WO2018038032A1 - Annular culture container, cell culture system, and cell culture method - Google Patents

Abstract

An annular culture container provided with an elongated tube (1) formed using a flexible member, in which an annular hermetically sealed space for culturing cells (2) is formed in the tube by connecting the ends of the tube (1) by one or more connecting members (11).

Description

Ring culture container, cell culture system, and cell culture method

The present invention relates to a cell culture method for culturing a large amount of cells in a closed container.

In recent years, in the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, and the like, it has been required to efficiently culture a large amount of cells, tissues, microorganisms, and the like in an artificial environment.
In such a situation, it is performed that a culture vessel (culture bag) is filled with cells and a culture solution, and a large number of cells are automatically cultured in a closed system.

When culturing adherent cells such as iPS cells in a closed container while forming an aggregate, it is necessary to stir the culture solution in the container so that the aggregate does not settle and adhere to the bottom surface.
However, when culturing cells using a closed container such as a culture bag, the agitated cells move randomly, so the agglomerates collide with each other and are damaged, or the size of the agglomerates increases or decreases. As a result, there is a problem that the aggregate size varies greatly.
As a technology that can solve such a problem, a culture container for culturing cells is manufactured by making a culture bag into a donut shape, vibrating on a rotary vibration table, and mixing the culture solution well in the bag. Proposed.

Special table 2007-522825 JP 2005-87114 A

However, when using a culture bag made in such a donut shape, cells may accumulate due to wrinkles occurring in the culture bag, and a large-capacity culture container for culturing cells in large quantities There was a problem that it was difficult to manufacture.

Therefore, the present inventors have intensively studied, using an elongated tube formed using a flexible member, connecting the ends of the tube with a connecting member, and forming a sealed space in the formed tube. The inventors have found that the above problems can be solved by culturing the cells while feeding the cells in one direction in a state where the cells and the culture solution are enclosed, and the present invention has been completed.
In other words, in an annular culture container formed using such a tube, adherent cells and floating cells that can be cultured by forming aggregates are sealed together with the culture solution, and a pump is attached to drive this to drive the tube. The cells can be cultured while feeding the culture medium and cells in one direction. By flowing the culture solution and the cells in the tube in one direction using such an annular culture vessel, it is possible to prevent the cell aggregates from colliding with each other and obtain a more uniform aggregate. Moreover, according to the annular culture container which consists of such a tube, it becomes possible to manufacture a culture container with a large capacity | capacitance easily.

Here, examples of the culture container formed using a tube include the continuous culture apparatus described in Patent Document 1 and the culture / recovery apparatus described in Patent Document 2.
However, the continuous culture apparatus described in Patent Document 1 cannot cultivate cells while feeding the culture solution and cells in the tube in one direction. In addition, the culture / recovery device described in Patent Document 2 is a device in which cells are attached to the inner surface of the tube and cultured, and the cultured cells are detached and recovered by deforming the tube, and the cells are attached to the container. It was not possible to culture the cells while feeding the culture solution and the cells in the tube in one direction.

The present invention has been made in view of the above circumstances, and can efficiently flow the culture solution and cells in the tube in one direction, thereby preventing the cell clumps from colliding with each other. An object of the present invention is to provide an annular culture vessel, a cell culture system, and a cell culture method capable of obtaining a uniform aggregate and easily producing a culture vessel having a large capacity.

In order to achieve the above object, the annular culture container of the present invention comprises an elongated tube formed using a flexible member, and the tube ends are connected by one or two or more connecting members. An annular sealed space in which cells are cultured is formed.

The cell culture system of the present invention includes the annular culture container and a peristaltic pump, and the pump is attached to the tube, and the culture medium and cells in the tube are driven by driving the pump. However, the culture is performed while being fed in one direction in the tube.

The cell culturing method of the present invention is a cell culturing method using the above cell culturing system, wherein an adherent cell or a floating cell that can be cultured by forming an aggregate is formed in the tube together with a culture solution, and the pump By driving the culture medium, the culture solution and cells in the tube are cultured while being fed in one direction in the tube.

According to the present invention, the culture solution and cells in the tube can be efficiently fed in one direction, thereby preventing the cell aggregates from colliding with each other and obtaining a uniform aggregate. In addition, a culture container having a large capacity can be easily produced.

FIG. 1 is a perspective view showing a schematic configuration of an annular culture vessel according to an embodiment of the present invention. FIG. 2 is a schematic diagram showing the configuration of the annular culture vessel according to the embodiment of the present invention. FIG. 3 is a schematic view showing a configuration of an annular culture vessel formed by connecting a plurality of annular spaces according to an embodiment of the present invention. FIG. 4 shows a step of adding a culture solution using an annular culture vessel formed by connecting a plurality of annular spaces according to an embodiment of the present invention. FIG. 5 shows a state in which the annular spaces of the annular culture vessel formed by connecting a plurality of annular spaces according to the embodiment of the present invention are stacked and arranged.

Hereinafter, embodiments of the annular culture vessel, the cell culture system, and the cell culture method of the present invention will be described in detail. However, the present invention is not limited to the specific contents of the following embodiments.

The annular culture container of this embodiment is composed of an elongated tube formed using a flexible member, and the end of the tube is connected by one or more connecting members to culture cells in the tube. A sealed space is formed.
Further, the cell culture system of this embodiment includes such an annular culture container and a peristaltic pump, and the pump is attached to the tube. By driving the pump, the culture medium and cells in the tube are The culture is performed while being fed in one direction in the tube.
Further, the cell culture method of the present embodiment is a cell culture method using such a cell culture system, and encloses adherent cells or floating cells that can be cultured by forming aggregates in a tube together with a culture solution, By driving the pump, the culture medium and the cells in the tube are cultured while being fed in one direction in the tube.

Specifically, as shown in FIG. 1, the annular culture container of the present embodiment has an elongated tube 1 made of a flexible member connected by a connecting member 11 to form an annular structure, and is sealed in the tube 1. A space is formed.
The term “annular” means that the tubes 1 are connected so as to form a ring, and are not limited to being connected in a circular shape, but are connected so as to form a rectangle or other shapes as shown in FIG. It may be. Further, the tube 1 in the annular culture container of the present embodiment may be formed by combining a plurality of tubes having different materials and rigidity using a connecting member or the like.

The tube 1 is preferably made of a gas permeable member. Specifically, the oxygen permeability coefficient is 400 ml · mm / m ² · day · atm (37 ° C-80% RH) or more, and the carbon dioxide permeability coefficient is 1200 ml · mm / m ² · day · atm (37 ° C-80%). RH) or more is preferable, the oxygen permeability coefficient is 1000 ml · mm / m ² · day · atm (37 ° C.-80% RH) or more, and the carbon dioxide permeability coefficient is 3000 ml · mm / m ² · day · atm. More preferably (37 ° C.-80% RH) or higher. If the tube 1 has such gas permeability, excellent cell growth efficiency can be obtained.

Moreover, it is preferable that the tube 1 is partly or entirely made of a member having elastic deformability. Since at least a part of the tube 1 is elastically deformable, the culture medium and cells in the tube 1 can be efficiently sent using a peristaltic pump (also referred to as a tube pump, a roller pump, or a squeezing pump). can do. Moreover, since the tube 1 can be bent freely, the annular culture vessel can be arranged with excellent space efficiency. Furthermore, it is not necessary to use an air discharge means such as a pump by extracting the air from the annular culture vessel and filling the culture solution after being in a vacuum state, and the number of members can be reduced.

Furthermore, it is preferable that part or all of the tube 1 has transparency so that the contents can be confirmed.
Examples of the material of the tube 1 include silicone rubber, soft vinyl chloride resin, polybutadiene resin, ethylene-vinyl acetate copolymer, chlorinated polyethylene resin, polyurethane-based thermoplastic elastomer, polyester-based thermoplastic elastomer, silicone-based thermoplastic elastomer, Styrenic elastomers such as SBS (styrene / butadiene / styrene), SIS (styrene / isoprene / styrene), SEBS (styrene / ethylene / butylene / styrene), SEPS (styrene / ethylene / propylene / styrene), polyolefin resin, fluorine A resin or the like can be used.
That is, it is preferable to use these as the material of the tube 1 and to have gas permeability, elastic deformability, and transparency as described above.

Also, the inner surface of the tube 1 is preferably a low adhesion surface. Thereby, the adherent cell etc. which can be cultured by forming an aggregate can be cultured suitably. Specifically, the low cell adhesion treatment can be performed on the inner surface of the tube 1 using, for example, MPC (2-Methacryloylethyl Phosphoryl Choline) polymer, hydrogel, or the like.

Furthermore, it is also preferable to provide a size adjusting filter for adjusting the size of the cell aggregate in the tube 1. As a result, the agglomerates that have become too large can be decomposed to a certain size or less, and the agglomerates can be uniformly arranged to a constant size.

The connecting member 11 forms an annular sealed space in the tube 1 by connecting the end portions of the tube 1. In FIG. 1, a T-shaped tube connector is used as the connecting member 11, and the end portion of the tube 1 is inserted and connected to the facing opening. Moreover, the opening part perpendicular | vertical to the tube 1 is used as the port 12, and can be connected to an external bag etc. via another tube. Note that the connection member 11 is not limited to such a T-shaped tube connector, and may be a tube connector such as a Y-type, an I-type, or an X-type, or other tube connection device.

Thus, it is preferable that the tube 1 is provided with one or more ports 12 for taking in and out substances from the outside of the annular culture container. This substance includes a liquid such as a culture solution, a gas such as air, and a solid matter such as a cell.
Specifically, as shown in FIG. 2, the port 12 is filled with a culture solution bag 5 for supplying the culture solution into the tube 1 or the air in the tube 1 to fill the tube 1 with the culture solution. An air exhaust bag 6 or an air vent filter for pouring can be connected. A sampling tube can also be connected to the port 12.

In FIG. 2, tube clamps are attached to the tubes connecting the tube 1 and the culture solution bag 5, and the tube 1 and the air exhaust bag 6, respectively. When filling the culture solution into the tube 1, the tube clamp between the tube 1 and the culture solution bag 5 is removed, the pump 4 is driven, and the culture solution in the culture solution bag 5 is filled into the tube 1. When air exists in the tube 1, the tube clamp between the tube 1 and the air exhaust bag 6 is removed, the pump 4 is driven, and the air in the tube 1 is discharged to the air exhaust bag 6. When the air discharge is completed, a tube clamp is attached between the tube 1 and the air exhaust bag 6. When the cells 1 and the culture solution 3 are filled in the tube 1, a tube clamp is attached between the tube 1 and the culture solution bag 5.

As described above, the sealed space in the tube 1 is filled with the cells 2 and the culture solution 3, and the cells 2 can be cultured while being fed in one direction in the tube 1. In FIG. 1, the cell 2 is schematically represented largely as an aggregate.

The flow of the cells 2 and the culture solution 3 in the tube 1 can be performed by driving the pump 4. As the pump 4, a peristaltic pump that can send the cells 2 and the culture solution 3 in the tube 1 by squeezing the tube 1 can be suitably used.

As the cell 2, an adherent cell or a floating cell that can be cultured by forming an aggregate (spheroid) can be preferably used. Specific examples include adherent cells such as iPS cells, neural stem cells, embryonic stem cells, hepatocytes, pancreatic islet cells, cardiomyocytes, corneal endothelial cells, and floating cells such as lymphocytes, monocytes, erythrocytes, and megakaryocytes. be able to.
By culturing these cells using the annular culture container of the present embodiment, it is possible to prevent the cell aggregates from colliding with each other, and it is possible to efficiently obtain uniform size aggregates. Become.
Moreover, the annular culture container of this embodiment can be easily manufactured by connecting the elongate tube 1 with the connection member 11, and can manufacture a culture container with a large capacity | capacitance easily.

Moreover, it is also preferable that the annular culture container of this embodiment is formed by connecting two or more tubes 1 with the connecting member 11 and connecting a plurality of annular spaces.
Specifically, for example, as shown in FIG. 3, three annular spaces may be connected and formed, but the number of annular spaces is not limited to this, and is two. May be four or more.
In the example of the figure, the tube 1 which comprises two cyclic | annular space is connected to the cyclic | annular culture container similar to FIG. For convenience, they are referred to as a first annular space, a second annular space, and a third annular space in order from the left. The culture medium bag 5 is connected only to the first annular space, and the pump 4 is attached. However, both or one of them may be connected to or attached to the second annular space and / or the third annular space. it can.

Cell culture can be performed by, for example, the method shown in FIG. 4 using the annular culture vessel formed by connecting a plurality of annular spaces of the present embodiment.
First, the tube clamp attached to the connecting member 11 connecting the first annular space and the second annular space is removed, and the tube clamps of the culture medium bag 5 and the air exhaust bag 6 are removed, and the pump 4 is driven. . Then, as described above with reference to FIG. 2, the tube 1 forming the first annular space is filled with the cells and the culture solution, and the cells are sent in one direction in the tube 1 to thereby perform the cell culture. Do.

When the cells grow in the first annular space to a certain density, they are then attached to the tube clamp attached to the tube 1 of the second annular space and the air exhaust bag 6 of the second annular space. Remove the tube clamp and remove the tube clamp of the culture medium bag 5. Moreover, a tube clamp is attached to the connection member 11 that connects the first annular space and the second annular space, and the pump 4 is driven. Then, the cell 1 is formed by filling the tube 1 forming the first annular space and the second annular space with the cells and the culture solution and feeding them in one direction in the tube 1.

Once the cells have grown to a certain density in the first and second annular spaces, then the tube clamp attached to the tube 1 in the third annular space and the air in the third annular space The tube clamp attached to the exhaust bag 6 is removed, and the tube clamp of the culture solution bag 5 is removed. Moreover, a tube clamp is attached to the connection member 11 that connects the second annular space and the third annular space, and the pump 4 is driven. Then, the cells 1 and the culture medium are filled in the tube 1 forming the first annular space, the second annular space, and the third annular space. Incubate. When there are four or more annular spaces, the same process is repeated.
By culturing cells in this way using an annular culture vessel formed by connecting a plurality of annular spaces of this embodiment, the culture space is appropriately increased in accordance with the proliferation of the cells and a culture solution is added. The cells can be efficiently propagated.

Moreover, the annular culture container formed by connecting a plurality of such annular spaces can be arranged by stacking the annular spaces on the support pillar 7 as shown in FIG. The figure shows an annular culture vessel formed by connecting four annular spaces.
Thus, according to the annular culture container formed by connecting a plurality of annular spaces of the present embodiment, the annular culture container can be three-dimensionally arranged, and high space efficiency can be obtained. That is, by producing an annular culture vessel having a large number of annular spaces, it is possible to easily produce a culture vessel having a large capacity and excellent space efficiency.

As described above, according to the annular culture container, the cell culture system, and the cell culture method of the present embodiment, the culture solution and the cells in the tube can be efficiently fed in one direction, whereby the cells The agglomerates can be prevented from colliding with each other to obtain a uniform agglomerate, and a large-capacity culture vessel can be easily produced.
In addition, by producing an annular culture vessel having a large number of annular spaces, it is possible to easily produce a culture vessel having a large capacity and excellent space efficiency.

It goes without saying that the present invention is not limited to the above embodiment, and that various modifications can be made within the scope of the present invention.
For example, it is possible to appropriately change such as connecting a plurality of culture solution bags to the annular culture container or connecting a cell collection bag.

The present invention can be suitably used for culturing a large amount of cells that can be cultured by forming aggregates.

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

DESCRIPTION OF SYMBOLS 1 Tube 11 Connection member 12 Port 2 Cell 3 Culture solution 4 Pump 5 Culture solution bag 6 Air exhaust bag 7 Support pillar

Claims (11)

1. An annular culture container comprising a tube formed using a flexible member, in which an end of the tube is connected by one or more connecting members, and an annular sealed space for culturing cells in the tube is formed .
2. The annular culture container according to claim 1, wherein the tube is provided with a port for taking in and out substances from the outside of the annular culture container.
3. The annular culture container according to claim 1 or 2, wherein the tube has gas permeability.
4. The annular culture container according to any one of claims 1 to 3, wherein at least a part of the tube has elastic deformability.
5. The annular culture container according to any one of claims 1 to 4, wherein the inner surface of the tube is a low adhesion surface.
6. The annular culture container according to any one of claims 1 to 5, further comprising a size control filter for adjusting a size of a cell aggregate in the tube.
7. The annular culture vessel according to any one of claims 1 to 6, wherein two or more tubes are connected by the connecting member and a plurality of annular spaces are connected.
8. The annular culture vessel according to any one of claims 1 to 7, wherein the connecting member comprises two or more ports.
9. The annular culture container according to any one of claims 1 to 8, wherein at least a part of the connecting member has elastic deformation.
10. An annular culture container according to any one of claims 1 to 7, and a pump,
    The cell culture system, wherein the pump is attached to the tube, and the culture medium and cells in the tube are cultured while being fed in one direction in the tube by driving the pump.
11. A cell culture method using the cell culture system according to claim 8,
    In the tube, encapsulated adherent cells or floating cells that can be cultured by forming an aggregate, together with a culture solution,
    A cell culture method comprising culturing the culture solution and cells in the tube while feeding the pump in one direction by driving the pump.

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