solution tends to adhere to the inner wall surface of the culture vessel. In the observation of the culture surface, the meniscus forms a shadow in the portion where it occurs, which hinders observation of the cultured cells present in the portion.

As a technique aiming at solving the problem of meniscus, Patent Document 1 discloses a layer A in which a solution that is in a gel state by containing a biological substance on the bottom of a culture vessel and being heated is frozen in an ungelled state. And a layer B frozen with an aqueous solution that does not become a gel state when heated, is superimposed on the layer A, and a cell culture device is disclosed. Patent Document 2 discloses a technique for flattening at least a part of a meniscus of a culture solution by floating a transparent flat plate on the culture solution.

JP 2001-340070 A Japanese Patent Laid-Open No. 5-181068

However, the technique described in Patent Document 1 requires a device for freezing and humidifying the solution, resulting in an increase in cost. In addition, for the technique described in Patent Document 2, it is necessary to prepare a flat plate separately, which increases costs, and there is a possibility that bubbles are formed between the flat plate and the solution or cause contamination. is there.

The present invention has been made in consideration of the above points, and an object of the present invention is to provide a culture vessel that enables high-precision observation without incurring an increase in cost.

According to the first aspect of the present invention, a bottom wall portion having a culture surface, a side wall portion having an inner peripheral surface disposed along a peripheral edge of the bottom wall portion, an outer peripheral end portion of the culture surface, and the inner portion A predetermined amount of liquid is used that is provided at a predetermined distance from the peripheral surface and is surrounded by a peripheral surface having a connecting portion with the inner peripheral surface, the culture surface, the side wall, and the peripheral surface. A culture chamber in which culture is performed, and a volume to the connection portion in the culture chamber is less than the predetermined amount, and the predetermined amount is a liquid level of the liquid accommodated in the culture chamber and the inner peripheral surface A culture container is provided in which the contact position is higher than the position of the connecting portion of the inner peripheral surface with the peripheral surface.

In the present invention, it is possible to provide a culture vessel that enables highly accurate observation.

1 is a diagram showing an embodiment of the present invention, and is a schematic external perspective view of a culture vessel 100. FIG. FIG. 2 is a cross-sectional view taken along line AA in FIG. FIG. 2 is a cross-sectional view taken along line AA in FIG. It is a figure which shows a mode that the culture surface 51 of the barrier surface 53 vicinity in the culture container 100 was observed. It is a figure which shows a mode that the culture surface of the side wall part vicinity was observed on the same conditions using the conventional 100mmDish.

Hereinafter, embodiments of the culture vessel of the present invention will be described with reference to FIGS. 1 to 5.
The following embodiment shows one aspect of the present invention and does not limit the present invention, and can be arbitrarily changed within the scope of the technical idea of the present invention. Moreover, in the following drawings, in order to make each configuration easy to understand, the actual structure is different from the scale and number of each structure.

Also, in the following description, the shape or positional relationship of each part will be described with the direction in which the culture surface is arranged as the horizontal direction and the direction perpendicular to the culture surface as the vertical direction. However, this merely defines the horizontal direction and the vertical direction for the convenience of explanation, and does not limit the orientation when the culture vessel according to the present invention is used.

FIG. 1 is a schematic external perspective view of the culture vessel 100. 2 is a cross-sectional view taken along line AA in FIG.
As shown in FIGS. 1 and 2, the culture vessel 100 is surrounded by the bottom wall portion 10, the side wall portion 30 disposed along the periphery of the bottom wall portion 10, and the bottom wall portion 10 and the side wall portion 30. And a culture chamber 50. The peripheral surface 55 is provided at a predetermined distance between the outer peripheral end of the culture surface 53 and the inner peripheral surface 31. The peripheral surface 55 is connected to the inner peripheral surface 31 via the connection portion 56. The outer peripheral edge is a boundary portion of the culture surface 53 region.

The bottom wall portion 10 has a plate shape arranged along a horizontal plane and is formed in a rectangular shape in plan view. The side wall portion 30 is arranged in a substantially rectangular frame shape (the rectangular four corner portions may be curved) on the periphery of the bottom wall portion 10b. Accordingly, the culture vessel 100 has a rectangular parallelepiped culture chamber 50 that opens upward.

As shown in FIG. 2, the side wall portion 30 is disposed on the outer peripheral side, as shown in FIG. And a step 43 formed between the first outer peripheral surface 41 and the second outer peripheral surface 42 in parallel with the horizontal plane. The first outer peripheral surface 41 constitutes a fitting portion into which the lid body 60 that closes the culture chamber 50 is fitted.

As shown in FIG. 1, the first outer peripheral surface 41 located on the long side of the side wall portion 30 and the first outer peripheral surface 41 located on one short side in the long side direction (the back side in FIG. 1). Are connected by an arcuate curved surface 44 in plan view. Moreover, the 1st outer peripheral surface 41 located in a long side among the side wall parts 30, and the 1st outer peripheral surface 41 located in the other short side (front side in FIG. 1) of a long side direction are circular arc by planar view. They are connected by a curved surface 45 having a shape and a curvature larger than that of the curved surface 44. That is, the first outer peripheral surface 41 is formed asymmetrically with respect to the long side direction with the short side direction as the center line. Accordingly, when the first outer peripheral surface 41 is fitted to the lid body 60, the lid body cannot be fitted unless it is in a prescribed direction, and the lid body is prevented from being assembled incorrectly.

The culture chamber 50 has a culture surface 51 at the bottom that is the inside of the side wall 30. At the edge of the culture surface 51, a barrier surface 53 that rises from the culture surface 51 at a height H1 is provided at a predetermined height. The barrier surface 53 is formed with a gradient (inclination angle) of about 2 degrees as an example. The barrier surface 53 is provided at a predetermined distance W from the inner peripheral surface 31 of the side wall portion 30. The predetermined distance W in the present embodiment is defined by the distance between the lower end of the inner peripheral surface 31 and the upper end of the barrier surface 53 in the side wall portion 30.

A peripheral surface 55 is provided at the upper end of the barrier surface 53. The peripheral surface 55 extends outward from the upper end of the barrier surface 53 and is connected to the inner peripheral surface 31 of the side wall portion 30. The peripheral surface 55 is inclined at an inclination angle θ gradually downward as it goes inward from the inner peripheral surface 31. A height H <b> 2 from the culture surface 51 at a position where the peripheral surface 55 is connected to the inner peripheral surface 31 of the side wall portion 30 is set according to the amount of the medium put into the culture chamber 50. More specifically, the height H <b> 2 is set such that the liquid level of the medium placed in the culture chamber 50 is above the connection portion 56 between the peripheral surface 55 and the inner peripheral surface 31. In other words, the volume up to the connection portion 56 in the culture chamber 50 is set to be less than the amount of the medium used for culture in the culture chamber 50.

The barrier surface 53 and the peripheral surface 55 are formed over the entire circumference inside the side wall 30 as shown in FIG. The peripheral surface 55 arranged inside the long side wall portion 30 and the peripheral surface 55 arranged inside the short side wall portion 30 are connected by a curved surface 57 at each intersection. The curved surface 57 is inclined at the same angle as the inclination angle θ of the peripheral surface 55 and is formed around an axis orthogonal to the culture surface 51. Since the barrier surface 53 and the peripheral surface 55 are formed on the inner side of the side wall portion 30 over the entire circumference, the culture surface 51 is surrounded by the barrier surface 53.

3 is a cross-sectional view taken along line AA in FIG. 1 in which a culture solution (medium) B is placed in the culture chamber 50. FIG. As shown in FIG. 3, a curved meniscus M is formed on the culture surface S of the culture solution B placed in the culture chamber 50 at the contact portion with the inner peripheral surface 31 of the side wall portion 30 due to the surface tension of the liquid. Arise. When observing the growth of observation objects such as cells to be cultured in the culture chamber 50 using a microscope or the like, observation with transmitted illumination (bright field, phase difference, differential interference, etc.) is essential, but to observe the observation object. May be refracted by the meniscus M, and the light may not reach the observation target. When this phenomenon occurs, the contrast of the object to be observed under the microscope is extremely deteriorated, and the illumination light does not reach the terminal part near the inner peripheral surface 31 at all, making observation difficult. In addition, if the appearance of the observation target is not uniform depending on the location, image analysis such as cell counting and morphological analysis becomes difficult, which may be a factor that hinders accurate analysis.

In the culture vessel 100 of the present embodiment, the boundary of the culture surface 51 by the barrier surface 53 is provided with a predetermined distance W from the inner peripheral surface 31. The predetermined distance W is set to be larger than the distance DM from the inner peripheral surface 31 to the end of the meniscus M. Therefore, the observation target cultured on the culture surface 51 can be illuminated with observation illumination light in a state where the influence of the meniscus M is suppressed, and high-precision observation is possible. In the culture vessel 100 of the present embodiment, since the barrier surface 53 having a height H1 rising from the culture surface 51 is provided at the periphery of the culture surface 51, the cultured cells from the culture surface 51 to the barrier surface 53 side are provided. Can be prevented.

In the culture vessel 100 of the present embodiment, the peripheral surface 55 provided between the upper end of the barrier surface 53 and the inner peripheral surface 31 of the side wall 30 is gradually inclined downward at an inclination angle θ as it goes inward. Therefore, it is possible to introduce cells or the like staying outside the culture surface 51 into the culture surface 51. Further, the peripheral surface 55 provided inside the long side of the side wall portion 30 and the peripheral surface 55 provided inside the short side of the side wall portion 30 are inclined at the same angle as the inclination angle θ of the peripheral surface 55. Since they are connected by the curved surface 57, it is possible to introduce cells or the like staying outside the culture surface 51 into the culture surface 51 even inside the portion where the long side and the short side of the side wall portion 30 intersect.

Further, by providing the curved surface 57, it is possible to prevent cells from collecting in the corner portion at the bottom of the container. In addition, operations such as sweeping out cells with a culture instrument such as a scraper (such as a brush) are facilitated.

As the size of the culture vessel 100, for example, a standardized outer shape having an outer length of 127.76 mm, an outer width of 85.48 mm, and a height of 14.35 mm can be adopted. This size is one of the standards developed by SBS (the Society Society for Biomolecular Screening) and approved by ANSI (American National Standards Institute) (so-called 100 mmDish). Incidentally, the thickness of the first outer peripheral surface 41 is about 2 to 3 mm.

The culture vessel 100 can be made of a transparent material. Examples of the material having transparency include inorganic substances typified by glass and quartz, and organic substances typified by synthetic resins. Synthetic resins include polystyrene (PS), polypropylene (PP), polymethylpentene (PMP), polycarbonate (PC), polymethyl methacrylate (PMMA), polymethylacrylmethylimide (PMMI), and cycloolefin copolymer (COC). And the like. Furthermore, the copolymer synthesize | combined from two or more of the monomer units of these polymers is also mentioned. In the case of a synthetic resin, it is preferable because it is easy to mold and is difficult to break.

The culture vessel 100 may be a structure formed by integral molding, or may be a structure formed by combining a member constituting the culture surface 51 and a member constituting the peripheral wall portion 30 and the barrier surface 53, for example. When the structure is a combination of members, each member may be made of different materials.

The culture surface 51, the barrier surface 53, the peripheral surface 55 and the inner peripheral surface 31 of the culture vessel 100 may be subjected to a surface treatment from the viewpoint of physical, chemical and / or biochemistry. Such a surface treatment can be appropriately selected by those skilled in the art. For example, at least one of the barrier surface 53, the peripheral surface 55, and the inner peripheral surface 31 may be subjected to a surface treatment for inhibiting cell culture.

Area of the culture surface 51 in the culture container 100, substantially 55cm ² or more, approximately 58cm ² or less. When the area of the culture surface 51 is about 55 cm ² or more and about 58 cm ² or less, a culture area equivalent to the SBS standard 100 mmDish can be secured. Many protocols such as the number of seeded cells, the amount of medium used, the amount of serum, the time to change the medium, the amount of various coatings, the time to confluence, the number of cells at confluence, etc. are built in accordance with the containers specified in the SBS standard. Yes. Therefore, when the culture vessel 100 of the present embodiment is used, it becomes possible to work while referring to the protocol constructed in the SBS standard vessel, and cell culture according to the purpose can be performed easily and without error. Is possible.

The predetermined distance W between the inner peripheral surface 31 and the barrier surface 53 in the side wall portion 30, that is, the predetermined distance W between the inner peripheral surface 31 and the culture surface 51 is approximately 10 mm or more and approximately 14 mm or less (when there is no barrier surface 53). However, when there is the barrier surface 53, approximately 13 mm or less is preferable. That is, the height of the liquid surface when the same amount of liquid is accommodated varies depending on whether the barrier surface 53 is present or not. Therefore, in consideration of changes in the liquid level, when the peripheral surface 55 is inclined, the height at which the liquid surface contacts the inner peripheral surface 31 can be adjusted by changing the length of the peripheral surface 55. It becomes possible. If the predetermined distance W is less than about 10 mm, the distance from the inner peripheral surface 31 to the end of the meniscus M is shorter than the distance DM from the culture surface 51, and part of the illumination light that illuminates the cells on the culture surface 51 Since it passes through the meniscus M, the observation accuracy may be adversely affected. On the other hand, if the predetermined distance W exceeds approximately 13 mm, the area of the culture surface 51 becomes small, and there is a possibility that the area of approximately 55 cm ² or more cannot be secured. In the culture container 100 of the present embodiment, the predetermined distance W is about 10 mm or more and about 14 mm or less, so that the culture surface 51 can be observed with high accuracy without a part of the illumination light passing through the meniscus M, and SBS. A culture area equivalent to the standard 100 mm dish can be secured.

It is preferable that the peripheral surface 55 in the culture vessel 100 is gradually inclined downward as it goes inward from the inner peripheral surface 31, and the inclination angle θ satisfies about 0 <θ ≦ about 5.5. When the inclination angle θ is 0 ° or less, that is, parallel to the horizontal surface or inclined upward in the direction from the inner peripheral surface 31 toward the inner side, the seedling is seeded and positioned outside the culture surface 51. A cell that stays on the peripheral surface 55 or moves in the direction toward the inner peripheral surface 31 due to its own weight at the time of sowing may cause an undesirable situation. In the culture container 100 of the present embodiment, since the inclination angle θ is larger than approximately 0 degrees, the cells that have been seeded and are located outside the culture surface 51 do not stay on the peripheral surface 55, and do not stay on the peripheral surface 55. It moves along the inclination of 55 and can be introduced into the culture surface 51.

On the other hand, when the inclination angle θ of the peripheral surface 55 is increased, the height H2 of the intersection between the peripheral surface 55 and the inner peripheral surface 31 is increased. Also, when the height H1 of the barrier surface 53 that defines the culture surface 51 is increased, the height H2 of the intersection between the peripheral surface 55 and the inner peripheral surface 31 that is inclined starting from the upper end of the barrier surface 53 is large. Become. Therefore, the barrier surface 53 needs to have a height that can prevent the cells grown on the culture surface 51 from growing on the peripheral surface 55. In addition, the amount of medium generally used in the SBS standard 100 mm dish is about 10 ml, but when about 10 ml of the culture medium B is put in the culture chamber 50, the liquid level of the culture liquid level S is higher than the height H2. If it is low, the culture liquid surface S is formed at a position in contact with the peripheral surface 55. In this case, the meniscus M is formed on the inner side than the case where the culture liquid surface S is in contact with the inner peripheral surface 31, and the inner end of the meniscus M is positioned above the culture surface 51 to illuminate the cells on the culture surface 51. There is a possibility that part of the illumination light to be refracted by the meniscus M.

In the present embodiment, the height H1 of the barrier surface 53 is approximately 0.5 mm, and the inclination angle θ of the peripheral surface 55 is defined as approximately 0 <θ ≦ approximately 5.5. The seeded cells can move along the peripheral surface 55 inclined toward the culture surface 51 and be introduced into the culture surface 51, and the liquid level of the culture liquid surface S becomes higher than the height H2 so that the culture liquid surface S is changed. Since it contacts with the inner peripheral surface 31, the meniscus M can be formed at a position outside the culture surface 51, and adverse effects on the observation accuracy of cells and the like on the culture surface 51 can be suppressed.
In addition, the present invention may have either a container having a barrier surface 53 whose culture surface is recessed with respect to the peripheral surface 55 and a configuration without the barrier surface 53. The configuration without the barrier surface 53 is a configuration in which the peripheral surface 53 is formed from the outer peripheral end of the culture surface. In the present invention, the position of the liquid surface of the liquid accommodated in the culture chamber is important. In the case of the configuration in which the barrier surface 53 is formed, the height of the barrier surface 53 is also an important factor. Further, it is possible to adjust by the combination of the inclination angle of the peripheral surface 55 and the length of the peripheral surface 55.

For example, when the culture vessel 100 is used for regenerative medicine, the final product is a cell in regenerative medicine, and the cell can be a product sold as a medicine. In this case, how to evaluate the quality of the cells as a product is a big issue. Unlike drugs (low molecular weight compounds), cells are alive and have different states, so sampling and testing cannot guarantee the quality of the entire product. Further, in the process of culturing, unexpected impurities (for example, those differentiated into cells different from the target cells) may easily appear. It is extremely difficult to control them to produce only a single cell. Therefore, it is very important in this field to provide a means for observing all the cells in the container in terms of improving the evaluation performance. In the culture vessel 100 of the present embodiment, as described above, since cells and the like located outside the culture surface 51 can be introduced into the culture surface 51 via the peripheral surface 55 during seeding, the influence of the meniscus M is suppressed. In this state, it is possible to observe all the cells in the culture vessel 100 with high accuracy. Therefore, when the culture container 100 of the present embodiment is used, it is possible to accurately perform image analysis such as cell counting and morphological analysis based on the result observed with high accuracy.

[Example]
Cells cultured using the culture vessel 100 were observed. The culture vessel 100 was manufactured by injection molding using polystyrene. The outer shape of the culture vessel 100 was 127.76 mm in the long side direction and 85.48 mm in the short side direction. The culture surface 51 in the culture vessel 100 has a shape in which the length in the long side direction is 100 mm, the length in the short side direction is 58 mm, and the corner portion is connected in an arc shape with a radius of 17.5 mm. In this case, the area of the culture surface 51 was about 5537 mm ² (55.37 cm ² ).

The width of the peripheral surface 55 in the culture vessel 100 was 10.5 mm (entire circumference) in both the long side direction and the short side direction from the lower end of the inner peripheral surface 31 to the upper end of the barrier surface 53. Further, the inclination angle θ of the peripheral surface 55 was set to 5 degrees. The height H2 at this time was approximately 1.42 mm.

10 ml of the culture solution B was put into the culture chamber 50 in the culture vessel 100. As the culture solution B, a basal medium DMEM (Dulbecco-Folkto modified Eagle's minimum essential medium) was used. In addition to the above, for example, in the case of iPS cells, the culture solution B uses “mTeSR1 (registered trademark)” manufactured by Veritas Co., Ltd. and “Stemfit (registered trademark)” manufactured by Ajinomoto Co., Inc. Can do.

When 10 ml of the culture broth B was put into the culture chamber 50, the culture liquid level S was formed at a position about 0.1 mm higher than the height H2. In this case, the inner end portion of the meniscus M was formed at a position outside the culture surface 51.

FIG. 4 is a view showing a state in which the culture surface 51 in the vicinity of the barrier surface 53 in the culture vessel 100 is observed. Moreover, FIG. 5 is a figure which shows a mode that the culture surface of the side wall part vicinity was observed on the same conditions using the conventional 100 mmDish. As shown in FIG. 4, when observed using the culture vessel 100, it was confirmed that a uniform image could be observed without being affected by the meniscus M. On the other hand, as shown in FIG. 5, when observed using a 100 mm dish, uneven illumination occurs between the container center and the side wall due to the influence of the meniscus M, and a wide range of cell observation is performed with high accuracy. It was difficult.

In addition, the following conditions for reducing the influence of the meniscus in this example are that when the liquid (approximately 10 ml to approximately 20 ml) is accommodated in the culture chamber in the culture container, the liquid level of the liquid is the inner peripheral surface and the peripheral edge of the container. It is a precondition that the surface is above the connecting portion to which the surface is connected. The following conditions are based on the preconditions when such a culture solution is accommodated in a container.
1) Configuration with a barrier surface
1-1) When the inclination angle of the peripheral surface is about 5 degrees, the culture area is 55 cm ² , and the barrier surface height (step) is about 0 mm <a = about 0.6 mm, the length of the peripheral surface is about 10.5 mm.
1-2) When the inclination angle of the peripheral surface is approximately 3 degrees, the culture area is approximately 55 cm ² , and the barrier surface height (step) is approximately 0 mm <a = approximately 1.0 mm, the peripheral surface length is approximately 10.5 mm.
1-3) When the inclination angle of the peripheral surface is about 1 degree, the culture area is about 55 cm ² , and the barrier surface height (step) is about 0 mm <a = about 1.5 mm, the peripheral surface length is about 10.5 mm.
The inclination angle of 1-4) peripheral surface about 5 degrees, the peripheral length when the inclination angle of the barrier surface and 0.5 degrees if about 10.5 mm, can be secured culture area of about 55 ~ 58cm ² 1 -5) If the height of the barrier surface is about 2.0 mm, the culture area is 55 cm ² , and the peripheral length is about 10.5 mm, the peripheral surface does not need to be inclined 2) Configuration without barrier surface
2-1) When the culture area is approximately 55 cm ² and the inclination angle of the peripheral surface is approximately 0 ° <b = approximately 7 °, the length of the peripheral surface is approximately 10.5 mm.
2-2) When the culture area is approximately 55 cm ² and the inclination angle of the peripheral surface is approximately 5 degrees, the length of the peripheral surface is approximately 0 mm <b = approximately 13 mm.
2-3) When the culture area is approximately 58 cm ² and the inclination angle of the peripheral surface is approximately 5 degrees, the length of the peripheral surface is approximately 0 mm <b = approximately 11.5 mm

As described above, the preferred embodiments according to the present invention have been described with reference to the accompanying drawings, but it goes without saying that the present invention is not limited to such examples. Various shapes, combinations, and the like of the constituent members shown in the above-described examples are examples, and various modifications can be made based on design requirements and the like without departing from the gist of the present invention.

In this example, the volume up to the connection in the culture chamber was less than about 10 ml. However, if the amount of liquid in the central portion of the liquid surface due to the meniscus effect is more strictly considered, the volume up to the connection in the culture chamber The volume can be about 11 ml or less.
Also. In the present invention, the basic condition is that approximately 10 ml of the culture solution is accommodated in the culture chamber, but it is also possible to accommodate 10 ml or more of liquid. The influence of the meniscus can be reduced. For example, when a 20 ml liquid storage container is assumed on the premise of storing 20 ml of liquid, the volume to the connection part of the culture chamber may be less than about 20 ml. That is, in the case of a dedicated container in which the amount of liquid to be stored is limited, it may be assumed that the volume up to the connection portion is set to be less than the amount of liquid to be used.

For example, in the said embodiment, although the barrier surface 53 and the peripheral surface 55 illustrated the structure formed over the perimeter along the side wall part 30, it is not limited to this structure, All the circumferences The structure provided only in one part may be sufficient. In this case, the circumferential lengths of the barrier surface 53 and the peripheral surface 55 are preferably at least twice the predetermined distance W. By making the length in the circumferential direction of the barrier surface 53 and the peripheral surface 55 more than twice the predetermined distance W, the influence of the meniscus M formed in the region where the barrier surface 53 and the peripheral surface 55 are not provided is eliminated. The culture surface 51 can be observed in the state.

Moreover, in the said embodiment, although the structure which the culture container 100 has a rectangular external shape was illustrated, it is good also as a culture container circular in planar view other than a rectangular shape. Also in this case, the size of the culture container is preferably a size based on the above-mentioned 100 mmDish and the area of the culture surface is about 55 cm ² or more and about 58 cm ² or less.

In the above embodiment, even when 10 ml of the culture solution is used, the equation of approximately 0 <θ ≦ approximately 5.5 is defined as the inclination angle θ at which the culture solution surface S does not contact the peripheral surface 55. In the case of using a culture solution that exceeds 1 °, the inclination angle may exceed the angle defined by the above formula. That is, the inclination angle θ may be increased within a range where the culture liquid surface S does not contact the peripheral surface 55. By increasing the inclination angle θ, it becomes possible to more reliably introduce cells seeded on the outside of the culture surface 51 into the culture surface 51.

DESCRIPTION OF SYMBOLS 10 ... Bottom wall part, 30 ... Side wall part, 31 ... Inner peripheral surface, 41 ... 1st outer peripheral surface (outer peripheral surface, fitting part), 50 ... Culture room, 51 ... Culture surface, 53 ... Barrier surface, 55 ... Perimeter Surface, 56 ... connection, 57 ... curved surface, 60 ... lid, W ... predetermined distance

Claims

A bottom wall having a culture surface;
A side wall portion arranged along the periphery of the bottom wall portion and having an inner peripheral surface;
A peripheral surface provided at a predetermined distance between the outer peripheral end of the culture surface and the inner peripheral surface, and having a connecting portion with the inner peripheral surface;
A culture chamber that is surrounded by the culture surface, the side wall, and the peripheral surface and in which culture using a predetermined amount of liquid is performed,
The volume to the connection part in the culture chamber is less than the predetermined amount,
The predetermined amount is a liquid amount in which the contact position between the liquid surface of the liquid accommodated in the culture chamber and the inner peripheral surface is higher than the position of the connecting portion between the inner peripheral surface and the peripheral surface. Culture container.
A barrier surface rising at a predetermined height from the outer peripheral edge of the culture surface;
The culture container according to claim 1, wherein the peripheral surface is provided at a predetermined distance between an end of the barrier surface and the inner peripheral surface.
The culture container according to claim 1 or 2, wherein the peripheral surface is gradually inclined downward as it goes inward from the inner peripheral surface of the culture surface.
The area of the culture surface is about 55 cm 2 or more and about 58 cm 2 or less,
The culture container according to any one of claims 1 to 3, wherein the predetermined distance is approximately 10 mm or more and approximately 14 mm or less.
The culture container according to any one of claims 1 to 4, wherein the predetermined amount is approximately 10 ml.
The culture container according to any one of claims 1 to 4, wherein the predetermined amount is approximately 20 ml.
The culture vessel according to claim 2, wherein the length of the barrier surface from the culture surface is greater than 0 mm and approximately 2.0 mm or less.
The culture vessel according to claim 3, wherein θ is greater than approximately 0 degrees and approximately 5.5 degrees or less, where θ is an inclination angle of the peripheral surface with respect to a horizontal plane.
The culture container according to any one of claims 1 to 8, wherein a circumferential length of the peripheral surface is at least twice the predetermined distance.
The culture chamber has a rectangular parallelepiped shape in which the side wall portion is arranged in a rectangular frame shape when viewed from above.
The culture according to any one of claims 1 to 9, wherein the barrier surface is provided on the inner side of each side of the rectangular side wall portion with the predetermined distance therebetween and surrounding the periphery of the culture surface. container.
The culture vessel according to claim 3 or 8, wherein the peripheral surface is connected by a curved surface formed around an axis that is inclined at the same angle as the inclination angle of the peripheral surface and orthogonal to the culture surface.
The outer peripheral surface of the side wall has a fitting part that fits a lid that closes the culture chamber,
The culture container according to any one of claims 1 to 11, wherein an outer shape of the fitting part is asymmetric with respect to at least one of a long side direction and a short side direction of the side wall part.