WO2017170335A1 - Cell culture vessel, support jig for cell culture vessel and cell culture method - Google Patents

Abstract

This cell culture vessel 1 is provided with: a vessel main body 2 comprising gas-permeable plastic films; and an injection/discharge port 3. The vessel main body 2 is sealed at a peripheral portion thereof, has a bulging shape on the upper face 2a side and is provided with a plurality of recesses 4 serving as cell culture parts on the bottom face 2b. In the cell culture vessel 1, cells to be cultured are injected with a culture medium into the vessel main body 2 through the injection/discharge port 3, and cultured. This enables cells to be efficiently cultured in the same vessel while maintaining an appropriate density of cells during culture and reducing the risk of contamination.

Description

Cell culture vessel, cell culture vessel support jig, and cell culture method

The present invention relates to a cell culture vessel, a cell culture vessel support jig, and a cell culture method for efficiently culturing various cells.

In recent years, in the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, etc., cells (including tissues, microorganisms, viruses, etc.) are required to be efficiently cultured and differentiated in large quantities in an artificial environment. It has been.

In such cell culture, it is necessary to maintain the cell density in the medium in an appropriate range. That is, as the cell density in the medium increases with cell proliferation, cell growth is inhibited due to depletion of medium components necessary for proliferation and accumulation of metabolites of the cell itself. It will decline. In addition, even if the cell density in the medium is too low, the cells cannot be efficiently induced to proliferate and differentiate. For this reason, when cells are cultured on a certain scale, the cells are usually cultured with repeated passages so that the cell density in the medium is maintained appropriately.

Conventionally, well plates and flasks are often used as culture vessels for subculture. For example, using a well plate, in order to obtain an appropriate cell density, add cells to the individual wells together with the culture medium, start culturing, allow the cells to grow sufficiently in the well, and then transfer to the flask. Incubate with additional culture medium according to the growth, and when it grows to a certain volume, transfer to a flask with a larger volume and add more culture medium. It is known (see, for example, paragraph [0027] of Patent Document 1).

Note that Patent Document 2 proposes a flask-type culture vessel in which a plurality of recesses are formed on one surface of a polyhedral shape such as a rectangular parallelepiped as a flask-type culture vessel. In Patent Document 2, an agglomerate formed by coalescence of single cells is formed in a recess that is a plurality of first culture portions provided in the first culture surface, and this is formed on the second culture surface in the container. It moves to the 2nd culture part with a larger area than a culture part, and is trying to become a bigger aggregate.

JP 2011-241159 A JP 2006-55069 A

However, when performing subculture in this way, it is necessary to repeat the pipetting operation many times when adding cells to each well of the well plate or when transferring cells from the well plate to the flask. . In addition, the cells must be transferred to a new culture container such as a flask every passage, which not only complicates work but also increases the risk of contamination with bacteria and viruses. .

In addition, in a flask-type culture vessel as in Patent Document 2, gas exchange is performed only when the lid that closes the opening is removed and the opening is opened. For this reason, there is a problem that not only a sufficient amount of oxygen cannot be supplied to cells in culture, but also there is a risk of contamination during gas exchange. Furthermore, in order to culture a large amount of cells on a certain scale beyond the laboratory level, a flask-type culture container having a limited capacity is not suitable.

The present invention has been made in view of the above circumstances, and efficiently cultures and induces differentiation of cells in the same container while reducing the risk of contamination while maintaining an appropriate cell density during culture. An object of the present invention is to provide a cell culture container, a cell culture container support jig, and a cell culture method.

The cell culture container according to the present invention includes a container main body made of a gas permeable plastic film and an injection port, and the container main body has a peripheral portion sealed and a container top surface side having a bulging shape. In addition, a plurality of recesses to be cell culture parts are provided on the bottom surface of the container body.

The cell culture container support jig according to the present invention is a jig for supporting the cell culture container, and is configured to support the bottom side of the container body in a non-contact manner with the recess.

The cell culture method according to the present invention is a cell culture method using the above-described cell culture container, wherein the bottom surface side of the container body is supported in a non-contact manner on the concave portion, and the cell is formed on the bottom portion of the concave portion. As a method of collecting and culturing

According to the present invention, cells can be efficiently cultured in the same container while reducing the risk of contamination while maintaining an appropriate cell density during culture.

It is explanatory drawing which shows the outline of the cell culture container which concerns on embodiment of this invention, (a) is a top view, (b) is a side view, (c) is a bottom view. It is explanatory drawing which shows the outline of the modification of the cell culture container which concerns on embodiment of this invention, (a) is a top view, (b) is a side view, (c) is a bottom view. It is explanatory drawing which shows the usage example of the cell culture container which concerns on embodiment of this invention.

Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.

A cell culture container 1 shown in FIG. 1 includes a container main body 2 made of a gas permeable plastic film and an injection port 3 made of a tubular member through which a culture medium, cells, and the like can circulate.

The container body 2 has a bulging shape in which the peripheral portion is sealed and the top surface 2a side bulges like a plateau, and the edge of the flat top surface 2a is inclined so as to be connected to the peripheral portion. Is formed. At the same time, the bottom surface 2b of the container body 2 is provided with a plurality of recesses 4 serving as cell culture parts.

The concave portion 4 provided on the bottom surface 2b of the container body 2 has an opening diameter (diameter) D of 0 in order to suppress the movement of cells in the container body 2 so that cells in culture remain in the single concave portion 4. It is preferably 3 to 10 mm, more preferably 0.3 to 5 mm, still more preferably 0.5 to 4 mm, particularly preferably 0.5 to 2 mm, and the depth d is 0.1 mm or more. Is preferred. The opening diameter of the recess 4 may be the same for all the recesses 4. For example, the bottom surface 2 b is divided into a plurality of regions, and the opening diameter of the recess 4 is made different for each region. The recessed part 4 to be provided may include two or more kinds of recessed parts having different opening diameters.
For example, in the present embodiment, the top surface 2a side of the container body 2 is bulged in a plateau shape, and the edge of the top surface 2a is inclined so as to be connected to the peripheral portion. In this way, since the height on the peripheral side becomes low, when the cells to be cultured are injected into the container body 2 together with the medium, the amount of the medium containing the cells decreases on the peripheral side and settles in the medium. The number of cells is also reduced. Then, if the opening diameters of all the recesses 4 are the same, the number of cells that settle into the recesses 4 on the peripheral side is reduced, so the same number of cells settle in all the recesses 4. As shown, it is preferable to increase the opening diameter of the recess 4 on the peripheral side.

Further, in the cell culture container 1 shown in FIG. 1, the shape of the recess 4 is a spherical crown so that cells can easily gather at the bottom of the recess 4, but the shape of the recess 4 is not limited to this. In order to facilitate the collection of cells at the bottom of the recess 4, the ratio d / D of the depth d to the opening diameter D of the recess 4 is preferably set to 0.05 to 1.

In addition, the occupied area of the concave portion 4 occupying the bottom surface 2b is preferably as large as possible within a range in which the moldability is not impaired in order to avoid cells from staying in the portion other than the concave portion 4 of the bottom surface 2b. Specifically, it is preferably 30 to 90% with respect to the area of the bottom surface 2b. The recesses 4 are preferably arranged in a zigzag pattern as shown in the figure so that the area occupied by the recesses 4 in the bottom surface 2b is as large as possible. However, the recesses 4 may be arranged in a lattice pattern as necessary.

Further, the size of the container body 2 is not particularly limited, but is preferably, for example, 50 to 500 mm in length and 50 to 500 mm in width.

Such a cell culture container 1 can be manufactured as follows, for example.
First, a top surface side plastic film that becomes the top surface 2 a side of the container body 2 and a bottom surface side plastic film that becomes the bottom surface 2 b side of the container body 2 are prepared. And the top surface side plastic film is shape | molded so that the peripheral part may be left and it may swell in plateau shape. On the other hand, a plurality of recesses 4 are formed in a predetermined arrangement in the bottom side plastic film. These can be formed by general vacuum forming, pressure forming, or the like, and can be formed so that the bulging shape or the shape of the concave portion 4 becomes a desired shape by appropriately adjusting a mold or the like. Can do.

Next, the top side plastic film and the bottom side plastic film molded as described above are overlapped, and the peripheral part is heat-sealed with a tubular member forming the injection port 3 at a predetermined position. And then trim the periphery if necessary. Thereby, the cell culture container 1 as shown in FIG. 1 can be manufactured.

The gas permeability of the plastic film forming the container body 2 is determined according to the gas permeability test method of JIS K 7126. The oxygen permeability measured at a test temperature of 37 ° C. is 5000 mL / (m ² · day · atm. ) Or more.
Moreover, it is preferable that a part or all of the plastic film has transparency so that the progress of cell culture and the state of cells can be confirmed.

The material used for the plastic film forming the container body 2 is not particularly limited as long as it has a desired gas permeability. Examples thereof include thermoplastic resins such as polyethylene, polypropylene, ethylene-vinyl acetate copolymer, polyester, silicone elastomer, polystyrene elastomer, and tetrafluoroethylene-hexafluoropropylene copolymer (FEP). These may be used as a single layer, or may be used by laminating the same or different materials, but has a layer that functions as a sealant layer in consideration of heat fusion when sealing the periphery. Is preferred.

In addition, the plastic film used for forming the container body 2 so as to have an appropriate shape retaining property capable of maintaining the bulging shape on the top surface 2a side and the shape of the concave portion 4 while having flexibility. The thickness is preferably 30 to 200 μm.

In addition, the injection port 3 is composed of a tubular member through which a culture medium, cells, and the like can circulate, as described above, but the tubular member forming the injection port 3 is, for example, polyethylene, polypropylene, It can be molded into a predetermined shape by injection molding, extrusion molding, or the like using a thermoplastic resin such as vinyl chloride, polystyrene-based elastomer, or FEP.

Moreover, in order to avoid that a port is obstruct | occluded by the sticking with the top | upper surface 2a side and the bottom face 2b side of the container main body 2 in the injection | pouring port 3, it protrudes in the container main body 2 from the base end. A port blocking prevention piece can be provided. When such a port blockage prevention piece is provided, it is preferably provided so as to be positioned on the top surface 2a side of the container body 2 so as not to hinder the sedimentation of cells into the recess 4 provided on the bottom surface 2b.

In order to perform cell culture using such a cell culture vessel 1, the cells to be cultured together with the culture medium are placed in the vessel body 2 while maintaining a closed system via a liquid feeding tube connected to the injection port 3. inject. Then, the cells injected into the container body 2 settle in the medium and are collected at the bottom of each recess 4.

Here, in a flat pouch-shaped container in which two plastic films are stacked and the periphery is sealed, the bottom surface is deformed so that the periphery is lifted as the container is filled with the content liquid. On the other hand, in this embodiment, the top surface 2a side of the container body 2 has a bulging shape, and by designing the bulging shape on the top surface 2a side in consideration of the injection amount, It is possible to suppress the deformation of the bottom surface 2b when the cells to be cultured are injected together with the medium. Accordingly, the cells can be uniformly accommodated in the respective recesses 4 without the recesses 4 provided on the bottom surface 2b being inclined.

It is preferable that a cell low adhesion treatment is applied to the bottom surface 2b including the recesses 4 so that the cells settled in the medium are likely to collect at the bottoms of the recesses 4 so that the cells do not adhere easily. Examples of the low cell adhesion treatment include, for example, a treatment for imparting hydrophilicity to the surface of the plastic film by a surface treatment such as a plasma treatment, a phospholipid polymer, a surfactant, a protein such as albumin, and the like. Treatment for inhibiting the adsorption of cell adhesion protein.

In addition, in order to avoid the cells from staying in the portion other than the concave portion 4 of the bottom surface 2 b, particularly the peripheral portion side of the bottom surface 2 b, As shown in FIG. 2, it is preferable that the bottom surface 2b side also has a bulging shape that bulges in a plateau like the top surface 2a side. By doing in this way, it is formed so that the edge of bottom face 2b may incline and continue to a peripheral part, and it can avoid that a cell retains in the peripheral part side of bottom face 2b. Furthermore, it is preferable that the bottom surface 2b side of the container body 2 also has a bulging shape in order to suppress deformation of the bottom surface 2b when the cells to be cultured are injected together with the culture medium.
In addition, when setting it as such an aspect, when manufacturing the cell culture container 1 as mentioned above, while making the bottom face side plastic film bulge leaving the peripheral part, it was bulged. What is necessary is just to make it form the recessed part 4 in a site | part.

As described above, according to the cell culture container 1 of the present embodiment, the cells injected into the container body 2 settle in the medium and gather at the bottom of each recess 4, and the cell density is increased in a state where the cell density is increased. It can induce culture and differentiation well. And since the container main body 2 consists of a plastic film which has gas permeability, it can supply sufficient quantity of oxygen to the cell in culture | cultivation. In particular, in this embodiment, when the concave portion 4 is formed on the bottom surface side plastic film as described above, the plastic film is stretched so that the thickness of the concave portion 4 is other than the concave portion 4 of the bottom surface 2b. It is preferable to make the thickness thinner than the thickness of the portion, whereby the gas permeability of the recess 4 can be increased, and sufficient oxygen can be supplied to the cells collected in the recess 4.

Moreover, when performing cell culture using the cell culture container 1, cells can be cultured under predetermined conditions in a culture vessel such as a CO ₂ incubator. At this time, as shown in FIG. 3, the cell culture vessel 1 is preferably installed on a frame in the incubator via a jig 10 that can support the bottom surface 2 b side of the concave portion 4 in a non-contact manner. By doing so, the concave portion 4 is surely prevented from being crushed and deformed, and the cells in culture collected in the concave portion 4 are prevented from flowing out around the concave portion 4. In addition, the gas permeability from the bottom surface 2b can be maximized.

As the jig 10 that can support the bottom surface 2b side of the cell culture container 1 in the recess 4 in a non-contact manner, for example, a perforation or a recess having a diameter that allows the recess 4 to be inserted in a non-contact manner corresponds to the arrangement of the recess 4 And a plurality of flat jigs, or a wire netting jig in which wires are assembled in a mesh shape so as not to be in contact with the concave portions 4 between the concave portions 4.

When the cells collected at the bottom of the recess 4 grow more than a certain level and the cell density exceeds a range suitable for culture, the top surface 2a is used as the culture surface with the top of the cell culture container 1 reversed. By using it, the culture area can be expanded. This makes it possible to continue cell culture while maintaining an appropriate cell density in the same container.
When the cells to be cultured are anchorage-dependent cells, when reversing the top and bottom of the cell culture container 1, an enzyme solution for peeling the cells from the bottom surface of the recesses 4 is added as necessary. In this case, the enzyme solution can be injected into the container body 2 while maintaining a closed system via the liquid feed tube connected to the injection port 3. .

In addition, cell culture usually requires a period of several days to several weeks, and during that period, it is necessary to add a medium or change a medium as necessary. These operations can be easily performed while maintaining the closed system by adding the medium or replacing the medium through the liquid feeding tube connected to the. Furthermore, after completion of the culture, the cells in the cell culture vessel 1 can be recovered while maintaining the closed system via the liquid feeding tube connected to the injection port 3.

As described above, by performing cell culture using the cell culture container 1 of the present embodiment, the cell density at the time of culture is appropriately maintained while maintaining a closed system without subculture, and the cells are efficiently used. It is possible to multiply it. Moreover, a complicated transfer operation is not required, and cells can be efficiently cultured in the same container while reducing the risk of contamination.

Furthermore, since the container body 2 is formed using a plastic film, it is lightweight even if the capacity is increased and is not bulky. Therefore, the container body 2 is suitable for large-scale cell culture. A sufficient amount of medium can be injected. In addition, the size of the container body 2 can be adjusted using a clamp or the like, and can be flexibly handled according to the number of cells and the amount of medium. On the other hand, in the flask type culture container as in Patent Document 2, it is necessary to replace the container with a different size.

Although the present invention has been described with reference to the preferred embodiment, the present invention is not limited to the above-described embodiment, and it goes without saying that various modifications can be made within the scope of the present invention.

For example, in the above-described embodiment, the container main body 2 has a rectangular shape and includes the injecting port 3 on one side of the short side, but is not limited thereto. The shape of the container body 2 may be a square shape, an oval shape, a circular shape, or the like, and can be various shapes as necessary. The position and the number of the injection ports 3 can be changed as appropriate.

All the contents of the documents described in this specification and the content of the Japanese application that forms the basis of the Paris priority of the present application are incorporated herein.

The present invention can be used as a technique for efficiently culturing various cells.

DESCRIPTION OF SYMBOLS 1 Cell culture container 2 Container main body 2a Top surface 2b Bottom surface 3 Port for injection / injection 4 Concavity 10 Jig

Claims (10)

1. A container body made of a plastic film having gas permeability and an injection port are provided.
   The container body is sealed at the periphery, and the container top surface has a bulging shape,
   A cell culture container, wherein a plurality of recesses serving as cell culture parts are provided on the bottom surface of the container body.
2. 2. The cell culture container according to claim 1, wherein the recess has an opening diameter of 0.3 to 10 mm and a depth of 0.1 mm or more.
3. The cell culture container according to claim 1 or 2, wherein an area occupied by the concave portion in the bottom surface is 30 to 90% with respect to an area of the bottom surface.
4. The cell culture container according to any one of claims 1 to 3, wherein the concave portion includes two or more types of concave portions having different opening diameters.
5. The cell culture container according to any one of claims 1 to 4, wherein the oxygen permeability of the plastic film is 5000 mL / (m ² · day · atm) or more.
6. The cell culture container according to any one of claims 1 to 5, wherein the thickness of the recess is thinner than the thickness of the bottom surface other than the recess.
7. The cell culture container according to any one of claims 1 to 6, wherein the bottom surface including the concave portion is subjected to a low cell adhesion treatment.
8. The cell culture container according to any one of claims 1 to 7, wherein a port blockage prevention piece protruding into the container main body from the base end of the injection port is provided so as to be positioned on the top surface side.
9. A jig for supporting the cell culture container according to any one of claims 1 to 8,
   A support jig for a cell culture vessel, wherein the bottom side of the vessel body can be supported in a non-contact manner on the recess.
10. A cell culture method using the cell culture container according to any one of claims 1 to 8,
    A cell culture method comprising: supporting a bottom surface of the container body in a non-contact manner with the recess; and collecting and culturing cells at the bottom of the recess.

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