JP2016077164A - Cell culture vessel - Google Patents

Abstract

An object of the present invention is to provide a cell culture container having gas permeability at the bottom and excellent shape stability at the bottom. The present invention provides a cell culture container having a bottom and an outer frame portion formed so as to be able to seal a culture medium together with the bottom, the bottom having a gas permeable film layer and a support layer, A medium is supported, and the gas permeable film layer is disposed so as to cover at least a part of a medium supporting surface that supports the medium at the bottom, and the support layer includes the gas permeable film. The object is achieved by providing a cell culture vessel characterized in that it is disposed on the opposite side of the medium support surface of the adhesive film layer and has an opening. [Selection] Figure 2

Description

  The present invention relates to a cell culture container having gas permeability at the bottom and excellent shape stability at the bottom.

In order to culture cells efficiently, it is necessary to culture them under conditions suitable for growth. For this reason, cells are usually cultured in a culture constant temperature bath that can be maintained at a predetermined temperature and humidity.
In addition, when observing the state of cells in culture, it is necessary to take out the cell culture container containing the cells from the thermostat. In order to prevent contamination when the cells are taken out from the thermostat, cell culture is usually performed. A cell culture container with a lid is used as the container.
On the other hand, in cell culture, it is necessary to supply gas such as oxygen and carbon dioxide into the cell culture container in order to supply oxygen to the cells and to supply carbon dioxide for adjusting the pH of the medium.
However, a polystyrene container with a lid, which is generally used as a cell culture container, has a problem that gas permeability is low and gas cannot be sufficiently supplied into the cell culture container. .
In order to solve such a problem, a cell culture container in which a gas permeable film is disposed at the bottom or the like has been disclosed (Patent Document 1, etc.).
By providing the gas permeable film in the cell culture container, it becomes possible to stably supply the gas into the cell culture container.

Special Table 2002-53983

Here, from the viewpoint of easy supply of oxygen and carbon dioxide to the culture medium, a location in direct contact with the culture medium is employed as the location of the gas permeable film. Specifically, the bottom of the cell culture container or the like is preferably used as the arrangement location.
However, since the gas permeable film is generally thin, when the gas permeable film is placed at the bottom of the cell culture container, the gas permeable film is bent due to the weight of the filled medium, and the culture is performed. There is a problem that the cells to be gathered in the bent part.
In addition, the larger the area of the bottom, the more the gas permeable film is bent, and the problem that the cells gather is conspicuous. Therefore, it is difficult to apply to a large-area cell culture container for mass culture of cells. There's a problem.

  The main object of the present invention is to provide a cell culture vessel having gas permeability at the bottom and excellent shape stability at the bottom.

  In order to achieve the above object, the present invention provides a cell culture container having a bottom and an outer frame portion formed so that a culture medium can be sealed together with the bottom, wherein the bottom includes a gas permeable film layer and a support layer. The gas permeable film layer is disposed so as to cover at least a part of a medium support surface that supports the medium at the bottom, and the support layer includes: A cell culture container is provided, which is disposed on the opposite side of the culture medium support surface of the gas permeable film layer and has an opening.

According to the present embodiment, the bottom portion has the gas permeable film layer and the support layer that supports the gas permeable film layer, so that the gas permeable film layer is less likely to be bent when the medium is filled. it can.
Therefore, the cell culture container can have gas permeability at the bottom and excellent shape stability at the bottom.

  In the present embodiment, the support layer preferably has a cell observation opening having a larger area than the opening. This is because the cell culture vessel can be easily observed.

  In this embodiment, it has an intermediate bottom part arranged in a space surrounded by the bottom part and the outer frame part, the intermediate bottom part has a second gas permeable film layer and a second support layer, and the culture medium The second gas permeable film layer is disposed so as to cover at least a part of the medium support surface of the intermediate bottom portion, and the second support layer is the second support layer. It is preferable that the gas permeable film layer is disposed on the opposite side of the medium support surface and has an opening. This is because the cell culture container can be efficiently cultured in a large area.

  In the present embodiment, the support layer is preferably formed integrally with the outer frame portion. This is because the cell culture container can be easily formed.

  The present invention has an effect of providing a cell culture container having gas permeability at the bottom and excellent shape stability at the bottom.

It is a schematic plan view which shows an example of the cell culture container of this invention. It is the sectional view on the AA line of FIG. It is a schematic sectional drawing which shows the other example of the cell culture container of this invention. It is a schematic sectional drawing which shows the other example of the cell culture container of this invention. It is explanatory drawing explaining the culture medium support surface in this invention. It is explanatory drawing explaining the culture medium support surface in this invention. It is explanatory drawing explaining the culture medium support surface in this invention. It is a schematic plan view which shows the other example of the cell culture container of this invention. It is a graph which shows the evaluation result of an Example and a comparative example.

The present invention relates to a cell culture container.
Hereinafter, the cell culture container of the present invention will be described in detail.

  The cell culture container of the present invention is a cell culture container having a bottom part and an outer frame part formed so that the medium can be sealed together with the bottom part, and the bottom part has a gas permeable film layer and a support layer, A medium is supported, and the gas permeable film layer is disposed so as to cover at least a part of a medium supporting surface that supports the medium at the bottom, and the support layer includes the gas permeable film. It is arrange | positioned on the opposite side of the said culture medium support surface of an adhesive film layer, and is provided with an opening part, It is characterized by the above-mentioned.

Such a cell culture container of the present invention will be described with reference to the drawings. FIG. 1 is a schematic plan view showing an example of the cell culture container of the present invention, which is observed from the support layer side of the cell culture container. 2 is a cross-sectional view taken along line AA in FIG. 3 and 4 are schematic sectional views showing other examples of the cell culture container of the present invention.
As illustrated in FIGS. 1 to 4, the cell culture container 10 of the present invention has a bottom portion 1 and an outer frame portion 3 formed so as to be able to seal a culture medium together with the bottom portion 1. The gas-permeable film layer 1a has a gas-permeable film layer 1a and a support layer 1b, and supports the medium. The gas-permeable film layer 1a has at least a part of a medium-supporting surface that supports the medium in the bottom portion 1. The support layer 1b is disposed on the opposite side of the gas permeable film layer 1a from the medium support surface and includes an opening 2a.
In this example, the gas permeable film layer 1a is disposed so as to cover the entire surface of the medium support surface. Further, the outer frame portion 3 surrounds the periphery of the bottom portion 1 and overlaps the side wall portion 3a disposed in a direction perpendicular (90 °) to the surface direction X of the cell culture container 10 and the bottom portion 1 in plan view. And an upper surface portion 3b disposed on the surface. The cell culture container 10 has an inlet 4 used for filling the medium and a lid 5 for sealing the inlet 4.
FIG. 3 shows the above-mentioned medium support surface, that is, the surface on the medium support surface side of the gas permeable film layer 1a when the cell culture container is placed on a horizontal table so that the base and the bottom face each other. An example having a plane parallel to the surface direction X, which is a direction parallel to the table, and a plane inclined with respect to the surface direction X is shown.
FIG. 4 includes a second gas permeable film layer 6a and a second support layer 6b, and an intermediate bottom portion 6 that supports the culture medium. The second gas permeable film layer 6a includes the intermediate bottom portion. 6, the second support layer 6b is disposed on the opposite side of the medium support surface of the second gas permeable film layer 6a, and has an opening. 6c.

According to the present embodiment, the bottom portion has the gas permeable film layer and the support layer that supports the gas permeable film layer, so that the gas permeable film layer is less likely to be bent when the medium is filled. it can.
The support layer has an opening, and the gas permeable film layer can be in direct contact with the outside air at the opening.
Therefore, the cell culture container can have a gas permeability at the bottom and excellent shape stability at the bottom.
In addition, since the bottom portion has the gas permeable film layer, gas can be efficiently supplied to the medium through the gas permeable film layer, so that cells cultured in the medium can be efficiently proliferated. . Furthermore, since the bottom portion is excellent in shape stability, cells can be cultured uniformly over the entire bottom portion. For this reason, the said cell culture container can be made into what can culture a cell efficiently in a large area.

The cell culture container of the present invention has a bottom part and an outer frame part.
Hereafter, each structure of the cell culture container of this invention is demonstrated in detail.

1. Bottom The bottom in this embodiment has a gas permeable film layer and a support layer.
The bottom portion supports the culture medium.
Here, supporting the culture medium constitutes a culture medium support surface that supports the culture medium when the culture medium is filled.
The medium support surface is a surface that supports the filled medium, that is, a surface that contacts the medium from below.
More specifically, such a medium support surface can be surrounded by the side wall portion of the outer frame portion of the surface of the bottom portion in contact with the medium.
5, 6, and 7 are explanatory diagrams for explaining the medium support surface, and shows a state in which the cell culture container 10 is filled with the medium 11. In FIG. 5 to FIG. 7, the culture medium support surface of the bottom portion 1 is indicated by a broken line, and is a portion that overlaps all of the culture media 11 in plan view among the surfaces of the bottom portion 1 in contact with the culture medium. Of the surface on the side in contact with the culture medium at the bottom, it is surrounded by the side wall 3a. FIGS. 5 to 7 show examples in which the cross-sectional shape of the cell culture container is the shape of FIGS. 2 to 4 described above, respectively, with respect to the surface direction of the surface of the side wall 3a in contact with the medium. An example in which the angle is vertical (90 °) is shown.
Moreover, the dotted line in FIG. 7 shows the culture medium support surface of the intermediate | middle bottom part 6. FIG. FIG. 7 shows an example in which the culture medium support surface of the intermediate bottom portion 6 is surrounded by the side wall portion 3a among the surfaces of the intermediate bottom portion 6 on the side in contact with the culture medium.
5 to 7 indicate the same members as those in FIGS. 2 to 4, and a description thereof will be omitted here.

(1) Support layer The support layer in this embodiment is arrange | positioned on the opposite side of the said culture medium support surface of the said gas-permeable film layer, and is provided with an opening part.
Moreover, the said opening part enables the surface on the opposite side of the said culture medium support surface of a gas-permeable film layer to contact with external air directly.
The said support layer supports the said gas-permeable film layer, and suppresses generation | occurrence | production of the bending by the culture medium of the said gas-permeable film layer.

Here, “suppressing the occurrence of bending” refers to suppressing deformation of the gas permeable film layer due to the weight of the medium to such an extent that no cell gathers.
As such a support layer, a portion where the position of the surface of the gas permeable film layer on the side of the medium support surface is lowered by 5 mm or more before and after filling of the medium can be prevented.
In addition, after filling of a culture medium, it can be set as the case where the distance from the culture medium support surface side surface of a gas-permeable film layer to a culture medium liquid surface is 2 cm. In addition, as a method for measuring such deflection, a method of filling a cell culture container with a medium and confirming it may be used. However, for easy visual recognition of the deflection of the gas permeable film layer, it is included in the cell culture container. A method may be used in which a gas permeable film having the same area as the gas permeable film layer is prepared as a measurement sample, and the periphery of the gas permeable film is fixed.

The shape of such an opening is not particularly limited as long as gas can be supplied to the gas permeable film layer and the occurrence of bending of the gas permeable film layer can be suppressed.
The shape can be, for example, a rectangular shape such as a triangular shape, a quadrangular shape, and a hexagonal shape, a circular shape, an elliptical shape, or the like.
In the present embodiment, it is particularly preferable that the shape is a square shape or a hexagonal shape. This is because the opening is easy to form due to the above shape. Moreover, it is easy to arrange the openings at regular intervals and to provide a support layer that can stably support the gas-permeable film layer.

The number of the openings may be 1 or more, but is usually 2 or more.
This is because the gas permeable film layer can be stably supported by the support layer arranged so as to partition the plurality of openings.
When the number of openings is 2 or more, the arrangement of the openings may be anything that can suppress the occurrence of bending of the gas permeable film layer, but the openings are arranged at regular intervals, It is preferable that the support layer has a lattice shape in plan view, or a honeycomb shape when the opening has a hexagonal shape. It is because it can be set as the support layer which can support a gas-permeable film layer stably by being the said arrangement | positioning.

The area of the opening is not particularly limited as long as the gas can be supplied to the gas permeable film layer and the occurrence of bending of the gas permeable film layer can be suppressed.
The area, for ^example, be in the range of 1mm ² ~10,000mm 2, among ^others, preferably in the range of 25mm ² ~2,500mm 2, within the range in particular of 100 mm ² 500 mm ² Preferably there is. When the area is within the above range, a gas can be sufficiently supplied to the gas permeable film layer, and a support layer capable of sufficiently suppressing the occurrence of bending of the gas permeable film layer is obtained. Because it can.
In addition, the said area says the area of the opening part per one.

The aperture ratio of the support layer is not particularly limited as long as gas can be supplied to the gas permeable film layer and the occurrence of bending of the gas permeable film layer can be suppressed.
The aperture ratio is preferably in the range of, for example, 10% to 90%, more preferably in the range of 30% to 85%, and particularly in the range of 60% to 80%. It is preferable. A support layer capable of sufficiently supplying gas to the gas permeable film layer and sufficiently suppressing the occurrence of bending of the gas permeable film layer when the aperture ratio is within the above range. Because it can be done.
The opening ratio is determined by the ratio of the total area of the opening to the total area of the gas permeable film layer (total area of the opening / total area of the gas permeable film layer × 100).
In addition, when the support layer has an opening for cell observation described later, the opening ratio includes the opening for cell observation as an opening.

The support layer preferably has a cell observation opening having a larger area than the opening. The support layer has the opening. When observing cells cultured on the gas permeable film layer, the cell may be easily observed depending on the size of the opening. There are cases where it is not possible. On the other hand, since the support layer has the cell observation opening, it becomes possible to observe cells without being blocked by the support layer through the cell observation opening. This is because it can be easily observed.
FIG. 8 shows an example in which the support layer has two circular cell observation openings 2b.
In addition, about the code | symbol in FIG. 8, since it shows the member same as FIG. 1, description here is abbreviate | omitted.

  The shape of the opening for cell observation is not particularly limited as long as cell observation can be performed, but it can be the same as the opening.

The area of the opening for cell observation is not particularly limited as long as the area is larger than that of the opening and the cell observation can be performed.
The area, for ^example, be in the range of 10mm ² ~2000mm 2, among ^others, is preferably in the range of 100mm ² ~1500mm 2, to be particularly in the range of 400mm ² ~900mm ² preferable. It is because the cells on the gas permeable film layer can be easily observed when the area is within the above range.
In addition, the said area says the area of the opening part for cell observation per.

The number of cell observation openings is not particularly limited as long as cell observation can be performed, and is appropriately set according to the area of the gas permeable film layer and the like.
The number can be two, for example.

The material constituting the support layer is not particularly limited as long as the bending of the gas permeable film layer can be suppressed.
Examples of the material include inorganic materials such as glass, metal materials, and resin materials. Among them, resin materials are preferable. This is because the support layer can be easily formed when the material is a resin material.
Specifically, as the resin material, polyethylene terephthalate (PET) resin, vinyl chloride resin, polystyrene (PS) resin, polypropylene (PP) resin, and the like can be preferably used. Is preferred. This is because the above materials are widely used in containers for cell culture.
In the present embodiment, two or more of the above materials may be used in combination.

The thickness of the support layer is not particularly limited as long as it can suppress the deflection of the gas permeable film layer. The area of the gas permeable film layer supported by the support layer, the aperture ratio of the support layer, and It depends on the constituent material of the support layer.
The thickness can be, for example, in the range of 1 mm to 5 mm, preferably in the range of 1 mm to 3 mm, and particularly preferably in the range of 1 mm to 2 mm.
More specifically, the thickness is within the range area of the gas permeable film layer of 15,000mm ² ~25,000mm ^2, the aperture ratio of the support layer is in the range of 70% to 80%, When the constituent material of the support layer is polystyrene, it can be in the range of 1 mm to 3 mm, and in particular, it is preferably in the range of 1 mm to 2 mm.
This is because when the thickness is within the above range, the support layer can stably support the gas permeable film layer.
In addition, the said thickness is specifically shown by a in FIG.

The support layer may have gas permeability, but usually does not have gas permeability.
Having gas permeability means that oxygen and carbon dioxide necessary for cell culture can be permeated into the cell culture container, and having no gas permeability means that the cell culture container is in a sealed state. In this case, it means that stable cell culture becomes difficult.
The oxygen permeability of the support layer can be less than 100 cm ³ / m ² · 24 hr · atm. The carbon dioxide permeability of the support layer can be less than 1,000 cm ³ / m ² · 24 hr · atm.
The gas permeability of the support layer does not include gas permeation through the opening and the cell observation opening.

The support layer is usually connected to the outer frame portion in order to support the gas permeable film layer at the bottom.
In the present embodiment, the support layer is preferably formed integrally with the outer frame portion. This is because the cell culture container can be easily formed.
1 and 2 described above show an example in which the support layer is formed integrally with the side wall portion of the outer frame portion.

(2) Gas-permeable film layer The gas-permeable film layer in this embodiment is arrange | positioned so that at least one part of the culture medium support surface which supports the said culture medium of a bottom part may be covered.
Moreover, the surface on the opposite side to the said culture medium support surface of a gas-permeable film layer is exposed from the opening part of the said support layer, and can be directly in contact with external air.

The gas permeable film layer is disposed so as to cover at least a part of the medium support surface.
Here, being arranged so as to cover the medium support surface means that the medium support surface side surface of the gas permeable film layer constitutes at least a part of the medium support surface. That is, the culture medium support surface side surface of the gas permeable film layer is a surface in contact with the culture medium from below.
The arrangement | positioning location of the said gas-permeable film layer should just cover at least one part of the said culture medium support surface. Especially in this embodiment, it is preferable to cover the whole surface of the said culture medium support surface. It is because it can be set as the cell culture container excellent in gas permeability by the said arrangement | positioning location being the whole surface of a culture medium support surface.

The gas permeable film layer has gas permeability.
Here, having gas permeability specifically means that oxygen and carbon dioxide necessary for cell culture can be permeated into the cell culture vessel.
The oxygen permeability of such a gas permeable film layer can be 100 cm ³ / m ² · 24 hr · atm or more. In addition, the carbon dioxide permeability of the gas permeable film layer can be 1,000 cm ³ / m ² · 24 hr · atm or more.
In the present embodiment, the transmittance of the oxygen of the gas permeable film layer is preferably ^{2,000cm 3 / m 2 · 24hr ·} atm~100,000cm 3 / m 2 · 24hr · atm in the range of, Among them, it is preferable that ^{3,500cm 3 / m 2 · 24hr ·} atm~60,000cm 3 / m 2 · 24hr · atm in the range of. This is because oxygen can be stably supplied when the transmittance is within the above-described range. Moreover, it is because a gas-permeable film layer can be made into a thing without a failure | damage.
It in the present embodiment, the transmittance of the carbon dioxide in the gas permeable film layer is in the range of ^{5,000cm 3 / m 2 · 24hr ·} atm~150,000cm 3 / m 2 · 24hr · atm is preferred, it is preferably ^{15,000cm 3 / m 2 · 24hr ·} atm~130,000cm 3 / m 2 · 24hr · atm in the range of. This is because when the transmittance is within the above range, carbon dioxide can be stably supplied, and the pH of the medium can be stably controlled. Moreover, it is because a gas-permeable film layer can be made into a thing without a failure | damage.

The material constituting the gas permeable film layer is not particularly limited as long as it has a desired gas permeability. For example, biaxially stretched polystyrene (OPS), ethylene-vinyl acetate copolymer Examples include coalescence (EVA), polystyrene, polyethylene, and the like. Among these, OPS can be preferably used in the present embodiment. It is because it can be made excellent in gas permeability by the above-mentioned material being the above-mentioned material. Further, it is not toxic to cells and can form a bottom suitable for cell culture.
In addition, as a commercial item of the said material, Asahi Kasei Chemicals OPS etc. can be mentioned.

The surface of the gas permeable film layer on the medium support surface side may be subjected to a surface treatment within a range not impeding gas permeability.
Examples of the surface treatment include plasma treatment and fine shaping treatment performed for the purpose of improving hydrophilicity, and fluorine processing treatment and fine shaping treatment performed for the purpose of improving water repellency.

  The layer structure of the gas permeable film layer is not particularly limited as long as it has a desired gas permeability, and may be a single layer or a laminate of a plurality of layers.

The thickness of the gas permeable film layer is not particularly limited as long as the gas permeable film layer has a desired gas permeability, and varies depending on the constituent material and the like, but can be within a range of 25 μm to 500 μm. In particular, it is preferably within the range of 50 μm to 250 μm, and particularly preferably within the range of 50 μm to 150 μm. This is because when the thickness is within the above range, the gas permeability can be improved.
In addition, when a gas-permeable film layer is what laminated | stacked multiple layers, it means the total thickness.

The planar view shape of the gas permeable film layer is appropriately set according to the type of cells to be cultured, and can be, for example, a square shape or a circular shape.
FIG. 1 which has already been described shows an example in which the gas-permeable film layer has a square shape in plan view.

Further, the shape of the gas permeable film layer may be a plane shape parallel to the plane direction, and has a plane shape parallel to the plane direction and a plane shape inclined with respect to the plane direction. May be.
FIG. 2 already described shows an example in which the shape of the gas permeable film layer is composed only of a planar shape parallel to the surface direction. Moreover, FIG. 3 already demonstrated shows the example in which the said shape has a planar shape parallel to a surface direction, and a planar shape which has an inclination with respect to a surface direction.

The area of the gas permeable film layer, although different depending on the type and Suto of cells to be cultured, for ^example, be in the range of 500mm ² ~300,000mm 2, among others, 10, preferably in the range of 000mm ² ~200,000mm ^2, preferably in the range especially of 15,000mm ² ~160,000mm ^2. It is because the effect of this invention that the bending of a bottom part can be suppressed even if it is a large area can be exhibited more effectively because the said area is in the above-mentioned range. Moreover, it is because a cell culture container can make a cell culture efficiently in a large area.
In addition, the said area is a total area of the area which comprises the culture medium support surface of the said gas-permeable film layer.

(3) Bottom portion The bottom portion has a gas permeable film layer and a support layer, but may have other configurations as necessary.
As said other structure, it can have the contact bonding layer which fixes the said gas-permeable film layer and a support layer, for example.
Moreover, when a gas-permeable film layer covers a part of culture medium support surface, it has a culture medium support part which comprises a culture medium support surface with a gas-permeable film layer normally.
The material, thickness, and gas permeability that constitute such a medium support portion may be any material that can stably support the medium and can be the same as the support layer. Moreover, the said culture medium support part shall be formed integrally with the said support layer.

2. Outer frame part The outer frame part in this embodiment is formed so that a culture medium can be sealed with the said bottom part.
Here, that the culture medium can be sealed together with the bottom means that the culture medium can be held without leaking by the culture support surface side surface of the bottom and the outer frame part.

Such an outer frame portion is disposed so as to surround the medium support surface of the bottom portion and to overlap with the side wall portion disposed in a direction substantially perpendicular to the surface direction of the cell culture container and the bottom portion in plan view. And an upper surface portion.
In the present embodiment, it is preferable that the side wall portion and the upper surface portion are integrally formed. This is because the outer frame portion can be easily formed.

The surface direction of the cell culture container in the present embodiment refers to a direction parallel to the base when the cell culture container is placed on a horizontal base so that the base and the bottom face each other. Specifically, the plane direction is a direction indicated by X in FIG.
Further, the side wall portion arranged in a direction substantially perpendicular to the surface direction of the cell culture container is within an angle range of 80 ° to 100 ° formed by the surface of the side wall portion in contact with the culture medium with respect to the surface direction. It is what is arranged so that.
Here, the angle formed by the surface of the side wall portion in contact with the culture medium with respect to the surface direction is specifically indicated by α in FIG.

The outer frame portion may have gas permeability or may have gas barrier properties.
It is because the cell culture container can be made excellent in gas permeability by having gas permeability. Moreover, it is because it is easy to make the thickness of an outer frame part thin, and it is easy to make a cell culture container into a bag shape like a cell culture bag.
Moreover, it is because it has gas barrier property, it is easy to make it thick, etc., and it can make a cell culture container excellent in intensity | strength. Moreover, it is because it is easy to make a cell culture container into a box shape like a flask and a bottle.

The material and thickness constituting the outer frame portion are not particularly limited as long as the medium can be sealed together with the bottom portion, and can be the same as the support layer, for example.
In addition, the said thickness says the thickness of the thickest location among the thickness of the said outer frame part, and is specifically shown by b in FIG.

3. Cell Culture Container The cell culture container of the present invention has the above-mentioned bottom part and outer frame part, but may have other configurations as necessary.
As such another configuration, for example, it is preferable to have an intermediate bottom portion that is disposed in a space surrounded by the bottom portion and the outer frame portion and supports the culture medium.
This is because the cell culture container can be used for cell culture in a large area.
FIG. 4 which has already been described is a schematic cross-sectional view showing an example of the cell culture vessel 10 having the intermediate bottom portion 6. In this example, each intermediate bottom portion 6 is connected to the side wall portion 3a of the outer frame portion 3 and is integrally formed.

(1) Intermediate bottom The intermediate bottom is arranged in a space surrounded by the bottom and the outer frame. Moreover, it has a 2nd gas-permeable film layer and a 2nd support layer, and supports the said culture medium.
Here, being arranged in a space surrounded by the bottom and the outer frame means that the intermediate bottom is surrounded by the bottom and the outer frame and is filled with the medium in the cell culture container. It means to be placed. The bottom part and the intermediate bottom part are arranged so as to overlap in plan view at a predetermined interval, and the cell culture container has a bottom part arranged in multiple stages. Moreover, the culture medium supported in a bottom part and each intermediate | middle bottom part is normally a separate body, and is filled without mixing.

The intermediate bottom is not particularly limited as long as it can stably support the filled medium and can stably culture the cells, but covers the medium support surface. It is preferable to have a second gas permeable film layer disposed and a second support layer disposed on the opposite side of the medium support surface of the second gas permeable film layer and having an opening. .
By having the intermediate bottom, the cell culture area of the cell culture container can be increased. Moreover, since the said intermediate | middle bottom part has the said 2nd gas-permeable film layer, supply of gas can be stably received from the bottom part side of an intermediate | middle bottom part. For this reason, it is because the said cell culture container can be made into what can carry out cell culture efficiently with a large area. Further, by having the second support layer, the shape stability of the intermediate bottom portion can be improved.

  In the present embodiment, the second gas permeable film layer and the second support layer used for the intermediate bottom are the same as the contents of the gas permeable film layer and the support layer described in the section “1. However, it is preferable that the second support layer does not have the cell observation opening, that is, the cell observation opening has only the support layer constituting the bottom. The intermediate bottom portion is laminated with the bottom portion, and it may be difficult to focus on a microscope or the like, and it is less necessary to arrange an opening for cell observation, and the area of the intermediate bottom portion is smaller than the opening portion. This is because the intermediate bottom can be made excellent in shape stability by not having a large cell observation opening.

  The number of the intermediate bottoms is appropriately set according to the amount of cells cultured in the cell culture container.

(2) Other configurations As the other configurations described above, an inlet used for filling the medium and a lid portion that seals the inlet can be provided.
Such an inlet and a lid can be the same as those generally used for cell culture containers such as flasks.
For example, the injection port may be formed integrally with the side wall portion or the upper surface portion of the outer frame portion, and the constituent material and thickness thereof are the same as the constituent material and thickness of the outer frame portion. Can do.

(3) Cell Culture Container The method for producing the cell culture container of the present invention is not particularly limited as long as it is a method capable of forming the cell culture container having the above configuration.
For example, a support layer is formed by forming an opening in the bottom surface of a commercially available cell culture container, and then a gas permeable film is disposed on the medium support surface side of the support layer, that is, on the inner surface side of the container. The method of fixing a film with respect to a container can be mentioned.
In addition, a method for forming a cell culture container having a support layer formed on the bottom surface is a method of forming an opening portion on the bottom surface after forming a container sealed with a bottom surface and an outer frame portion not including the opening portion. Alternatively, the bottom surface including the opening may be formed simultaneously with the outer frame using an injection molding method or the like.

  The present invention is not limited to the above embodiment. The above-described embodiment is an exemplification, and the present invention has substantially the same configuration as the technical idea described in the claims of the present invention, and any device that exhibits the same function and effect is the present invention. It is included in the technical scope of the invention.

  Hereinafter, the present invention will be specifically described using examples and comparative examples.

[Example 1]
Opening ratio 70% (30 openings of 18 mm × 22 mm) on the bottom of a flask molded with PS resin (culture area 170 cm ² , bottom, side and top thickness 3 mm), and the opening spacing is 2 mm Thus, an opening was formed to form a lattice-like support layer.
Next, an OPS film having a thickness of 20 μm is arranged as a gas permeable film layer on the inside of the flask of the support layer, and the cell permeable film layer is fixed with the flask, the support layer and an adhesive to fix the cell culture container. Obtained.
In addition, the gas permeable film layer was arrange | positioned so that the whole surface of a culture medium support surface might be covered.
The culture area refers to the total area of the medium support surface, and refers to the area of the bottom surface of the flask.

[Examples 2 to 4]
As a gas permeable film layer, an OPS film having a thickness of 50 μm (Example 2) and 250 μm (Example 3), and an OPS film having a thickness of 20 μm and having a plasma treatment applied to the surface on the medium support surface side (Example 4) ) Was used in the same manner as in Example 1 except that a cell culture vessel was obtained.

[Comparative Example 1]
A flask (culture area: 170 cm ² ) molded from PS resin before opening was used as a cell culture vessel.

[Comparative Example 2]
A cell culture container was obtained in the same manner as in Example 1 except that the bottom surface of the flask was completely removed, that is, the support layer was not provided.

[Evaluation]
Human skeletal myoblasts (seeding density 100 cells / cm ² ) were cultured using the cell culture vessels obtained in Examples and Comparative Examples, and the number of cells (cells / cm ² ) was counted on the 10th day. The results for Examples 1 to 4 and Comparative Example 1 are shown in FIG.

[Summary]
As shown in FIG. 9, the cell culture container of the example had a cell number more than twice that of the comparative example.
Moreover, in Examples 1-4 and the comparative example 1, the bending of the bottom part was not observed.
In Examples 1 to 4, the cell culture was performed with the thickness of the OPS film changed. However, the number of cell cultures did not change greatly depending on the thickness. If the OPS film was used, cell culture was performed even at a thickness of 250 μm. It was found that the increase in the number was effective.
On the other hand, in Comparative Example 2, when the cell culture container was filled with human skeletal myoblasts together with the culture medium, the bottom, that is, the gas permeable film layer was bent. Further, the number of cell cultures was about 40% of Comparative Example 1. The reason why the number of cell cultures is so small is presumed as follows. That is, the cells are collected in the central portion of a part of the container bottom (about 50% of the whole) due to the inclination due to the bending of the gas permeable film. As a result, cell culture within a small area, and further, cells become confluent within that narrow area. As a result of the decrease in the number of cultures, it is assumed that the number of cell cultures decreases.

DESCRIPTION OF SYMBOLS 1a ... Gas-permeable film layer 1b ... Support layer 1 ... Bottom part 2a ... Opening part 2b ... Opening part for cell observation 3a ... Side wall part 3b ... Upper surface part 3 ... Outer frame part 4 ... Inlet 5 ... Lid part 6 ... Middle bottom part 6a ... Gas permeable film layer 6b ... Support layer 6c ... Opening 10 ... Cell culture vessel

Claims (4)

A cell culture container having a bottom and an outer frame part formed so as to be able to seal a culture medium together with the bottom,
The bottom has a gas permeable film layer and a support layer, and supports the medium.
The gas permeable film layer is disposed so as to cover at least a part of a medium support surface that supports the medium at the bottom,
The cell culture container, wherein the support layer is disposed on the opposite side of the medium permeable surface of the gas permeable film layer and has an opening.   The cell culture container according to claim 1, wherein the support layer has a cell observation opening having a larger area than the opening. An intermediate bottom portion disposed in a space surrounded by the bottom portion and the outer frame portion;
The intermediate bottom portion has a second gas permeable film layer and a second support layer, and supports the medium.
The second gas permeable film layer is arranged so as to cover at least a part of the medium support surface of the intermediate bottom part,
The cell according to claim 1 or 2, wherein the second support layer is disposed on the opposite side of the medium support surface of the second gas permeable film layer and has an opening. Culture container.   The cell culture container according to any one of claims 1 to 3, wherein the support layer is formed integrally with the outer frame portion.