JP6942448B2 - Cell culture container, cell culture system using this, and cell culture method - Google Patents

Description

The present invention relates to a cell culture vessel for culturing cells, a cell culture system using the cell culture container, and a cell culture method.

Recent studies of human pluripotent stem cells such as human ES cells and human iPS cells have increased the possibility of practical application of regenerative medicine, and these pluripotent stem cells have the ability to proliferate indefinitely and nerve cells. Since it has the ability to differentiate into various cells such as myocardial cells, blood cells, and retinal cells, it is expected to fundamentally change the treatment methods for intractable diseases, lifestyle diseases, etc. ..

One of the challenges for the practical application of medical technology for regenerating various organs using pluripotent stem cells is to produce the number of cells required for organ regeneration and to make the quality of the produced cells uniform. There is a problem that it does not become. For example, the regeneration of the liver requires about 2 × 10 ¹¹ cells, the culturing the number of cells on a flat substrate takes 10 ⁶ cm ² or more culture area. Since this culture area is equivalent to about 20,000 cells in a general 10 cm dish, it is difficult to supply the number of cells required for organ regeneration using a flat substrate such as a dish which is a conventional technique. In addition, since the culture environment in each substrate is different, the quality of cells varies.

Therefore, in order to solve the above problems, techniques for culturing using various culture containers (Patent Documents 1 to 5) are disclosed. For example, in Patent Document 1, a columnar cell culture vessel is developed by stacking disc-shaped culture plates having a large number of holes, and spaces are provided at the center, top, and bottom of the cell culture vessel. A technique is disclosed in which the concentration of nutrients, oxygen, etc. in a cell culture vessel is made uniform by stirring the medium with a stirrer or the like at the bottom. Further, Patent Document 2 discloses a technique for uniformly seeding cells by providing a perforated plate in a cell suspension supply section. Patent Document 3 discloses that a cell culture apparatus including a cell culture chamber is provided with a medium uniform distribution means for uniformly distributing the medium flowing into the cell culture chamber. Patent Document 4 discloses that in a high-density culture method for animal cells, a partition plate such as a baffle plate is used to divide and flow the medium so that the flow of the medium is not biased. On the other hand, in Patent Document 5, by devising the microspace structure, a large number of cell culture carrier substrates having a porous structure portion in which polygonal through holes having a side length or diameter of less than 50 μm to less than 500 μm are formed are laminated. The cell culture reactor is disclosed.

Special Table 2013-526269A Japanese Unexamined Patent Publication No. 02-5850 Japanese Unexamined Patent Publication No. 10-191961 Japanese Unexamined Patent Publication No. 11-225751 Japanese Unexamined Patent Publication No. 2011-172533

As a result of studies by the present inventors on the above-mentioned problems, in order to culture a large amount of cells and to make the quality of the cultured cells uniform, a fresh medium is always supplied and nutrients in the cell culture vessel are supplied. It is important to maintain the culture medium sufficiently and to disperse the nutrients uniformly, and for that purpose, we have found a new problem that the flow velocity distribution of the medium supplied into the cell culture vessel must be uniform. Therefore, the present inventors have examined the techniques described in Patent Documents 1 to 5 in order to solve the above problems.

However, in the cell culture vessel described in Patent Document 1, the flow velocity gradually slows toward the upper part, making it difficult to maintain a uniform culture state, and in the cell culture vessel described in Patent Document 2, a perforated plate and a net plate are used. However, in the case of a net plate, the void ratio is too large, so that the flow of the medium supplied into the cell culture vessel is not stable, and the flow velocity of the medium flowing into the culture vessel is not stable. It was found that the distribution was difficult to be uniform. Further, Patent Documents 3 and 4 do not describe or suggest a means for uniformly distributing the medium and a specific configuration of the partition plate. Further, Patent Document 5 merely discloses a cell culture reactor for stacking a large number of cell culture carrier substrates having a porous structure portion and adhering cells to the porous structure portion of each substrate for culturing. It does not suggest any means for homogenizing the flow of the feed solution supplied into the culture vessel.

In view of the above problems and the like, an object of the present invention is to provide a cell culture container capable of making the flow velocity distribution of the supply liquid supplied into the cell culture container uniform.

That is, the present invention provides as follows.
(1) A cell culture container for culturing cells.
A container body with an inlet and outlet,
It is provided with a dispersion which is arranged in the container body and has a through hole formed therein.
In the container body, a first space in which the supply liquid flowing from the introduction port stays is formed on the inlet side of the dispersion, and the dispersion is on the outlet side of the dispersion. A second space is formed in which the supply liquid flowing out from the through hole of the above flows.
When the cross-sectional area of the first space adjacent to the dispersion is Sa (mm ² ) and the total opening area of the through holes of the dispersion is St (mm ² ), the area ratio St / A cell culture vessel that satisfies the relationship of Sa ≤ 0.58.
(2) The cell culture vessel according to (1), wherein a plurality of the through holes are formed in the dispersion.
(3) The diameter of the maximum inscribed circle inscribed in the inner circumference of the introduction port is larger than the diameter of the maximum inscribed circle inscribed in the inner circumference of the through hole of the dispersion in the narrowest portion (1). Or the cell culture vessel according to (2).
(4) In the dispersion, a plurality of the through holes are arranged along a predetermined direction.
The hole spacing between the adjacent through holes among the arranged through holes on the side of the first space is D (μm or mm), and the width of the dispersion in the predetermined direction is L (μm or mm). The cell culture vessel according to (1) or (2), which satisfies the relationship of 0.0167 ≤ D / L.
(5) The cell culture container according to any one of (1) to (4), further comprising a rectifier arranged between the dispersion in the container body and the outlet.
(6) The cell culture container according to (5), wherein a third space in which the supply liquid flowing out of the rectifying body stays is formed in the container main body on the outlet side of the rectifying body.
(7) The cell culture vessel according to (5) or (6), wherein a through hole is formed in the rectifying fluid.
(8) The cell culture vessel according to (7), wherein the rectifier has the same number and shape of through holes as the dispersion.
(9) When the cross-sectional area of the space inside the container body at a position adjacent to the rectifying body is Sb (mm ² ) and the total opening area of the through holes of the rectifying body is Sh (mm ² ). The cell culture vessel according to (7) or (8), which satisfies the relationship of Sh / Sb ≦ 0.58.
(10) The diameter of the maximum inscribed circle inscribed in the inner circumference of the outlet is larger than the diameter of the maximum inscribed circle inscribed in the inner circumference of the through hole of the rectifying body at the narrowest portion (7). The cell culture vessel according to any one of (9).
(11) In the rectifying body, a plurality of through holes are arranged along a predetermined direction.
0.0167 when the hole spacing between adjacent through holes among the arranged through holes is E (μm or mm) and the width of the rectifying body in the predetermined direction is Z (μm or mm). The cell culture vessel according to any one of (7) to (9), which satisfies the relationship of ≦ E / Z.
(12) The cell culture container according to any one of (1) to (11) and
A medium supply source that supplies a medium as a supply liquid to the cell culture container, and
A pumping unit that pumps the medium from the medium supply source to the inlet of the cell culture vessel, and
A cell culture system characterized in that it comprises at least.
(13) In the cell culture system, the outlet of the cell culture container is connected to the medium supply source so that the medium that has passed through the cell culture container is collected by the medium supply source (12). The cell culture system according to.
(14) In the cell culture system, the outlet of the cell culture container is connected to the waste liquid container so that the medium that has passed through the cell culture container is collected in a waste liquid container different from the medium supply source. The cell culture system according to (12).
(15) A method for culturing cells using the cell culture system according to any one of (12) to (14).
The process of seeding cells,
The process of culturing cells and the process of exfoliating cells,
Cell culture method including.

According to the cell culture vessel of the present invention, the flow velocity of the supply liquid supplied into the cell culture vessel can be made uniform. In particular, in the cell culture vessel of the present invention, when the cell-containing supply liquid is supplied from the introduction port, it is unlikely that the cells are clogged in the through holes of the dispersion, and the cells that flow in the second space. Since the flow velocity of the contained feed solution is unlikely to fluctuate, cells can be uniformly dispersed and seeded. Further, when the culture solution is supplied using the cell culture vessel of the present invention, the nutrients are sufficiently maintained in the cells adhering to the cell culture vessel, and the nutrients are uniformly dispersed in the cell culture vessel. Therefore, it is possible to culture a large amount of cells while maintaining uniform cell quality.

According to the cell culture system of the present invention, a medium for culturing can be circulated and supplied to the cell culture vessel of the present invention, and a large amount of cells can be cultured while maintaining uniform cell quality. Can be done.

According to the cell culture method of the present invention, since a cell culture container having a large culture area and capable of uniformly supplying nutrients and the like is used, a large amount of cells can be cultured while maintaining uniform cell quality.

It is a schematic perspective view of the cell culture container which concerns on 1st Embodiment of this invention. It is a schematic perspective view which showed the space in the container body of the cell culture container shown in FIG. FIG. 1 is a cross-sectional view taken along the line AA of FIG. FIG. 3 is a cross-sectional view taken along the line BB of FIG. FIG. 3 is a cross-sectional view taken along the line CC of FIG. (A) is a diagram for explaining the cross-sectional area Sa at a position adjacent to the dispersion in the first space of the container body of the cell culture container according to the first embodiment, and (b) is a diagram for explaining the first. It is a figure for demonstrating the total opening area St of the through hole of the dispersion of the cell culture container which concerns on embodiment. It is a figure for demonstrating the length ratio D / L of the dispersion of the cell culture vessel which concerns on 1st Embodiment. (A) is an enlarged cross-sectional view of the vicinity of the through hole in FIG. 3, and (b) to (d) are enlarged cross-sectional views showing a modified example thereof. It is a figure which showed the dispersion which concerns on the modification of the dispersion of 1st Embodiment corresponding to FIG. It is an enlarged sectional view of the dispersion which concerns on another modification of the dispersion of 1st Embodiment. It is a schematic perspective view which showed the space in the container body of the cell culture container which concerns on 2nd Embodiment corresponding to FIG. It is a schematic cross section of the cell culture vessel which concerns on 2nd Embodiment corresponding to FIG. It is a schematic conceptual diagram of the cell culture system provided with the cell culture container which concerns on 1st or 2nd Embodiment. It is a figure which showed the result of analysis (1)-(4). It is a figure which showed the result of analysis (5)-(8). It is a figure which showed the result of analysis (9)-(11). It is a figure which showed the result of analysis (12)-(14). It is a figure which showed the result of analysis (15)-(17). It is a figure which showed the result of analysis (18) and (19). It is a figure which showed the dispersion which concerns on the modification of the dispersion of 1st Embodiment corresponding to FIG. It is a figure which showed each dimension (mm) in the enlarged view of the region 9X in FIG. 9 in the specific example of FIG. It is a figure which showed the result of analysis (20).

The cell culture vessel according to the two embodiments of the present invention will be described below with reference to FIGS. 1 to 12.
[First Embodiment]
1. 1. Overall Structure of Cell Culture Container 1 The overall structure of the cell culture vessel 1 according to the first embodiment will be described below with reference to FIGS. 1 to 5. FIG. 1 is a schematic perspective view of the cell culture container 1 according to the first embodiment of the present invention, and FIG. 2 is a schematic perspective view showing the space inside the container body 10 of the cell culture container 1 shown in FIG. It is a figure. 3 is a cross-sectional view taken along the line AA of FIG. 1, FIG. 4 is a cross-sectional view taken along the line BB of FIG. 3, and FIG. 5 is a cross-sectional view taken along the line CC of FIG. It is a figure.

The cell culture container 1 according to the present embodiment is a container for culturing cells. The material of the cell culture vessel 1 is not particularly limited, and specific examples thereof include inorganic materials such as metal, glass, silicon, and plastic, and more preferably plastic. Examples of the plastic include polystyrene resin, polyethylene resin, polypropylene resin, ABS resin, acrylic resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin and the like. The cell culture vessel according to the present embodiment can be produced by a method well known and commonly used by those skilled in the art. For example, when the cell culture container 1 made of a plastic material is manufactured, it can be manufactured by an injection molding method, an extrusion molding method, or the like.

The cell culture container 1 includes a container body 10. The container body 10 includes an introduction port 12 for introducing a supply liquid such as a medium into the cell culture container 1 and an outlet 13 for drawing out the supply liquid from the cell culture container 1. A dispersion 40, which will be described later, is arranged between the introduction port 12 and the outlet port 13 in the container body 10. As a space in the container body 10, a first space 16 in which the supply liquid flowing from the introduction port 12 stays is formed on the side of the introduction port 12 of the dispersion 40, and is formed on the side of the outlet 13 of the dispersion 40. A second space 14 is formed in which the supply liquid flowing out from the through hole 41 described later in the dispersion 40 flows. The first space 16 is formed by the inner wall surface 15 of the container body 10, the upstream wall surface 17, and the facing wall surface 42 of the dispersion 40 facing the introduction port 12. The second space 14 is formed by the inner wall surface 15 of the container body 10, the downstream wall surface 43 of the dispersion 40 facing the outlet 13, and the downstream wall surface 18 of the container body 10. In the second space 14, cells are cultured. A multi-layer culture substrate may be arranged in the second space 14 so as not to obstruct the passage of liquid, and the material of the multi-layer culture substrate is not particularly limited, but for example, an inorganic substance such as metal, glass, silicon, and plastic. Materials and the like can be mentioned, and plastic is more preferable. Examples of the plastic include polystyrene resin, polyethylene resin, polypropylene resin, ABS resin, acrylic resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin and the like. Instead of the multi-layer culture substrate, a porous cell culture carrier or the like can also be used.

Although the volume of the internal space of the container body is not particularly limited, for example, at 1,000mm ³ or more, preferably 10,000 mm ³ or more, 100,000 mm ³ or more, 1,000,000 mm ³ or more. It is particularly suitable for cell culture when the volume of the internal space is in this range. The upper limit of the volume of the internal space is not particularly limited, but is preferably ^{100,000,000 mm 3 or less, for example.}

Since the second space 14 is a space where cell culture is mainly performed, the volume of the second space 14 is preferably larger than the volume of the first space 16 and is preferably 2 times or more or 3 times or more. More preferred. The volume of the second space 14 is, for example, 1,000 times or less the volume of the first space 16. The volume of the second space 14 is not limited as long a volume suitable for cell culture, typically 100 mm ³ or more, such as 1,000mm ³ or more, 10,000 mm ³ or more, 100,000 mm ³ or more, or 1,000,000 mm ³ or more. When the volume of the second space 14 is in this range, it is particularly suitable for cell culture. The upper limit of the volume of the second space 14 is not particularly limited, but is preferably ^{100,000,000 mm 3 or less, for example.}

The introduction pipe 20 and the outlet pipe 30 are connected to the container body 10. The introduction pipe 20 is formed with an introduction flow path 21 for introducing the supply liquid into the first space 16, and an introduction port 12 is arranged downstream of the introduction flow path 21. On the other hand, the lead-out pipe 30 is formed with a lead-out flow path 31 for leading out the supply liquid from the space inside the container body 10, and a lead-out port 13 is arranged upstream of the lead-out flow path 31. In the present embodiment, the opening area of the introduction port 12 and the opening area of the outlet port 13 are the same, but are not particularly limited. For example, the opening area of the outlet 13 may be different from the opening area of the introduction port 12, and when the opening area of the outlet 13 is larger than the opening area of the introduction port 12, the inside of the cell culture vessel 1 It is preferable because it is considered that the pressure of the cells is reduced and the stress on the cells is reduced.

In the present embodiment, the introduction port 12 is formed in the center of the flat upstream wall surface 17, and the outlet port 13 is formed in the center of the flat downstream wall surface 18. The introduction flow path 21 of the introduction pipe 20 and the outlet flow path 31 of the outlet pipe 30 are formed along the center line P (same linear shape) of the container body 10, and the center of the introduction port 12 and the outlet port 13 are formed. The center of the container body 10 is arranged on the center line P of the container body 10, but is not particularly limited.

In the present invention, the positions and relative arrangements of the introduction port 12 and the outlet port 13 in the container body 10 are not limited to the embodiments shown in the present embodiment. The introduction port 12 and the outlet port 13 are preferably formed on the opposite wall surfaces (upstream wall surface 17 and downstream wall surface 18 in the present embodiment) of the container body 10, respectively, but the introduction port 12 is upstream of the container body 10. It does not have to be provided in the center of the side wall surface, and may be provided at a position near the peripheral edge of the wall surface, or may be provided at another position of the container body 10. The outlet 13 does not have to be provided in the center of the wall surface on the downstream side of the container body 10, and may be provided at a position near the peripheral edge of the wall surface, or may be provided at another position of the container body 10. You may. The introduction port 12 and the outlet port 13 do not have to be provided so as to face each other on one axis, and may be provided on different axes. However, as in the present invention, the introduction port 12 is provided near the center of the wall surface on the upstream side of the container body 10, the outlet 13 is provided near the center of the wall surface on the downstream side of the container body 10, and the introduction port is provided. In the embodiment in which the outlet 12 and the outlet 13 are provided so as to face each other on one axis, when the supply liquid flows into the container body 10, the flow velocity distribution on the cross section of the flow path is point-symmetrical with respect to the axis. This is preferable because the flow velocity is less likely to be biased.

The shape of the openings of the inlet 12 and the outlet 13 is not particularly limited, and may be circular, polygonal (triangle, quadrangle, pentagon, hexagon, etc.), elliptical, or slit, preferably. It is circular. In the present invention, the range of a circle, a polygon, and an ellipse is not limited to a perfect circle, a polygon, and an ellipse, but also includes a shape similar to these.

2. Dispersion 40
The cell culture container 1 is arranged inside the container body 10, and includes a dispersion 40 in which a through hole 41 through which a supply liquid supplied to the cell culture container 1 passes is formed. The supply liquid supplied to the cell culture vessel 1 stays in the first space 16, passes through the through hole 41 of the dispersion 40, flows out to the second space 14, flows, and is led out from the outlet 13. .. In the present embodiment, the shape of the dispersion 40 is not particularly limited, but it is preferably plate-shaped. It is preferable that the peripheral edge of the dispersion 40 is connected to the inner wall surface 15 of the container body 10. The dispersion 40 may be integrally molded with the container body 10 of the cell culture container 1. Further, a member to be the dispersion 40 may be attached to the container body 10 to form the dispersion 40. In the present embodiment, the dispersion 40 is arranged perpendicular to the flow path from the introduction port 12 to the outlet port 13 in the container body 10, but is not limited to this, and is not limited to the flow path. It may be arranged so as to be inclined.

The material of the dispersion 40 is not particularly limited, and specific examples thereof include inorganic materials such as metal, glass, silicon, and plastic, and more preferably plastic. Examples of the plastic include polystyrene resin, polyethylene resin, polypropylene resin, ABS resin, acrylic resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin and the like. In the embodiment in which the dispersion 40 is integrally molded with the container body 10 of the cell culture container 1, the material of the dispersion 40 may be the same as that of the container body 10. Further, in the embodiment in which the member to be the dispersion 40 is attached to the container body 10 to form the dispersion 40, the material of the dispersion 40 may be the same as or different from the material of the container body 10. ..

Only one dispersion 40 is arranged inside the container body 10 of the cell culture container 1 of the present embodiment shown in the figure. When there is only one dispersion 40, the internal structure of the container body 10 is simplified, so that it is easy to realize cell culture in a uniform environment.

A through hole 41 is formed in the dispersion 40, and it is preferable that the through hole 41 is formed so as to penetrate in one direction. As a result, the supply liquid from the introduction port 12 to the second space 14 passes through each through hole 41 formed so as to penetrate along a certain direction, so that the non-woven fabric, foam material, or the like has an irregular shape. Unlike the flow of the supply liquid passing through the through hole, the flow can be stabilized, the flow velocity can be made uniform, and the cells can be easily dispersed and adhered uniformly to the second space 14. In addition, a supply liquid such as a medium can be supplied at a uniform flow rate. Further, when supplying a supply liquid such as a medium, the difference between the positions of the flow velocity in the second space 14 can be reduced. Therefore, the cells cultured in the second space 14 can be easily exfoliated uniformly after the release agent treatment. A plurality of through holes 41 may be formed in the dispersion 40, and the number of through holes 41 may be designed to satisfy, for example, the area ratio St / Sa ≦ 0.58, and is not particularly limited. The details of the area ratio St / Sa will be described later.

As described above, it is preferable that the through hole 41 is formed so as to penetrate in one direction, and the "one direction" in this preferred embodiment is the direction in which the supply liquid in the second space 14 flows. .. Further, in the present embodiment, it is preferable that a plurality of through holes 41 are formed in the dispersion 40, and all the through holes are formed along the same direction. As a result, the directions of the flow of the supply liquid passing through the plurality of through holes 41 from the introduction port 12 are all aligned in the same direction (direction along the center line P), so that the through holes 41 to the second through holes 41 are aligned along the same direction. The supply liquid can flow into the space 14. In this way, the flow of the supply liquid in the second space 14 can be easily made into a laminar flow, and the flow velocity distribution of the supply liquid supplied in the second space 14 can be made uniform. In the present invention, when the introduction port and the outlet are formed on the opposite wall surfaces of the container body (upstream wall surface 17 and downstream wall surface 18 in the present embodiment), the introduction port of the container body is formed. It is preferable that the through hole of the dispersion is formed so that the direction from the wall surface to the wall surface on which the outlet of the container body is formed is the penetration direction.

The cross-sectional shape of the through hole 41 (the shape of the cross section by a plane perpendicular to the penetrating direction) is not particularly limited, and may be, for example, circular, polygonal (triangular, quadrangular, pentagonal, hexagonal, etc.), elliptical, or slit. It may be present, preferably circular. In the present invention, the range of a circle, a polygon, and an ellipse is not limited to a perfect circle, a polygon, and an ellipse, but also includes a shape similar to these.

In a preferred embodiment in which a plurality of through holes 41, which will be described later, are formed in the dispersion 40, more preferably, when the dispersion 40 is viewed from the side of the first space 16, the first space 16 of the dispersion 40 On the side surface, a plurality of through holes 41 are arranged along a predetermined direction. Here, examples of the "predetermined direction" include the height direction and the width direction of the dispersion 40. More preferably, the plurality of through holes 41 are arranged at equal intervals along the predetermined direction on the surface on the side of the first space 16.

3. 3. First space 16
In the present embodiment, the dispersion 40 is arranged in the container body 10 at a position facing the introduction port 12 and away from the introduction port 12. As a result, a first space 16 for retaining the supply liquid flowing in from the introduction port 12 is formed between the introduction port 12 and the dispersion 40.

By forming the first space 16, the supply liquid introduced from the introduction port 12 can be retained in the first space 16 before heading to the second space 14, and the hydraulic pressure of the supply liquid can be stabilized. In the present embodiment, the cross-sectional area of the container body 10, specifically, the cross-sectional area of the first space 16 at the position 16a adjacent to the dispersion 40 and the total opening area of the through hole 41 of the dispersion 40 are obtained. By having a predetermined area ratio, which will be described later, the flow velocity distribution of the supply liquid from each through hole 41 toward the second space 14 can be made more uniform.

In the present invention, the shape of the first space is not particularly limited as long as the supply liquid introduced from the introduction port can be retained before heading to the second space, and is not particularly limited, for example, a rectangular parallelepiped or a pyramid (cone). It can be a body, a truncated cone, a pyramid, a pyramid, etc.), a column (a cylinder, a prism, etc.), a sphere, and / or a hemisphere, or a combination of two or more of these. In the present embodiment shown in FIGS. 3 and 4, among the wall surfaces forming the first space 16, the facing wall surface 42 of the dispersion 40 facing the introduction port 12 and the introduction port 12 facing the facing wall surface 42 are formed. It is parallel to the upstream wall surface 17. Further, the cross section (cross-sectional area) of the first space 16 from the upstream wall surface 17 toward the facing wall surface 42 is constant without being enlarged or reduced. As a result, the first space 16 becomes a rectangular parallelepiped shape.

In the present embodiment, by providing the rectangular parallelepiped first space, the hydraulic pressure of the supply liquid can be applied more uniformly to the facing wall surface 42 of the dispersion 40, so that the second through holes 41 to the second space can be acted on more uniformly. The flow velocity distribution of the supply liquid toward the space 14 can be further made uniform.

In the present invention, the shape of the second space is also not particularly limited as long as the supply liquid flowing out from the through hole 41 of the dispersion 40 can flow, and is not particularly limited, for example, a rectangular parallelepiped or a pyramid (cone, cone). It can be a pedestal, a pyramid, a pyramid pedestal, etc.), a column (cylinder, a pyramid, etc.), a sphere, and / or a hemisphere, or a combination of two or more of these.

4. Area ratio St / Sa (porosity of dispersion 40)
FIG. 6A is a diagram for explaining the cross-sectional area Sa of the first space 16 of the container body 10 at the position 16a adjacent to the dispersion 40 in the cell culture vessel 1 according to the first embodiment. 6 (b) is a diagram for explaining the total opening area St of the through hole 41 of the dispersion 40 of the cell culture container 1 according to the first embodiment. In the present embodiment, the area of the hatched portion shown in FIG. 6A corresponds to the ^{cross-sectional area Sa (mm 2) of the first space 16 at the position 16a adjacent to the dispersion 40.} The area of the hatched portion shown in FIG. 6B corresponds to ^{the total opening area St (mm 2} ) of the through hole 41 of the dispersion 40 adjacent to the first space 16. At this time, it is preferable to satisfy the relationship of area ratio St / Sa ≦ 0.58.

In the present invention, the area ratio and the length ratio are both calculated after unifying the units of the area and the length to be compared.

In the present invention, the cross-sectional area of the first space 16 at the position 16a adjacent to the dispersion 40 is cut off by the surface of the dispersion 40 along the side surface (opposing wall surface 42) of the first space 16. It refers to the area of the cross section of one space 16. For example, when the flat plate-shaped dispersion 40 is provided as in the cell culture vessel 1 according to the first embodiment, it is located on the downstream side of the first space 16 and on the side of the first space 16 of the flat plate-shaped dispersion 40. The area of the cross section cut out by the plane along the surface (opposing wall surface 42) is the cross-sectional area of the first space 16 at the position 16a adjacent to the dispersion 40. Further, as in the example shown in FIG. 10, when the cell culture vessel includes the dome-shaped dispersion 40D, it is located on the downstream side of the first space 16 and on the side of the first space 16 of the dome-shaped dispersion 40D. The area of the cross section cut out by the hemispherical surface along the surface (opposing wall surface 42) is the cross-sectional area of the first space 16 at the position 16a adjacent to the dispersion 40. Preferably, the surface of the dispersion 40 along the side surface (opposing wall surface 42) of the first space 16 is a plane perpendicular to the direction of the flow of the supply liquid in the first space 16.

In the present invention, the opening area of the individual through-holes 41 of the dispersion 40 refers to the smallest cross-sectional area of the cross-sectional areas of the individual through-holes 41 (cross-sectional area perpendicular to the penetrating direction). The total opening area is the total opening area of each through hole 41. In the present embodiment, as shown in FIG. 8A, since the cross-sectional area of the through hole 41 does not change in the through hole and is constant, the opening area of the through hole 41 is an arbitrary position in the through hole. The cross-sectional area of the through hole 41 at 411 can be adopted. As a modification, as shown in FIG. 8B, when the cross-sectional area of the through hole 41 decreases from the side of the first space 16 toward the side of the second space 14, the opening area of the through hole 41 As a result, the cross-sectional area of the through hole 41 at the end portion 412 on the side of the second space 14 can be adopted. Further, as another modification, as shown in FIG. 8C, when the cross-sectional area of the through hole 41 becomes smaller from the side of the second space 14 toward the side of the first space 16, the through hole 41 is formed. As the opening area of, the cross-sectional area of the through hole 41 at the end portion 413 on the side of the first space 16 can be adopted. Further, as another modification, as shown in FIG. 8D, when the cross-sectional area of the through hole 41 becomes smaller from the side of the first space 16 and the side of the second space 14 toward the diaphragm 414. As the opening area of the through hole 41, the cross-sectional area of the through hole 41 at the position of the diaphragm 414 can be adopted. As shown in FIGS. 8A to 8D, the through hole 41 is not limited to a form in which the cross-sectional area changes regularly along the penetrating direction, and the cross-sectional area changes irregularly along the penetrating direction. In any case, the smallest cross-sectional area of the through-hole 41 can be used as the opening area of the through-hole 41.

By satisfying the above-mentioned area ratio relationship, the dispersion 40 acts as a resistance to the flow of the supply liquid, and the supply liquid can be dispersed and flowed into the second space 14, and is formed at a position facing the introduction port 12. The flow velocity of the supply liquid flowing through the through hole 41 is suppressed, and the flow velocity of the supply liquid locally increasing in the second space 14 is reduced. As a result, the flow velocity distribution of the supply liquid in the second space 14 can be made uniform. Here, when the area ratio St / Sa> 0.58, the ratio of the through hole 41 to the dispersion 40 is large, so that the flow of the supply liquid from the introduction port 12 is easily affected in the second space 14. There is.

In order to further exert this effect, as shown in the analyzes (1) to (8) described later, the area ratio St / Sa ≦ 0.50 is more preferably satisfied, and the area ratio St / Sa ≦ 0. 47, Area ratio St / Sa ≤ 0.40, Area ratio St / Sa ≤ 0.34, Area ratio St / Sa ≤ 0.20, Area ratio St / Sa ≤ 0.19, Area ratio St / Sa ≤ 0. 10. The area ratio St / Sa ≦ 0.09, the area ratio St / Sa ≦ 0.05, and the area ratio St / Sa ≦ 0.03 are satisfied. Alternatively, the area ratio St / Sa ≦ 0.01 may be satisfied. (See FIGS. 14a, 14b and Table 1). When the area ratio St / Sa is in the range listed here, the above-mentioned effect that the dispersion 40 makes the flow velocity distribution of the supply liquid in the second space 14 uniform is particularly enhanced. The numerical range given here may be set to this range when St / Sa is calculated and the third place of the minority is rounded off. The lower limit of St / Sa is not particularly limited, and St / Sa> 0 may be used, and for example, St / Sa ≧ 0.001 can be set.

The range of the cross-sectional area St is not particularly limited as long as the relationship of the area ratio St / Sa (St / Sa ≦ 0.58 or the above-mentioned more specific range) is satisfied, but is, for example, 10,000 mm ² or less. preferably 9,000Mm ² or less, 8,639Mm ² or less, 8,000 mm ² or less, 7,069Mm ² or less, 7,000 mm ² or less, 6,000 mm ² or less, 5,026Mm ² or less, 5,000 mm ² or less , 4,000 mm ² or less, 3,000 mm ² or less, 2,898mm ² below, 2,827mm ² below, 1,500mm ² below, 1,335mm ² below, 1,000mm ² or less, 500 mm ² or less, 423mm ² or less Is. The lower limit of the cross-sectional area St is not particularly limited as long as the relationship of the area ratio St / Sa is satisfied, but is, for example, 5 mm ² or more, 10 mm ² or more, 20 mm ² or more, 50 mm ² or more, 94 mm ² or more.

The range of the total opening area Sa is not particularly limited as long as the relationship of the area ratio St / Sa (St / Sa ≦ 0.58 or the above-mentioned more specific range) is satisfied, but for example, 100,000 mm ² or less. , and the preferably 50,000 mm ² or less, 30,000 mm ² or less, 20,000 mm ² or less, 15,000 mm ² or less. The lower limit of the total opening area Sa is not particularly limited as long as the relationship of the area ratio St / Sa is satisfied, but is, for example, 10 mm ² or more, preferably 100 mm ² or more, 200 mm ² or more, 300 mm ² or more, 400 mm ^2. The above is 500 mm ² or more.

5. Introductory port 12 and through hole 41
As shown in FIG. 5, in the embodiment of the present invention, the diameter of the maximum inscribed circle inscribed in the inner circumference of the introduction port 12 is β (for example, μm or mm), and the narrowest of the through holes 41 of the dispersion 40. The diameter of the maximum inscribed circle inscribed in the inner circumference 41a of the portion is α (for example, μm or mm). Here, the inner circumference 12a of the introduction port 12 refers to the inner circumference of the introduction port 12 when viewed from the side of the first space 16. The "inner circumference" of the through hole 41 of the dispersion 40 refers to the inner circumference on the cross section (cross section perpendicular to the penetration direction) of the through hole 41 of the dispersion 40. The "narrowest portion" in the through hole 41 of the dispersion 40 refers to the portion of the through hole 41 in which the diameter of the maximum inscribed circle inscribed in the inner circumference of the through hole 41 is the smallest. In the present embodiment, as shown in FIG. 8A, the diameter of the maximum inscribed circle inscribed in the inner circumference of the through hole 41 does not change in the penetration direction and is constant. Any position 411 can be the "narrowest part". As a modification, as shown in FIG. 8B, when the diameter of the maximum inscribed circle inscribed in the inner circumference of the through hole 41 decreases from the side of the first space 16 toward the side of the second space 14. The end portion 412 of the through hole 41 on the side of the second space 14 corresponds to the "narrowest portion". Further, as another modification, as shown in FIG. 8C, the diameter of the maximum inscribed circle inscribed in the inner circumference of the through hole 41 is directed from the side of the second space 14 to the side of the first space 16. When it becomes smaller, the end portion 413 of the through hole 41 on the side of the first space 16 corresponds to the "narrowest portion". Further, as another modification, as shown in FIG. 8D, the diameter of the maximum inscribed circle inscribed in the inner circumference of the through hole 41 is narrowed down from the side of the first space 16 and the side of the second space 14. When it becomes smaller toward 414, the throttle 414 of the through hole 41 corresponds to the "narrowest portion". As shown in FIGS. 8A to 8D, the through hole 41 is not limited to a form in which the diameter of the maximum inscribed circle inscribed in the inner circumference of the through hole 41 changes regularly along the penetration direction. The diameter may change irregularly along the penetration direction, and in any case, the portion having the smallest diameter can be the narrowest portion of the through hole 41. In the present invention, the "diameter of the maximum inscribed circle inscribed in the inner circumference of the introduction port" refers to the diameter of the circle when the inner circumference of the introduction port is a circle as shown in FIG. In the case of the shape of, it refers to the diameter of the maximum inscribed circle. Similarly, the "diameter of the maximum inscribed circle inscribed in the inner circumference of the through hole of the dispersion" is the diameter of the circle when the inner circumference of the through hole of the dispersion is a circle as shown in FIG. Refers to the diameter of the maximum inscribed circle C when it has another shape as shown in FIG.

In the following description, for convenience, "the diameter of the maximum inscribed circle inscribed in the inner circumference of the introduction port" is referred to as "the diameter of the introduction port", and "the maximum inscribed circle inscribed in the inner circumference of the narrowest portion of the through hole". The "diameter of the circle" may be referred to as the "hole diameter of the through hole".

The diameter β of the introduction port 12 is, for example, 10 μm or more, preferably larger than 50 μm, larger than 100 μm, larger than 200 μm, larger than 300 μm, larger than 400 μm, and 500 μm because cells need to pass through the introduction port 12. Larger, greater than 600 μm, greater than 700 μm, greater than 800 μm, greater than 900 μm, greater than 1 mm and greater than or equal to 4 mm. The upper limit of the diameter β of the introduction port 12 is not particularly limited, but is, for example, 100 mm or less, preferably 90 mm or less, 80 mm or less, 70 mm or less, 60 mm or less, 50 mm or less, 40 mm or less, 30 mm or less, 20 mm or less, 10 mm or less. ..

Further, the pore diameter α of the through hole 41 is, for example, 10 μm or more, preferably 50 μm or more, 100 μm or more, 200 μm or more, 300 μm or more, 400 μm or more, 500 μm or more, 600 μm because it is necessary to allow cells to pass through the through hole 41. These are 700 μm or more, 800 μm or more, 900 μm or more, 1 mm or more, 5 mm or more, and 10 mm or more. When the pore diameter α of the through hole 41 is 500 μm or more, the supply liquid containing cells is unlikely to be clogged in the through hole 41, and the flow of the supply liquid supplied to the second space 14 through the through hole 41 is not obstructed. It is preferable because it is easily dispersed. The upper limit of the hole diameter α of the through hole 41 is not limited, but is, for example, 100 mm or less, preferably 90 mm or less, 80 mm or less, 70 mm or less, 60 mm or less, 50 mm or less, 40 mm or less, 30 mm or less, 20 mm or less.

6. Relationship between the diameter β of the introduction port 12 and the hole diameter α of the through hole 41 As shown in FIG. 5, in one of the embodiments of the present invention, the diameter β (for example, μm or mm) of the introduction port 12 is a dispersion. It is larger than the hole diameter α (for example, μm or mm) of the through hole 41 of 40. Further, in the embodiment shown in FIG. 17, the diameter β of the maximum inscribed circle inscribed in the inner circumference of the introduction port 12 is the maximum inscribed circle inscribed in the inner circumference 41a of the through hole 41 of the dispersion 40 in the narrowest portion. This is an example larger than the diameter α of C.

In the embodiment of the present invention, the introduction port 12 and the through hole 41 are formed so that the diameter β of the introduction port 12 of the dispersion 40 is larger than the hole diameter α of the through hole 41 of the dispersion 40. , Regardless of the distance between the through holes 41, the dispersion 40 acts as a resistance to the flow of the supply liquid from the introduction port 12, so that the dispersion 40 is placed upstream of the dispersion 40 before the supply liquid passes through the through holes 41. The supply liquid can be dispersed on the facing wall surface 42 located.

As a result, in the embodiment in which the dispersion 40 has a plurality of through holes 41, in particular, the flow velocity of the supply liquid flowing through the through holes 41 formed at the position facing the introduction port 12 and the supply liquid flowing through the other through holes 41. It is possible to suppress the variation with the flow velocity of. As a result, it is possible to reduce the increase in the flow velocity of the supply liquid locally in the second space 14. Even when the introduction port 12 and the through hole 41 are formed so that the diameter β of the introduction port 12 is larger than the pore diameter α of the dispersion 40, the through hole is used to make the flow of the supply liquid more uniform. The spacing between the 41s may be further designed.

In this preferred embodiment, as long as the diameter β of the introduction port 12 is larger than the hole diameter α of the through hole 41 of the dispersion 40, the ratio α / β is not particularly limited, but for example, the relationship of α / β <1 is satisfied. .. For example, α / β ≦ 0.9, α / β ≦ 0.8, α / β ≦ 0.7, α / β ≦ 0.6, α / β ≦ 0.5, α / β ≦ 0.4, α The relationships of / β ≦ 0.3, α / β ≦ 0.25, α / β ≦ 0.2, and α / β ≦ 0.1 may be satisfied. When calculating the ratio α / β, the units of the hole diameter α of the through hole 41 of the dispersion 40 and the diameter β of the introduction port 12 are unified. For example, when the unit of the hole diameter α is μm, the unit of the diameter β is also unified to μm, and when the unit of the hole diameter α is mm, the unit of the diameter β is also unified to mm.

The diameter β and the pore diameter α in the above-mentioned α / β ratio are not particularly limited, but may be appropriately designed within the range of the numerical values described in the present specification, for example.

In order to more clearly exhibit the above effect due to the diameter β of the introduction port 12 being larger than the hole diameter α of the through hole 41 of the dispersion 40, as in the embodiment shown in FIG. 5, the introduction port 12 When the inner circumference 12a and the inner circumference 41a at the narrowest portion of the through hole 41 of the dispersion 40 are overlapped with each other, it is preferable that the former completely encloses the entire latter, and in this case, the introduction port 12 It is more preferable that the inner circumference 12a of the above and the inner circumference 41a of the through hole 41 of the dispersion 40 have a similar shape.

7. Length ratio D / L in hole spacing D and dispersion width L (spacing of through holes 41)
FIG. 7 is a diagram for explaining the hole spacing and the length ratio D / L of the through holes 41 of the dispersion 40 of the cell culture vessel 1 according to the first embodiment on the side of the first space 16. ..

In this embodiment, in the dispersion 40, a plurality of through holes 41 are arranged along a predetermined direction (direction X in FIG. 7) on the surface of the dispersion 40 on the side of the first space 16. The "predetermined direction" may be one direction (vertical direction in FIG. 7) as shown in FIG. 7, or may be a plurality of directions intersecting each other as shown in FIG. 5 and the like. Of the through holes 41 arranged in the dispersion 40, the hole spacing between adjacent through holes on the side of the first space 16 is D (for example, μm or mm), and the dispersion 40 in a predetermined direction X Let the width be L (eg, μm or mm). Here, the hole spacing D is the shortest along the predetermined direction X of the inner circumference 41b of the adjacent through hole 41 on the side of the first space 16 when the dispersion 40 is viewed from the side of the first space 16. Refers to the distance. The width L refers to the width along a predetermined direction X when the dispersion 40 is viewed from the side of the first space 16. The hole spacing D between the through holes 41 arranged in one direction is preferably the same (including substantially the same), but may be different. Further, the inner circumferences 41b of the plurality of through holes 41 arranged in one direction preferably have similar shapes to each other, but are not limited to this. Further, the plurality of through holes 41 arranged in one direction preferably have the same hole diameter, but may be different. The definition of the hole diameter of the through hole is as described above.

In the embodiment in which a plurality of through holes 41 are arranged along a predetermined direction (direction X in FIG. 7) on the surface of the dispersion 40 on the side of the first space 16, the range of the hole spacing D is particularly large. Although not limited, it is, for example, 1 μm or more, preferably 10 μm or more, 50 μm or more, 100 μm or more, 500 μm or more, 1 mm or more, 3 mm or more, 5 mm or more, 10 mm or more. The upper limit of the hole spacing D is not limited, but is preferably 250 mm or less, for example.

The range of the width L of the dispersion 40 is not particularly limited, but is, for example, 60 μm or more, preferably 100 μm or more, 500 μm or more, 1 mm or more, 10 mm or more, 50 mm or more, 60 mm or more. The range of the width L of the dispersion 40 is not particularly limited, but is preferably 250 mm or less, for example.

As shown in FIGS. 5 and 17, it is preferable that the introduction port 12 and the through hole 41 are formed so that the diameter β of the introduction port 12 is larger than the hole diameter α of the through hole 41 of the dispersion 40. However, the effect of the present invention can also be obtained by satisfying the relationship of 0.0167 ≦ D / L (length ratio). In particular, when the diameter β of the introduction port 12 is equal to or less than the hole diameter α of the through hole 41 of the dispersion 40, it is preferable to satisfy the relationship of 0.0167 ≦ D / L (length ratio). When calculating the ratio D / L, the units of the hole spacing D and the width L of the dispersion 40 are unified. For example, when the unit of the hole spacing D is μm, the unit of the width L of the dispersion 40 is also unified to μm, and when the unit of the hole spacing D is mm, the width of the dispersion 40 is used. The unit of L is also unified to mm.

Here, when an array consisting of a plurality of through holes 41 is included on one dispersion 40, D / L, which is a length ratio for at least one array, may satisfy the above relationship. For example, as shown in FIG. 5, the dispersion 40 has a rectangular shape when viewed from the first space 16, and a plurality of through holes 41 have two or more through holes 41 in a direction (longitudinal direction) along the long side of the rectangle. In a mode in which rows are formed, two or more rows are formed in a direction along the short side of the rectangle (short side direction), and D in each row is the same, at least. The D / L, which is the length ratio in the lateral direction, may satisfy the above relationship.

When the dispersion 40 satisfies the above-mentioned relationship of 0.0167 ≦ D / L (length ratio), the distance between the through holes can be secured. As a result, even when the diameter β of the introduction port 12 is equal to or smaller than the hole diameter α of the through hole 41 of the dispersion 40, the supply liquid can be uniformly dispersed on the facing wall surface 42 of the dispersion 40. As a result, it is possible to reduce the local increase in the flow velocity of the supply liquid in the second space 14, and the supply liquid can be uniformly dispersed in the second space 14 from the plurality of through holes 41, and the supply liquid can be uniformly dispersed in the second space 14. The flow velocity distribution of the feed solution can be made uniform. In this aspect, the hole diameter α of the through hole 41 of the dispersion 40 (defined above) is compared with the diameter β of the introduction port 12 (defined above), and the hole diameter of the through hole 41 of the dispersion 40 is compared. α may be equal to or larger than the diameter β of the introduction port 12, and the ratio thereof is not particularly limited, but for example, α / β ≧ 1, α / β ≧ 1.2, α / β ≧ 1.25, α / β. The relationship of ≧ 2 and α / β ≧ 2.5 may be satisfied.

When the diameter β of the introduction port 12 is equal to or less than the hole diameter α of the through hole 41 of the dispersion 40 and D / L <0.0167, the hole spacing D is smaller than the width L of the dispersion 40. Therefore, the supply liquid may easily pass through the dispersion 40, and it may be difficult for the supply liquid to be uniformly dispersed in the second space 14.

In order to further exhibit this effect, as shown in the analyzes (9) to (17) described later, more preferably 0.0833 ≦ D / L is satisfied, and further preferably 0.1667 ≦ D / L. (See Table 2). When the diameter β of the introduction port 12 is equal to or less than the hole diameter α of the through hole 41 of the dispersion 40, it is particularly preferable that the D / L is in this range.

The hole spacing D and the width L of the dispersion in the above-mentioned D / L ratio are not particularly limited, but are appropriately designed within the range of the numerical values described in the present specification, for example. May be good.

8. Shape of through hole 41 If the flow velocity distribution of the supply liquid flowing in the second space 14 can be made uniform, the shape of the inner circumference of the through hole 41 (inner circumference on the cross section of the through hole 41) is circular. It is not particularly limited to. For example, the shape may be a polygonal shape (triangular shape, quadrangular shape, pentagonal shape, hexagonal shape, etc.), an elliptical shape, or a slit shape. When the shape of the inner circumference of the through hole 41 is a shape surrounded by smoothly continuous lines such as a circle, an ellipse, or a flat shape thereof, a shape surrounded by smoothly continuous curves is particularly necessary. In the case of, it is particularly preferable because the flow velocity of the supply liquid passing through the through hole 41 is unlikely to be biased. In particular, it is preferable that the shape of all the inner circumferences of the plurality of through holes 41 is a shape surrounded by smoothly continuous lines.

8 (a) is an enlarged cross-sectional view of the vicinity of the through hole 41 of FIG. 3, and FIGS. 8 (b) to 8 (d) are enlarged cross-sectional views showing a modified example thereof.

In the present embodiment, as shown in FIG. 8A, the cross-sectional area of the through hole 41 and the maximum inscribed circle inscribed in the inner circumference of the through hole 41 from the first space 16 to the second space 14 The diameter of the is constant without changing in the penetrating direction. In addition to this, for example, as shown in FIG. 8B, as the first space 16 to the second space 14 progress, the cross-sectional area of the through hole 41 and the maximum inner circumference inscribed in the inner circumference of the through hole 41 The diameter of the tangent circle may be reduced.

Further, as another modification, as shown in FIG. 8C, the cross-sectional area of the through hole 41 and the maximum inscribed in the inner circumference of the through hole 41 as the first space 16 progresses to the second space 14. The diameter of the inscribed circle may be increased.

As another modification, as shown in FIG. 8D, a through hole 41 in which a diaphragm 41b is formed may be formed between the first space 16 and the second space 14. In this modification, in the aperture 41b, the cross-sectional area of the through hole 41 and the diameter of the maximum inscribed circle inscribed in the inner circumference of the through hole 41 are the smallest.

9. Other Modifications FIG. 9 is a diagram showing a dispersion 40A according to a modification of the dispersion 40 of the first embodiment, which corresponds to FIG. As shown in FIG. 5, in the above-described embodiment, a plurality of through holes have the same shape and are formed at equal intervals in the dispersion 40, but these may be formed irregularly. In addition to this, as shown in FIG. 9, for example, through holes 41A to 41C having a plurality of types of pore diameters may be formed in the dispersion 40A.

Specifically, as shown in FIG. 9, in this modified example, the hole diameters of the through holes 41A to 41C become smaller from the outside of the dispersion 40A toward the center thereof. More specifically, in the present embodiment, a plurality of through holes 41A are formed along the outer edge of the dispersion 40A, and a plurality of through holes 41B smaller than the hole diameter of the through holes 41A are formed inside the through holes 41A. .. Further, in the center of the dispersion 40A surrounded by the plurality of through holes 41B, a plurality of through holes 41C smaller than the hole diameter of the through holes 41B are formed. At least, it is preferable that the hole diameter of the through hole 41C facing the introduction port 12 is formed to be smaller than the diameter β of the introduction port 12.

Even in such a case, the supply liquid can be uniformly dispersed on the facing wall surface 42 of the dispersion 40A, and the supply liquid can flow from the through holes 41A to 41C into the second space 14 at a uniform flow velocity.

FIG. 17 is a diagram showing a dispersion 40E according to a modified example of the dispersion 40 of the first embodiment, which corresponds to FIG. As shown in FIG. 5, in the above-described embodiment, each through hole 41 has an inner circumference 41a of an independent circle, but the shape of the inner circumference is not particularly limited, and for example, a plurality of figures are fused. It may be formed so as to have an inner peripheral shape. Specifically, in the modified example shown in FIG. 17, the through hole 41E formed in the dispersion 40E has an inner circumference 41a having a shape in which a plurality of circles are arranged in one direction and fused with each other. Even in such a case, the supply liquid can be uniformly dispersed on the facing wall surface 42E of the dispersion 40E, and the supply liquid can flow from each through hole 41E into the second space 14 at a uniform flow rate.

Further, in the above-described embodiment, the shape of the dispersion 40 is plate-shaped, but the shape is not particularly limited as long as the above-mentioned through hole 41 can be formed, but the shape is not particularly limited, but is block-shaped, dome-shaped, or the like. Can be mentioned. Further, the plate-shaped dispersion 40 may have a flat plate shape or a curved plate shape.

FIG. 10 is an enlarged cross-sectional view of the dispersion 40D according to another modification of the dispersion 40 of the first embodiment. As shown in FIG. 10, a dome-shaped dispersion 40D is arranged so as to cover the introduction port 12, and a plurality of through holes 41D are formed in the dispersion 40D so that the supply liquid flows radially from the introduction port 12. You may. In this modification, the through holes 41D are formed in a fixed direction, but each through hole 41D is formed in a different direction (radial). Even in this case, the supply liquid can flow from the through hole 41D of the dispersion 40D into the second space 14 at a uniform flow rate as compared with the case where the dispersion is not provided. ^{Further, also in this modification, the cross-sectional area Sa (mm 2} ) at the position 16a adjacent to the dispersion 40D in the first space 16 is set, and the total opening area of the through hole 41D of the dispersion 40D is St (mm ² ). , It is preferable that the relationship of the area ratio St / Sa ≦ 0.58 is satisfied, and more preferably, the diameter β of the introduction port 12 is larger than the hole diameter α of the through hole 41D of the dispersion 40.

[Second Embodiment]
FIG. 11 is a schematic perspective view showing the space inside the container body 10 of the cell culture container 1A according to the second embodiment, which corresponds to FIG. FIG. 12 is a schematic cross section of the cell culture vessel 1A according to the second embodiment, which corresponds to FIG.

The difference between the cell culture vessel 1A according to the second embodiment and that of the first embodiment is that the rectifying fluid 50 is newly provided. Therefore, the same portion as the cell culture vessel of the first embodiment is designated by a reference numeral, and detailed description thereof will be omitted.

10. Rectifier 50
As shown in FIGS. 11 and 12, in the cell culture vessel 1A, a rectifying body 50 that regulates the flow of the supply liquid flowing through the second space 14 is arranged on the outlet 13 side of the second space 14, and is a rectifying body. A through hole is formed in 50.

If the rectifying body 50 can regulate the flow of the supply liquid flowing through the second space 14, it is not always necessary to form the through hole 51. For example, it may be a resistor or the like that acts as a resistance to the flow of the supply liquid in the vicinity of the outlet 13, or the number of through holes 51 of the rectifying body 50 may be one, and is illustrated in the first embodiment. It may be the same as the viewpoint of the dispersion. Further, the shape of the through hole 51 of the rectifying body 50 may be the same as that of the dispersion shown in the first embodiment.

Further, in the present embodiment, a third space 19 for retaining the supply liquid flowing out of the rectifying body 50 is formed between the rectifying body 50 and the outlet 13. In the present embodiment, the first space 16, the second space 14, and the third space 19 are formed inside the container body 10. By providing the third space 19, the supply liquid flowing out from the outlet 13 is temporarily secured in the third space 19. Therefore, the flow velocity of the supply liquid in the vicinity of the outlet 13 can be suppressed, and the flow of the supply liquid flowing out from the second space 14 can be adjusted. Only one rectifying fluid 50 is arranged inside the container body 10 of the cell culture container 1A of the present embodiment shown in the figure.

In the present invention, the shape of the third space is not particularly limited as long as it can retain the supply liquid flowing out of the rectifying body 50, and for example, a rectangular parallelepiped, a cone (cone, a truncated cone, a pyramid, etc.) It can be a pyramid cone, etc.), a column (cylinder, a rectangular parallelepiped, etc.), a sphere, and / or a hemisphere, or a combination of two or more of these. In the present embodiment shown in FIGS. 11 and 12, among the wall surfaces forming the third space 19, the facing wall surface 53 of the rectifying body 50 facing the outlet 13 and the outlet 13 facing the facing wall 53 are formed. It is parallel to the downstream wall surface 18. Further, the cross section (cross-sectional area) of the third space 19 from the facing wall surface 53 toward the downstream wall surface 18 is constant without being enlarged or reduced. As a result, the third space 19 becomes a rectangular parallelepiped shape.

Similar to the dispersion 40, the through hole 51 of the rectifying body 50 has a cross-sectional area of Sb (mm ² ) in the space inside the container body 10 of the cell culture container 1A at a position adjacent to the rectifying body 50, and is a rectifying body. When the total opening area of the through holes 51 of 50 is set to Sh (mm ² ), it is preferable to satisfy the relationship of Sh / Sb ≦ 0.58. As a result, similarly to the dispersion 40, in the rectifier 50, it is possible to suppress the local increase in the flow of the supply liquid flowing out from the second space 14 toward the outlet 13. More preferably, the area ratio Sh / Sb ≦ 0.47 is satisfied, and more preferably, the area ratio Sh / Sb ≦ 0.40, the area ratio Sh / Sb ≦ 0.34, the area ratio Sh / Sb ≦ 0.20, and the area. Satisfy the ratio Sh / Sb ≦ 0.19, the area ratio Sh / Sb ≦ 0.10, the area ratio Sh / Sb ≦ 0.09, the area ratio Sh / Sb ≦ 0.05, and the area ratio Sh / Sb ≦ 0.03. .. Alternatively, the area ratio Sh / Sb ≦ 0.01 may be satisfied. The numerical range given here may be set to this range when Sh / Sb is calculated and the third digit of the minority is rounded off. The lower limit of Sh / Sb is not particularly limited, and Sh / Sb> 0 may be used, and for example, Sh / Sb ≧ 0.001 can be set. When the area ratio Sh / Sb is in the range listed here, the above-mentioned effect of suppressing the local increase in the flow of the supply liquid flowing out from the second space 14 toward the outlet 13 by the rectifying body 50 is particularly effective. Increase. Here, "the cross-sectional area of the space inside the container body 10 of the cell culture container 1A at a position adjacent to the rectifying fluid 50" is defined as at least one of the second space 14 and the third space 19, more preferably the first. 3 Refers to the cross-sectional area of the space 19 at a position adjacent to the rectifying body 50. That is, one or both of the cross-sectional area of the second space 14 at the position 14a adjacent to the rectifying body 50 and the cross-sectional area of the third space 19 at the position 19a adjacent to the rectifying body 50 are Sh / Sb. It is preferable that the condition of Sh / Sb is satisfied, and it is more preferable that the cross-sectional area of the third space 19 at the position 19a adjacent to the rectifying body 50 satisfies the above condition of Sh / Sb.

The range of the cross-sectional area Sh is not particularly limited as long as the relationship of the area ratio Sh / Sb (Sh / Sb ≦ 0.58 or the above-mentioned more specific range) is satisfied, but is, for example, 10,000 mm ² or less. preferably 9,000Mm ² or less, 8,639Mm ² or less, 8,000 mm ² or less, 7,069Mm ² or less, 7,000 mm ² or less, 6,000 mm ² or less, 5,026Mm ² or less, 5,000 mm ² or less , 4,000 mm ² or less, 3,000 mm ² or less, 2,898mm ² below, 2,827mm ² below, 1,500mm ² below, 1,335mm ² below, 1,000mm ² or less, 500 mm ² or less, 423mm ² or less Is. The lower limit of the cross-sectional area Sh is not particularly limited as long as the relationship of the area ratio Sh / Sb is satisfied, but is, for example, 5 mm ² or more, 10 mm ² or more, 20 mm ² or more, 50 mm ² or more, 94 mm ² or more.

The range of the total opening area Sb is not particularly limited as long as the relationship of the area ratio Sh / Sb (Sh / Sb ≦ 0.58 or the above-mentioned more specific range) is satisfied, but is usually 100,000 mm ² or less. , and the example 50,000 mm ² or less, 30,000 mm ² or less, 20,000 mm ² or less, 15,000 mm ² or less. The lower limit of the total opening area Sb is not particularly limited as long as the relationship of the area ratio Sh / Sb is satisfied, but is, for example, 10 mm ² or more, preferably 100 mm ² or more, 200 mm ² or more, 300 mm ² or more, 400 mm ² or more. , 500 mm ² or more.

In the present invention, the cross-sectional area of the second space 14 at the position 14a adjacent to the rectifying body 50 is a cross section of the second space 14 cut out by a surface along the side surface of the rectifying body 50 on the side of the second space 14. Refers to the area of. The surface of the rectifying body 50 along the side surface of the second space 14 is preferably a plane perpendicular to the flow direction of the supply liquid in the second space 14. In the present invention, the cross-sectional area of the third space 19 at the position 19a adjacent to the rectifying body 50 is a cross section of the third space 19 cut out by a surface along the surface of the rectifying body 50 on the side of the third space 19. Refers to the area of. The surface of the rectifying body 50 along the surface on the side of the third space 19 is preferably a plane perpendicular to the flow direction of the supply liquid in the third space 19.

In the present invention, the opening area of the individual through-holes 51 of the rectifying body 50 refers to the smallest cross-sectional area of the cross-sectional areas of the individual through-holes 51 (cross-sectional area perpendicular to the penetrating direction). The total opening area is the total opening area of each through hole 51. The smallest cross-sectional area of the individual through-holes 51 of the rectifying body 50 can be obtained in the same manner as the smallest cross-sectional area of the individual through-holes 41 of the dispersion 40.

Similar to the dispersion 40, the diameter of the maximum inscribed circle inscribed in the inner circumference of the outlet 13 is larger than the diameter of the maximum inscribed circle inscribed in the inner circumference of the through hole 51 of the rectifying body 50 at the narrowest portion. It is preferable that the outlet 13 and the through hole 51 of the rectifying body 50 are formed so as to increase the size.

Further, in another preferred embodiment of the present embodiment, when the rectifying body 50 is viewed from at least one side of the second space 14 and the third space 19 (preferably, the rectifying body 50 is the third space 19). When viewed from the side), a plurality of the through holes 51 are arranged in a predetermined direction in the rectifying body 50, and the hole spacing of the adjacent through holes among the arranged through holes 51 is E (). For example, when μm or mm) and the width of the rectifying fluid in the predetermined direction is Z (for example, μm or mm), the range of the hole spacing E is not particularly limited, but is, for example, 1 μm or more. It is preferably 10 μm or more, 50 μm or more, 100 μm or more, 500 μm or more, 1 mm or more, 3 mm or more, 5 mm or more, and 10 mm or more. The upper limit of the hole spacing E is not limited, but is preferably 250 mm or less, for example. The range of the width Z of the rectifying body 40 is not particularly limited, but is, for example, 60 μm or more, preferably 100 μm or more, 500 μm or more, 1 mm or more, 10 mm or more, 50 mm or more, 60 mm or more. The range of the width Z of the rectifying body 40 is not particularly limited, but is preferably 250 mm or less, for example. The hole spacing E is the hole spacing on the surface of the rectifying body 50 when viewed from the surface of at least one of the second space 14 and the third space 19, and is more preferably adjusted. It is the hole spacing on the surface when the fluid 50 is viewed from the surface on the side of the third space 19.

Further, in the embodiment in which a plurality of the through holes 51 are arranged along a predetermined direction in the rectifying body 50, the hole spacing E of the adjacent through holes among the arranged through holes 51 and the predetermined direction are provided. When the width Z of the rectifying fluid in the above satisfies the relationship of 0.0167 ≦ E / Z (length ratio), the rectifying fluid 50 also has the lead-out port 13 from the second space 14 as in the dispersion 40. It is possible to suppress a local increase in the flow of the supply liquid flowing out toward it. In order to effectively exhibit this effect, 0.0833 ≦ E / Z is more preferably satisfied, and 0.167 ≦ E / Z is more preferably satisfied. Satisfying the relationship of 0.0167 ≤ E / Z means that the diameter of the maximum inscribed circle inscribed in the inner circumference of the outlet 13 is inscribed in the inner circumference of the through hole 51 of the rectifying body 50 at the narrowest portion. It is particularly preferable when the diameter is equal to or smaller than the diameter of the maximum inscribed circle. When calculating the ratio E / Z, the unit of the hole spacing E and the width Z of the rectifying body 50 is unified. For example, when the unit of the hole spacing E is μm, the unit of the width Z of the rectifying body 50 is also unified to μm, and when the unit of the hole spacing E is mm, the width of the rectifying body 50. The unit of Z is also unified to mm.

The hole spacing E and the width Z of the rectifying fluid in the above-mentioned E / Z ratio are not particularly limited, but are appropriately designed within the range of the numerical values described in the present specification, for example. May be good.

Here, the inner circumference of the outlet 13 refers to the inner circumference of the outlet 13 when the outlet 13 is viewed from the side of the third space 19. On the other hand, the "inner circumference" of the through hole 51 of the rectifying body 50 refers to the inner circumference on the cross section (cross section perpendicular to the penetrating direction) of the through hole 51 of the rectifying body 50. The "narrowest portion" of the through hole 51 of the rectifying body 50 refers to the portion of the through hole 51 in which the diameter of the maximum inscribed circle inscribed in the inner circumference of the through hole 51 is the smallest. In the present invention, the "diameter of the maximum inscribed circle inscribed in the inner circumference of the outlet" refers to the diameter of the circle when the inner circumference of the outlet is a circle as shown in FIG. In the case of the shape of, it refers to the diameter of the maximum inscribed circle. Similarly, the "diameter of the maximum inscribed circle inscribed in the inner circumference of the through hole of the rectifying body" is the diameter of the circle when the inner circumference of the through hole of the rectifying body is a circle as shown in FIG. Refers to the diameter of the maximum inscribed circle when it has another shape (not shown). A preferred embodiment of the relative relationship between the inner circumference of the lead-out port 13 and the inner circumference of the through hole 51 of the rectifying body 50 in the narrowest portion is the inner circumference 12a of the introduction port 12 and the through hole 41 of the dispersion 40. Can be selected from the same range as the preferred embodiment of the relative relationship with the inner circumference 41a in the narrowest portion.

In the following description, for convenience, "the diameter of the maximum inscribed circle inscribed in the inner circumference of the outlet" is referred to as "the diameter of the outlet", and "inside the inner circumference of the through hole of the rectifying body at the narrowest part". The "diameter of the maximum inscribed circle in contact" may be referred to as "the diameter of the through hole of the rectifying body".

The diameter of the outlet 13 is, for example, 10 μm or more, preferably greater than 50 μm, greater than 100 μm, greater than 200 μm, greater than 300 μm, greater than 400 μm, greater than 500 μm, because cells need to pass through the outlet 13. Greater than 600 μm, greater than 700 μm, greater than 800 μm, greater than 900 μm, greater than 1 mm and greater than or equal to 4 mm. The upper limit of the diameter β of the introduction port 12 is not limited, but is, for example, 100 mm or less, preferably 90 mm or less, 80 mm or less, 70 mm or less, 60 mm or less, 50 mm or less, 40 mm or less, 30 mm or less, 20 mm or less.

Further, the pore diameter of the through hole 51 is, for example, 10 μm or more, preferably 50 μm or more, 100 μm or more, 200 μm or more, 300 μm or more, 400 μm or more, 500 μm or more, 600 μm or more because it is necessary to allow cells to pass through the through hole 51. , 700 μm or more, 800 μm or more, 900 μm or more, 1 mm or more, 5 mm or more, 10 mm or more. When the pore diameter α of the through hole 51 is 500 μm or more, the supply liquid containing cells is unlikely to be clogged in the through hole 51, and the flow of the supply liquid supplied to the third space 19 through the through hole 51 is not obstructed. It is preferable because it is easily dispersed. The upper limit of the hole diameter of the through hole 51 is not limited, but is, for example, 100 mm or less, preferably 90 mm or less, 80 mm or less, 70 mm or less, 60 mm or less, 50 mm or less, 40 mm or less, 30 mm or less, 20 mm or less.

In the embodiment in which the plurality of through holes 51 are arranged in the rectifying body 50 along a predetermined direction, the size of the diameter of the outlet 13 with respect to the through hole 51 is not particularly limited, but the diameter of the outlet is limited. When the diameter of the through hole 51 in the rectifying body 50 is larger than that of the through hole 51, the hydraulic pressure of the supply liquid flowing out of the rectifying body 50 and staying in the third space 19 can be reduced.

The hole spacing D and the width L of the dispersion in relation to the size of the diameter of the outlet 13 with respect to the above-mentioned through hole 51 are not particularly limited, but are, for example, the numerical values described in the present specification. Within the range, it may be designed as appropriate.

Here, when the through hole 51 of the rectifying body 50 is formed under the same conditions as the dispersion 40, a surface perpendicular to the flow path direction at a position intermediate between the dispersion 40 and the rectifying body 50 is particularly formed. When the disperse 40 and the rectifier 50 have a plane-symmetrical relationship when the planes of symmetry are used, it is not necessary to distinguish between the introduction port 12 and the outlet port 13 in the cell culture vessel 1A. That is, the cell culture vessel 1A can be used by selecting either one as the introduction port 12 and the other as the outlet port 13.

11. Cell culture system 100
FIG. 13 is a schematic conceptual diagram of the cell culture system 100 provided with the cell culture containers 1 and 1A according to the first and second embodiments.

As shown in FIG. 13, in the cell culture system 100, the cell culture container 1 (1A), the medium supply source 91 for supplying the medium as the supply liquid, and the medium from the medium supply source 91 are supplied to the cell culture container 1 (1A). It is provided with at least a pumping unit 92 such as a pump that pumps the culture medium 12 to the introduction port 12. Specifically, the cell culture vessel 1 (1A) is connected to the medium supply source 91 via the pumping unit 92. A valve 91a is provided between the medium supply source 91 and the pumping unit 92, whereby the medium can be fed.

Preferably, in the cell culture system 100 according to the present embodiment, the medium that has passed through the cell culture container 1 (1A) is collected in the medium supply source 91 via the three-way valve 91b. The outlet 13 of 1A) is connected to the medium supply source 91. As a result, the medium can be circulated in the cell culture system 100.

Preferably, in the cell culture system 100 according to the present embodiment, the outlet 13 of the cell culture container 1 (1A) is a waste liquid container in which the medium that has passed through the cell culture container 1 (1A) is different from the medium supply source 91. It is connected to the waste liquid container 94 so that it can be collected in 94. As a result, the medium can be collected in the waste liquid container 94.

In the present embodiment, the cell culture system 100 is provided with a three-way valve 91b, and the medium can be selectively circulated and the medium can be recovered in the cell culture system 100. However, for example, when the medium is not circulated, the outlet 13 of the cell culture container 1 (1A) may be connected to the waste liquid container 94 without using the three-way valve 91b. When the medium is not drained, the outlet 13 of the cell culture container 1 (1A) may be connected to the medium supply source 91 without using the three-way valve 91b.

More preferably, the cell culture system 100 includes a suspension source 95 for supplying a suspension containing cells and a stripping solution supply source 96 for exfoliating cells adhering to the cell culture vessel 1 (1A) after culturing. , Is equipped. The suspension supply source 95, the stripping liquid supply source 96, the buffer solution supply source 97, and the coating agent supply source 98 are connected to the pumping unit 92 via valves 95a, 96a, 97a, 98a. By selecting and opening and closing these valves 95a, 96a, 97a, 98a, a suspension, a stripping solution, a buffer solution, or a coating agent can be selectively supplied to the cell culture vessel 1 (1A) as a supply solution. Can be done. It is preferable that the three-way valve 91b and the valves 95a, 96a, 97a, 98a can be connected aseptically. In the cell culture system 100, when only the cells are cultured in the medium, the suspension supply source 95, the stripping solution supply source 96, and the buffer solution supply source 97 can be omitted.

Further, in the cell culture system 100 according to the present embodiment, a temperature sensor and a temperature control device can be connected to the medium supply source 91. As a result, the temperature of the medium supplied to the cell culture container 1 (1A) and the medium circulating in the cell culture system 100 can be kept constant. A dialysis system can also be linked to a cell culture system 100 (eg, medium source 91, etc.) to remove small molecule waste products and nutrients required for culture (eg glucose, vitamins, etc.). Small molecules such as lipids and amino acids and media containing these small molecules) can also be supplied. Further, the cell culture system 100 can be equipped with a system for adjusting the pH. It is also possible to install a system for adjusting the oxygen concentration and carbon dioxide in the medium. It is also possible to install a system for newly supplementing proteins such as growth factors. In addition, a cell separation device can be connected to the cell culture system 100 (for example, suspension source 95 or the like), whereby a series of steps from isolation of adherent cells to culture is performed in a closed system. be able to. As the cell separation device, for example, CellEffic BM (registered trademark) can be used.

12. Cell culture method The cell culture method will be described below. In this embodiment, cells are adhered to the inner wall surface 15 of the cell culture vessel 1 (1A) shown in FIGS. 1, 11 and the like, and the cells are cultured by bringing the adhered cells into contact with a medium. First, cells and a medium will be illustrated below.

12-1. Cell The cell used in the present invention is not particularly limited as long as it is an adhesive cell (adhesive cell), and may be an animal-derived cell or the like, preferably a mammalian animal-derived cell or the like. It can be preferably a living tissue-derived cell, a cell derived from a living tissue-derived cell, or the like, and particularly preferably from an epithelial tissue-derived cell, a cell derived from an epithelial tissue cell, or the like, or a binding tissue-derived cell or a binding tissue-derived cell. It can be a derived cell or the like, a muscle tissue-derived cell or a cell derived from a muscle tissue-derived cell or the like, or a nerve tissue-derived cell or a cell derived from a nerve tissue-derived cell or the like. The cells derived from the muscle tissue-derived and muscle tissue-derived cells are not particularly limited, and examples thereof include myocardial cells, myoblasts, endothelial cells, and wall cells, which are derived from the nerve tissue-derived and nerve tissue-derived cells. The cells are not particularly limited, but for example, 293FT cells, primary nerve cells, established nerve cells (for example, human neuroblastoma SH-SY5Y), ReproNeuro (ReproCELL), Human Neuronal Kit (Xcell Science). , ICell Nurons (Cellular Dynamics International's), HEK293 cells, BHK-21 cells, etc.

Further, the adhesive cell can be an animal-derived stem cell, a cell differentiated from an animal-derived stem cell, or the like, preferably a cell differentiated from a mammalian animal-derived stem cell or a mammalian-derived stem cell, and more preferably. Can be a human-derived stem cell, a cell differentiated from a human-derived stem cell, or the like. The human-derived stem cells and cells differentiated from human-derived stem cells are not particularly limited, and examples thereof include human-derived mesenchymal stem cells and cells differentiated from human-derived mesenchymal stem cells. The tissue from which the mesenchymal stem cells are derived is not particularly limited, but may be cells isolated from various tissues such as bone marrow, adipose tissue, dental pulp, umbilical cord blood, placenta, and sheep membrane, and human-derived pluripotent stem cells. It may be a cell that has been induced to differentiate from.

Further, the adherent cell can be an animal-derived pluripotent stem cell, a cell differentiated from an animal-derived pluripotent stem cell, or the like, and more preferably from a mammalian animal-derived pluripotent stem cell and a mammalian animal-derived pluripotent stem cell. It can be a differentiated cell or the like, and most preferably a human-derived pluripotent stem cell or a cell differentiated from a human-derived pluripotent stem cell or the like.

The cells in the present invention may have a single adherent cell, or may exist as a cell population by adhering and / or aggregating a plurality of adherent cells.

A "pluripotent stem cell" is a cell having pluripotency that can differentiate into all types of cells constituting the living body, and is in vitro under appropriate conditions. A cell that can continue to grow indefinitely while maintaining pluripotency in the culture of. Specific examples of pluripotent stem cells include embryonic stem cells (ES cells) and EG cells (Shamblott MJ et al., Proc. Natl. Acad), which are pluripotent stem cells derived from fetal primordial germ cells. Sci. USA. (1998) 95, p. 13726-13731), GS cells (Conrad S., Nature (2008) 456, p. 344-349), which are pluripotent stem cells derived from testis, derived from somatic cells. Examples include, but are not limited to, iPS cells (induced pluripotent embryo cells), which are induced pluripotent stem cells. The pluripotent stem cells used in the present invention are particularly preferably ES cells or iPS cells. ES cells are cultured cells derived from undifferentiated cells collected from an inner cell mass existing inside an early embryo called a blastocyst. iPS cells are cultured cells that have been imparted with pluripotency by reprogramming somatic cells into an undifferentiated state by introducing a reprogramming factor into the somatic cells. As reprogramming factors, for example, OCT3 / 4 and KLF4 and SOX2 and c-Myc can be used (Yu J, et al. Science. 2007; 318: 1917-20.), For example, OCT3 / 4 and SOX2 and LIN28. And Nanog can be used (Takahashi K, et al. Cell. 2007; 131: 861-72.). The form of introduction of these factors into cells is not particularly limited, and examples thereof include gene transfer using a plasmid, introduction of synthetic RNA, and direct introduction as a protein. Further, iPS cells prepared by a method using microRNA, RNA, a small molecule compound or the like may be used. In addition, iPS cells prepared from cells derived from healthy volunteers having an HLA (Human Leukocyte Antigen) type combination (HLA homozygote), which is said to be less likely to cause rejection, may be used. As the pluripotent stem cells such as ES cells and iPS cells, commercially available cells or cells that have been sold may be used, or newly prepared cells may be used. As iPS cells, for example, 253G1 strain, 201B6 strain, 201B7 strain, 409B2 strain, 454E2 strain, HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, Nippons-B2 strain, TkDN4-M strain. , TkDA3-1 strain, TkDA3-2 strain, TkDA3-4 strain, TkDA3-5 strain, TkDA3-9 strain, TkDA3-20 strain, hiPSC 38-2 strain, MSC-iPSC1 strain, BJ-iPSC1 strain, etc. be able to. As ES cells, for example, KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SEES1, SEES2, SEES3, HUES8, CyT49, H1 and H9. , HS-181 strain and the like can be used. Newly prepared clinical grade iPS cells or ES cells may be used. The origin of the cells when producing iPS cells is not particularly limited, but for example, fibroblasts or lymphocytes can be used.

The cells used in this embodiment may be of any animal origin, for example, rodents such as mice, rats, hamsters, primates such as humans, gorillas, chimpanzees, and dogs, cats, rabbits, etc. It may be derived from domestic animals such as cows, horses, sheep, goats, pigs, or mammals such as pets, but human-derived cells are particularly preferable.

12-2. Medium The medium used in the present invention can be prepared by using an arbitrary liquid medium for culturing animal cells as a basal medium and appropriately adding other components (serum, serum substitute reagent, growth factor, etc.) as needed. .. In the embodiment in which the growth factor is added to the basal medium, a reagent (such as heparin) for stabilizing the growth factor in the medium may be prepared by adding the growth factor in addition to the growth factor. , The growth factor may be stabilized in advance with a gel, polysaccharide or the like, and then the stabilized growth factor may be added to the basal medium to prepare the growth factor.

As the basal medium, BME medium, BGJb medium, CMRL1066 medium, Glasgo MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscover's Modified Dulvecco's Medium), Medium 199 medium, Eagle's medium, Eagle's medium, Eagle's medium, Eagle's medium, Eagle's medium, Eagle's medium, Eagle's medium. (Dulvecco's Modified Eagle's Medium), ham F10 medium, ham F12 medium, RPMI 1640 medium, Fisher's medium, and a mixed medium thereof (for example, DMEM / F12 medium (Dulvecco's Modified Eagle's Medium)). A medium such as Nutrient Mixture F-12 Ham))) can be used, but is not particularly limited.

In the present embodiment, the cell culture method includes a step of seeding cells, a step of culturing cells, and a step of exfoliating cells. When the above cell culture system 100 is used, specifically, first, the valve 95a is opened, and the introduction port 12 of the cell culture container 1 (1A) is first opened from the suspension supply source 95 containing cells to the pumping unit 92. The cells are seeded by introducing a suspension containing the cells into the cells. The suspension is sent from the introduction port 12 through the through hole 41 of the dispersion 40 to the second space 14. The suspension is taken out from the outlet 13, the three-way valve 91b is switched, and the suspension is discharged to the waste liquid container 94. The step of seeding the cells can further include adhering the cells. Specifically, when the cell culture vessel 1 (1A) is filled with the suspension, the valve 95a and the three-way valve 91b are closed, the driving of the pumping unit 92 is stopped, and the cells are adhered. In order to adhere the cells, the inflow of the supply liquid into the cell culture vessel 1 (1A) is stopped for a certain period of time, although the time is not limited, the cells settle, and the cells adhere to the inner wall surface 15 or the multilayer culture substrate. It is preferable to include a step of allowing the cells to stand until they are allowed to stand. However, depending on the type of cells, a step of opening the valve 98a first and introducing a coating agent before introducing the suspension into the cell culture vessel 1 (1A) may be included. The coating agent is not particularly limited, and examples thereof include laminin, fibronectin, matrigel, gelatin, collagen, and cadherin.

Next, a step of culturing the cells is performed. The step involves culturing the cells while supplying medium to the cell culture vessel in which the cells have been seeded. Specifically, the valve 91a and the three-way valve 91b are opened, the pumping unit 92 is driven, and the medium is introduced from the medium supply source 91 into the introduction port 12 of the cell culture vessel 1 (1A). If necessary, the suspension remaining in the system of the cell culture system 100 is discharged together with the medium into the waste liquid container 94. When cells are adhered to the inner wall surface 15 or the like in advance with a suspension, the above-mentioned series of steps is omitted.

Next, the three-way valve 91b is switched, the medium from the outlet 13 is collected in the medium supply source 91, and the medium is circulated to supply the medium into the cell culture container 1 (1A). Thereby, the medium can be temporarily or constantly supplied into the cell culture vessel 1 (1A), and the cells can be cultured.

After the culture is completed, the valve 91a is closed, the valve 97a is opened, the buffer solution is introduced from the buffer solution supply source 97 into the introduction port 12 of the cell culture container 1 (1A), and the buffer solution is discharged to the waste liquid container 94. As a result, the inhibitor of the stripping solution remaining in the cell culture vessel 1 (1A) is removed. Then, the valve 96a is opened, and the stripping liquid is introduced into the cell culture vessel 1 (1A) from the stripping liquid supply source 96. By this step, the adhered cells can be exfoliated, and the cultured cells can be collected from the outlet 13 together with the exfoliating solution. More preferably, the valve 91a can be further opened, the medium can be introduced from the medium supply source 91 into the introduction port 12 of the cell culture vessel 1 (1A), and the cells cultured with the release agent and the medium can be collected from the outlet port 13. .. As a method for collecting the cultured cells, for example, the outlet 13 may be removed from the liquid feeding line, and the cultured cells may be poured directly into the collection container together with the release agent and the medium, or between the outlet 13 and the three-way valve 91b. A new three-way valve may be provided in the three-way valve, and a new collection container may be provided in the three-way valve. In the case where a new three-way valve is provided between the outlet 13 and the three-way valve 91b and the three-way valve is equipped with a new collection container, the above-mentioned three-way valve is switched toward the collection container to enable the outlet. The cultured cells can be recovered together with the release agent and the medium without removing 13 from the liquid feeding line.

The release agent is not particularly limited, but for example, trypsin, Accutase (registered trademark), TrypLE ^TM Express Enzyme (Life Technologies Japan Co., Ltd.), TrypLE ^TM Select Enzyme (Life Technologies Japan Co., Ltd.), Dispase (registered trademark), collagenase. And so on. In the present embodiment, the cells can be uniformly cultured by making the flow velocity distribution of the supply liquid such as the medium in the second space 14 uniform.

13. Fluid analysis 13-1. Confirmation of Effect of Area Ratio St / Sa In order to confirm the effect explained in "3. Area Ratio St / Sa (Porosity of Dispersion 40)", the inventors used the cell culture vessel shown in FIG. 1 and the like described above. As a basic model, the flow velocity distribution of the supply liquid flowing inside was analyzed. The flow velocity distribution was analyzed using the fluid analysis software ANSYS Fluent (http://www.ansys.com/ja-JP/Products/Fluids) provided by Ansys Japan Co., Ltd. In the analysis shown below, the space inside the container body 10 of the cell culture vessel 1 was set to a rectangular parallelepiped having a width of 250 mm, a length of 350 mm, and a height of 60 mm, and the diameters of the inlet and outlet were set to 4 mm. The inlet, outlet, through hole of the dispersion, and through hole of the rectifying body, which will be described later, are all set so that the inner circumference is circular and the diameter and cross-sectional area are the same at any position in the penetration direction. Since the diameter of the maximum inscribed circle inscribed in the inner circumference of the through hole 41 does not change in the penetration direction and is constant, any position in the penetration direction of the through hole 41 can be set as the "narrowest portion". In the description of all analysis conditions and results, the term pore diameter or caliber refers to the diameter of a circle. Next, a flat-plate dispersion having a thickness of 2 mm having both main surfaces flat was set at a position 25 mm from the introduction port so as to be perpendicular to the center line P. The cross section of the first space 16 at the position 16a adjacent to the dispersion 40 has a width of 250 mm and a height of 60 mm, and its area Sa is 15,000 mm ² . Then, in the dispersion 40, a plurality of through holes 41 having a circular flow path cross section were formed at equal intervals along the width direction and the height direction of the container body 10. The through holes 41 have the same hole spacing in the height direction and the hole spacing in the width direction of the dispersion 40. In the description of all the analysis conditions and results, the hole spacing of the through-holes of the dispersion is the same on both the first space side and the second space side of the dispersion, and similarly, the through-holes of the rectifying body described later. The hole spacing is the same on both the second space side and the third space side of the rectifying body.

In the model of analysis (1), the hole diameter of the through hole was set to 1 mm, the hole spacing was set to 10 mm, and the area ratio St / Sa was set to 0.01.
In the model of analysis (2), the hole diameter of the through hole was set to 1 mm, the hole spacing was set to 5 mm, and the area ratio St / Sa was set to 0.03.
In the model of analysis (3), the hole diameter of the through hole was set to 5 mm, the hole spacing was set to 10 mm, and the area ratio St / Sa was set to 0.09.
In the model of analysis (4), the hole diameter of the through hole was set to 5 mm, the hole spacing was set to 5 mm, and the area ratio St / Sa was set to 0.19.
In the model of analysis (5), the hole diameter of the through hole was set to 10 mm, the hole spacing was set to 5 mm, and the area ratio St / Sa was set to 0.34.
In the model of analysis (6), the hole diameter of the through hole was set to 5 mm, the hole spacing was set to 1 mm, and the area ratio St / Sa was set to 0.47.
In the model of analysis (7), the hole diameter of the through hole was set to 10 mm, the hole spacing was set to 1 mm, and the area ratio St / Sa was set to 0.58.
The model of analysis (8) is a case where the dispersion is not provided in the container body.

The value of St / Sa was rounded off to the third place in the minority. For these models, the velocity distribution of the fluid was analyzed using the Navier-Stokes equation at a flow velocity of 10 m / s. In the analysis, the density of the fluid was 1050 kg / m ³ , the viscosity coefficient was 3.5 × 10 ^-3 Pa · s, and the flow velocity on the inner wall surface of the container was 0, which was a non-slip condition. The results are shown in FIGS. 14a and 14b. 14a is a diagram showing the results of the analyzes (1) to (4), and FIG. 14b is a diagram showing the results of the analyzes (5) to (8).

In addition, in the analysis results of FIGS. 14a and 14b and subsequent FIGS. 15 to 16, the positions of the introduction port 12, the outlet port 13, and the dispersion 40 are also shown. In the figure, the shade indicates the flow velocity, the portion where the flow velocity is high is indicated by "H", and the portion where the flow velocity is low is indicated by "L".

By visually observing the images of the analysis results shown in FIGS. 14a and 14b, the uniformity of the flow velocity distribution in the second space 14 between the dispersion 40 and the outlet 13 is set to 0 for non-uniform cases and 0 for uniform cases. It was set to +5, and the state close to 0 to the state close to +5 was evaluated as +1, +2, +3, and +4 in order. The evaluation results are shown in the following table.

Compared with the analysis (8), by providing the dispersion as in the analyzes (1) to (7), the dispersion effect of the flow velocity flowing in from the introduction port could be confirmed. Further, from the results of the analyzes (1) to (7), it was clarified that the smaller the St / Sa, the more uniform the flow velocity distribution of the fluid in the second space. That is, it was found that if the relationship of St / Sa ≦ 0.58 is satisfied, the flow velocity of the fluid in the second space tends to be uniform, and the smaller the value of St / Sa, the more uniform the flow of the fluid.

13-2. Relationship between the diameter of the introduction port and the hole diameter of the through hole, and confirmation of the effect of the length ratio D / L "4. Relationship between the diameter of the introduction port and the hole diameter of the through hole" and "5. Length ratio D / L (through) In order to confirm the effect described in "Interval of holes 41)", the inventors analyzed the flow velocity distribution in the cell culture vessel. The following settings were made based on the model and conditions described in "13-1. Confirmation of effect of area ratio St / Sa". The diameters of the inlet and outlet were set to 4 mm. The analysis of the flow velocity distribution was performed using the fluid analysis software ANSYS Fluent provided by Ansys Japan Co., Ltd., in the same manner as in "13-1. Confirmation of the effect of the area ratio St / Sa".

In the model of analysis (9), the hole spacing was set to 1 mm and the hole diameter of the through hole was set to 1 mm.
In the model of analysis (10), the hole spacing was set to 1 mm and the hole diameter of the through hole was set to 5 mm.
In the model of analysis (11), the hole spacing was set to 1 mm and the hole diameter of the through hole was set to 10 mm.
In the model of analysis (12), the hole spacing was set to 5 mm and the hole diameter of the through hole was set to 1 mm.
In the model of analysis (13), the hole spacing was set to 5 mm and the hole diameter of the through hole was set to 5 mm.
In the model of analysis (14), the hole spacing was set to 5 mm and the hole diameter of the through hole was set to 10 mm.
In the model of analysis (15), the hole spacing was set to 10 mm and the hole diameter of the through hole was set to 1 mm.
In the model of analysis (16), the hole spacing was set to 10 mm and the hole diameter of the through hole was set to 5 mm.
In the model of analysis (17), the hole spacing was set to 10 mm and the hole diameter of the through hole was set to 10 mm.

For these models, the velocity distribution of the fluid was analyzed using the Navier-Stokes equation at a flow velocity of 10 m / s. In the analysis, the density of the fluid was 1050 kg / m ³ , the viscosity coefficient was 3.5 × 10 ^-3 Pa · s, and the flow velocity on the inner wall surface of the container was 0, which was a non-slip condition. The results are shown in FIGS. 15a-15c. 15a is a diagram showing the results of analysis (9) to (11), FIG. 15b is a diagram showing the results of analysis (12) to (14), and FIG. 15c is a diagram showing analysis (15) to (17). In the figure, the shade indicates the flow velocity, the portion where the flow velocity is high is indicated by "H", and the portion where the flow velocity is low is indicated by "L".

By visually observing the images of the analysis results of the analyzes (9) to (17) shown in FIGS. 15a to 15c, the uniformity of the flow velocity in the second space 14 was determined by the above-mentioned "13-1. Area ratio St / Sa. Evaluation was made according to the same criteria as "confirmation of effect". The evaluation results are shown in the following table. D / L was determined by setting the hole spacing of the through holes formed in the dispersion in the height direction of the dispersion as D (mm) and the dimension in the height direction of the dispersion as L (mm).

In FIGS. 15a to 15c, the smaller the pore diameter of the through hole, the more uniform the flow velocity distribution of the fluid in the second space.

Further, in the model of the analysis (9), the hole diameter of the through hole of the dispersion is smaller than the diameter of the introduction port, and the flow velocity distribution of the fluid in the second space is remarkably compared with the other analyzes (10) and (11). Was confirmed to be uniform. Similarly, when the hole spacing is 5 mm (FIG. 15b) and 10 mm (FIG. 15c), the flow velocity distribution of the fluid in the second space becomes uniform when the hole diameter of the through hole of the dispersion is smaller than the diameter of the introduction port. It became clear.

On the other hand, when the hole diameter of the through hole of the dispersion is 5 mm, which is larger than the diameter of the introduction port, the flow velocity distribution of the fluid in the second space is in the order of the models of analysis (10), (13), and (16). The uniformity of is improved. Similarly, when the hole diameter of the through hole of the dispersion is 10 mm, the uniformity of the flow velocity distribution of the fluid in the second space is improved in the order of the models of analysis (11), (14), and (17). .. From this, even when the hole diameter of the through hole is larger than the diameter of the introduction port, if the relationship of 0.0167 ≦ D / L is satisfied, the flow velocity distribution of the fluid in the second space tends to be uniform. It could be confirmed. It was revealed that more preferably 0.0833 ≦ D / L, and even more preferably 0.1667 ≦ D / L, the flow of the fluid in the second space becomes more uniform.

13-3. Confirmation of effect of rectifier In order to confirm the effect explained in "10. Rectifier", the inventors analyzed the flow velocity distribution in the cell culture vessel. The following settings were made based on the model and conditions described in "13-1. Confirmation of effect of area ratio St / Sa". The diameters of the inlet and outlet were set to 4 mm.

In the model of analysis (18), the hole diameter of the through hole of the dispersion is 1 mm, the hole spacing is 5 mm, and the rectifying body having the same size and shape as the dispersion is centered at a position 25 mm from the outlet on the outlet side. It is provided so as to be perpendicular to the line P.

In the model of analysis (19), the hole diameter of the through hole of the dispersion is 1 mm, the hole spacing is 5 mm, and no rectifying body is provided.

For these models, the velocity distribution of the fluid was analyzed using the Navier-Stokes equation at a flow velocity of 10 m / s. In the analysis, the density of the fluid was 1050 kg / m ³ , the viscosity coefficient was 3.5 × 10 ^-3 Pa · s, and the flow velocity on the inner wall surface of the container was 0, which was a non-slip condition. The result is shown in FIG. In the figure, the shade indicates the flow velocity, the portion where the flow velocity is high is indicated by "H", and the portion where the flow velocity is low is indicated by "L". In FIG. 16, it was confirmed that the flow velocity of the fluid in the second space was uniform and uniform in the model of the analysis (18) with the rectifying body as compared with the analysis (19) without the rectifying body. .. That is, it was clarified that the fluid flows more uniformly in the second space by providing the rectifying body in addition to the dispersion.

13-4. Dispersions and rectifiers having through-holes of different sizes In order to confirm the effects described in "9. Other Modifications", the inventors analyzed the flow velocity distribution in the cell culture vessel. The following settings were made based on the model and conditions described in "13-3. Confirmation of effect of rectifying fluid" above. The diameters of the inlet and outlet were set to 4 mm.

In the analysis (20), a flat-plate dispersion and a rectifier having the same shape and the same diameter and having a flat surface on both main surfaces and a thickness of 2 mm are arranged, and each of them has a circular arrangement shown in FIG. A large through hole 41A (diameter 10 mm), a medium through hole 41B (diameter 5 mm), and a small through hole 41C (diameter 2 mm) are arranged. In order to explain the hole spacing and diameter of each through hole, the region 9X of FIG. 9 is enlarged and the dimensions of each part are shown in FIG. For these models, the velocity distribution of the fluid was analyzed using the Navier-Stokes equation at a flow velocity of 10 m / s. In the analysis, the density of the fluid was 1050 kg / m ³ , the viscosity coefficient was 3.5 × 10 ^-3 Pa · s, and the flow velocity on the inner wall surface of the container was 0, which was a non-slip condition. The result is shown in FIG. In the figure, the shade indicates the flow velocity, and the portion where the flow velocity is high is indicated by "H".

Similar to the analysis (14), the analysis (20) has a through-hole portion having a through-hole hole diameter of 10 mm and a hole spacing of 5 mm. It was confirmed that the flow velocity of the fluid in the second space immediately after passing through was adjusted and became uniform. That is, it was clarified that the hole diameter of the through hole facing the introduction port is smaller than the diameter of the introduction port, so that the flow resistance of the supply liquid is generated and the supply liquid can be dispersed.

Although the embodiments of the present invention have been described in detail above, the present invention is not limited to the above-described embodiments, and various designs are designed without departing from the spirit of the present invention described in the claims. You can make changes.

1,1A: Cell culture container, 10: Container body, 12: Introduction port, 12a: Inner circumference of introduction port, 13: Outlet port, 14: Second space, 14a: Second space 14, adjacent to rectifier 50 Position, 15: Inner wall surface, 16: First space, 16a: First space 16, position adjacent to the dispersion 40, 17: Upstream wall surface, 18: Downstream wall surface, 19: Third space, 19a: Third Position of space 19 adjacent to the rectifying body 50, 20: introduction tube, 21: introduction flow path, 30: lead-out tube, 31: lead-out flow path, 40, 40A, 40D, 40E: dispersion, 41, 41A to 41D : Through hole spacing, 41a: Inner circumference of through hole, 411-414: Narrowest part of through hole 41, 42: Opposing wall surface, 43: Downstream wall surface, 50: Rectifier, 51: Through hole, 53: Opposing Wall surface, 91: medium supply source, 91a: valve, 91b: three-way valve, 92: pumping unit, 94: waste liquid container, 95: suspension supply source, 95a: valve, 96: stripping fluid supply source, 96a: valve, 97: Buffer fluid source, 97a: Valve, 98: Coating agent source, 98a: Valve, 100: Cell culture system, P: Center line, X: Predetermined direction.

Claims (14)

A cell culture container for culturing adherent cells.
A container body with an inlet and outlet,
It is provided with a dispersion which is arranged in the container body and has a plurality of through holes formed therein.
The container body has a pair of opposing wall surfaces, an upstream wall surface and a downstream wall surface, and a cell-adhesive inner wall surface connecting the upstream wall surface and the downstream wall surface.
The inlet to the upstream wall surface, the outlet is on the downstream wall surface, is provided so as to face onto uniaxially each other,
In the container body, a first space in which the supply liquid flowing from the introduction port stays is formed on the inlet side of the dispersion, and the dispersion is on the outlet side of the dispersion. A second space having a volume larger than that of the first space is formed in which the supply liquid flowing out from the through hole of the above flows.
The cross-sectional area of the first space cut out by the surface of the dispersion on the side of the first space was defined as Sa (mm ² ), and the total opening area of the through holes of the dispersion was ^{defined as St (mm 2} ). A cell culture vessel that sometimes satisfies the relationship of area ratio St / Sa ≤ 0.47. The cell according to claim 1, wherein the diameter of the maximum inscribed circle inscribed in the inner circumference of the introduction port is larger than the diameter of the maximum inscribed circle inscribed in the inner circumference in the narrowest portion of the through hole of the dispersion. Culture container. A plurality of the through holes are arranged in a predetermined direction in the dispersion.
The hole spacing between the adjacent through holes among the arranged through holes on the side of the first space is D (μm or mm), and the width of the dispersion in the predetermined direction is L (μm or mm). The cell culture vessel according to claim 1, wherein the cell culture vessel satisfies the relationship of 0.0167 ≦ D / L. The cell culture container according to any one of claims 1 to 3, further comprising a rectifier in which only one is arranged between the dispersion and the outlet in the container body. The cell culture container according to claim 4, wherein a third space in which the supply liquid flowing out of the rectifying body stays is formed in the container body on the outlet side of the rectifying body. The cell culture vessel according to claim 4 or 5, wherein a through hole is formed in the rectifying body. The cell culture vessel according to claim 6, wherein the rectifying body is formed with the same number and shape of through holes as the dispersion. When the cross-sectional area of the space inside the container body at a position adjacent to the rectifying body is Sb (mm ² ) and the total opening area of the through holes of the rectifying body is Sh (mm ² ), Sh The cell culture vessel according to claim 6 or 7, which satisfies the relationship of / Sb ≦ 0.58. The diameter of the maximum inscribed circle inscribed in the inner circumference of the outlet is larger than the diameter of the maximum inscribed circle inscribed in the inner circumference in the narrowest portion of the through hole of the rectifier, The cell culture container according to any one item. A plurality of through holes are arranged in a predetermined direction in the rectifying body.
0.0167 when the hole spacing between adjacent through holes among the arranged through holes is E (μm or mm) and the width of the rectifying body in the predetermined direction is Z (μm or mm). The cell culture container according to any one of claims 6 to 8, which satisfies the relationship of ≦ E / Z. The cell culture vessel according to any one of claims 1 to 10 and
A medium supply source that supplies a medium as a supply liquid to the cell culture container, and
A pumping unit that pumps the medium from the medium supply source to the inlet of the cell culture vessel, and
A cell culture system characterized in that it comprises at least. The eleventh claim, wherein in the cell culture system, the outlet of the cell culture container is connected to the medium supply source so that the medium that has passed through the cell culture container is collected by the medium supply source. Cell culture system. In the cell culture system, the outlet of the cell culture container is connected to the waste liquid container so that the medium that has passed through the cell culture container is collected in a waste liquid container different from the medium supply source. Item 11. The cell culture system according to Item 11. A method for culturing adherent cells using the cell culture system according to any one of claims 11 to 13.
Cells are seeded by introducing a suspension containing adherent cells from the inlet of the cell culture vessel according to any one of claims 1 to 10 provided in the cell culture system , and the cell culture is performed. The process of adhering to the inner wall surface of the container,
The process of culturing cells and the process of exfoliating cells,
Cell culture method including.

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