JP2018033318A - Cell culture vessel, cell culture method, and method of using cell culture vessel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell culture vessel capable of carrying out cell culturing in an airtight system and capable of easy retrieval of cultured cell sheets and tissue-shaped products, a cell culture method and method of using cell culture vessel.SOLUTION: A cell culture vessel is provided with a frame, an internal space formed by an upper face and a lower face adhered to the front and back of the frame, with at least one of the upper face and the lower face being made of a gas permeable film, and a substrate for culturing adhesive cells within the internal space, and the frame being provided with at least one access port. A culture medium is included therein, the substrate is inoculated with the adhesive cells through the access port, and the adhesive cells are cultured. Furthermore, as the substrate is transplanted together with the adhesive cells into a living organism, the substrate onto which the adhesive cells are attached is recovered.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a cell culture technique, and more particularly to a cell culture container, a cell culture method, and a method of using a cell culture container for culturing adherent cells.

  In the field of regenerative medicine and the like, when a cell sheet or tissue is produced, cell culture is performed using a substrate (scaffold) for adhering cells to produce a sheet or tissue. In such a case, in general, an open-type container having a large opening, such as a petri dish, is used to put in and out the base material and culture work. May cause problems. For this reason, cell culture for producing cell sheets, tissues, and the like needs to be performed under very strict management in facilities such as a cell processing center (CPC), resulting in high costs.

Therefore, in order to solve problems such as contamination with germs, a method of manufacturing a cell sheet or the like using a closed cell culture container has been proposed.
For example, FIG. 4 of Patent Document 1 describes a cell culture container in which a closed culture chamber is formed by a frame, a gas permeable membrane, and a surface facing the frame. 1 and 2 of Patent Document 2 describe a cell culture container in which a closed space is formed by a container body having an opening and a gas-permeable film that seals the opening.
According to these cell culture containers, it is possible to cultivate cells by attaching them on a gas permeable membrane or film in a closed system space, and it is possible to peel and collect the cell sheet from the gas permeable membrane or film. It has become.

Japanese Patent No. 4897752 JP 2008-022715 A

However, these conventional cell culture containers have a problem that they are not suitable for work in a medical field because they are intended to peel and collect a cell sheet from a gas permeable membrane or film.
For example, when the cells to be cultured are corneal cells, it is desirable to culture corneal epithelial (or endothelial) cells on the amniotic membrane and transplant the obtained cells together with the amniotic membrane. However, the conventional cell culture container requires a post-treatment step such as culturing by attaching cells on a gas permeable membrane or film, and treating with a solution such as trypsin in order to separate the cells from the film. Complicated work was required.
For this reason, it is not suitable for use in a medical field such as an operating room, and depending on the cultured cells, the cells may be damaged in the post-treatment process.

  Therefore, the present inventors have intensively studied, and can perform aseptic operations such as medium exchange while maintaining a closed system, and can easily take out the cultured cell sheets, tissue, etc. from the sealed space A cell culture vessel was devised. If such a cell culture container is used, it becomes possible to culture cell sheets, tissue-like materials and the like at low cost without using a large-scale facility such as a cell processing center.

  The present invention has been made in view of the above circumstances, and can perform cell culture in a closed system, and can easily take out a cultured cell sheet, tissue, etc., a cell culture container, a cell culture method, and a cell It aims at providing the usage method of a culture container.

In order to achieve the above object, in the cell culture container of the present invention, an internal space is formed by a frame and upper and lower surfaces bonded to the front and back of the frame, and at least one of the upper and lower surfaces is a gas permeable film. And a base material for culturing adhesive cells in the internal space, and the frame body having at least one access port.
In addition, the cell culture method of the present invention is a method of culturing the adherent cells by enclosing a medium in such a cell culture container and seeding the adherent cells on the substrate through an access port.
Furthermore, the method for using the cell culture container of the present invention is a method for culturing adhesive cells using such a cell culture container and transplanting the adhesive cells together with the base material into a living body. This is a method of collecting the adhered base material.

  According to the present invention, there are provided a cell culture container, a cell culture method, and a method for using the cell culture container, which can perform cell culture in a closed system and can easily take out the cultured cell sheet or tissue. It becomes possible.

It is a perspective view which shows the cell culture container which concerns on 1st embodiment of this invention. It is the top view and front view which show the cell culture container which concerns on 1st embodiment of this invention. It is a figure which shows the state which took out the holding member provided with the base material from the cell culture container which concerns on 1st embodiment of this invention. It is a perspective view which shows the cell culture container which concerns on 2nd embodiment of this invention. It is a perspective view which shows the cell culture container which concerns on 3rd embodiment of this invention.

  Hereinafter, embodiments of the cell culture container, the cell culture method, and the method of using the cell culture container of the present invention will be described in detail with reference to the drawings.

<First embodiment>
[Cell culture vessel]
First, the cell culture container of this embodiment is demonstrated with reference to FIGS. FIG. 1 is a perspective view showing a cell culture container of the present embodiment, and FIG. 2 is a plan view and a front view showing the cell culture container of the present embodiment. Moreover, FIG. 3 is a figure which shows the state which took out the holding member provided with the base material from the cell culture container of this embodiment.

As shown in FIGS. 1 and 2, the cell culture container 10 of the present embodiment has a frame 11 and an upper surface 14 and a lower surface 15 bonded to the front and back of the frame 11, and an internal space is formed by these. ing.
The frame 11 is a frame composed of hard side walls. Although it does not specifically limit as a material of this frame 11, From the viewpoint of adhere | attaching the gas permeable film mentioned later, polyolefin resin, such as polyethylene and a polypropylene, can be used suitably.
The shape of the frame 11 is not particularly limited as long as the internal space can be formed. For example, the frame 11 can have a substantially rectangular parallelepiped shape as shown in FIGS. Further, it may be a cylindrical shape or a polygonal column shape.

At least one of the upper surface 14 and the lower surface 15 is made of a gas permeable film. The gas permeable film has gas permeability necessary for cell culture, and specifically, preferably has an oxygen permeability of 5000 ml / m ² · day · atm (37 ° C.-80% RH) or more. . In addition, this gas permeable film preferably has low cytotoxicity, low elution properties, and suitability for radiation sterilization in order to achieve high cell growth efficiency. Further, in order to make it possible to observe the cultured cells from the outside of the cell culture vessel 10, it is preferable to use a transparent gas permeable film.
Moreover, if both the upper surface 14 and the lower surface 15 are comprised with a gas permeable film, while being able to improve gas permeability more, not only observation from an up-and-down surface is attained, but the detailed observation by a microscope is also attained. Therefore, it is preferable.

  As a material for the gas permeable film, a polyethylene resin is preferably used. Examples of the polyethylene resin include polyethylene, copolymers of ethylene and α-olefin, copolymers of ethylene and vinyl acetate, ionomers using ethylene and acrylic acid or methacrylic acid copolymers and metal ions. it can. Further, polyolefin, styrene elastomer, polyester thermoplastic elastomer, silicone thermoplastic elastomer, silicone resin and the like can also be used.

Moreover, as shown in FIG. 1, in the cell culture container 10 of this embodiment, when peeling a gas-permeable film from the frame 11, it is preferable to provide the easy peeling part used as a handle. The easy peeling portion can be formed on one or both of the upper surface 14 and the lower surface 15. In FIG. 1, an easy peel portion 141 is provided on the upper surface 14.
The upper surface 14 and / or the lower surface 15 on which the easy peeling portion is formed are bonded to the frame body 11 so as to have a relatively weak adhesive force so as to be easily peeled off.

Moreover, the cell culture container 10 of this embodiment is equipped with the base material 12 for culture | cultivating an adhesive cell in internal space.
The cells to be cultured are not particularly limited as long as they are adherent cells. For example, skin cells, corneal epithelial cells, corneal endothelial cells, vascular endothelial cells, epidermal cells, mesenchymal stem cells, chondrocytes and the like are preferably used. it can.

  The substrate 12 is used as a scaffold for the cells to adhere and proliferate when culturing adhesive cells. Although the base material 12 can select an appropriate thing according to the cell to culture, For example, amniotic membrane, collagen, etc. can be used suitably. By using amniotic membrane as the base material 12, rejection can be made difficult to occur during transplantation.

The holding member 13 is a member that stably supports the base material 12 in the cell culture container 10.
As the holding member 13, a transwell is shown in FIG. The transwell has a bottom surface formed of a gas permeable membrane, and the base material 12 is disposed on the bottom surface. The transwell includes a hole 131 through which the medium can move in the cell culture container 10. For this reason, it is possible to stably arrange the holding member 13 in the cell culture container 10 as compared with the case where there is no hole 131.
In FIG. 1, the transwell has a substantially cylindrical shape at the bottom and a substantially truncated cone shape at the top, so that cultured cells can be formed in a circular shape.
The holding member 13 may be any member as long as it can hold the substrate 12, and other members such as a dish may be used in addition to the transwell.

The cell culture container 10 of this embodiment includes at least one access port in the frame 11. The number is not particularly limited, and two or more may be provided.
In FIG. 1, three access ports are provided, one as a cell injection access port 111 and the other two as a medium transfer access port 112.
The cell injection access port 111 is made of an elastic body such as rubber or silicone, and a cell suspension containing cells is injected into the cell culture container 10 through the cell injection access port 111 using a syringe or the like. Can do.

Further, tubes 16 are connected to the two medium transfer access ports 112, respectively, and one is used for adding cells and the other is used for collecting cells via the tubes 16. The medium can be quickly exchanged inside.
Furthermore, in the cell culture container 10 of this embodiment, a sample port and other ports may be provided, and a plurality of various ports may be provided.
In addition, when the cell culture container 10 of this embodiment is provided with only one access port, all the movements related to the contents of the cell culture container 10 such as cell injection, medium transfer, and sampling are performed by one access port.

  According to such a cell culture container 10 of this embodiment, adhesive cells can be cultured in a closed system, and the cultured cells can be easily and safely collected.

[Cell culture method and method of using cell culture container]
In the cell culture method and the method of using the cell culture container of the present embodiment, the medium is sealed in the cell culture container 10 described above, and the adherent cells are seeded on the base material 12 via the access port. Culture is performed. Specifically, for example, it can be used in the following environment.

  First, in the operating room in an aseptic environment, the cell culture container 10 of the present embodiment is taken out from the sterilization bag, the end of one tube 16 is connected to a medium supply container, and the end of the other tube 16 is connected to a waste liquid container. Connect to. Thereby, the internal space of the cell culture container 10 is maintained in a closed system. Hereinafter, the whole of the cell culture container 10, the medium supply container, and the waste liquid container may be referred to as a cell culture system.

  Next, the patient is operated to collect corneal epithelial cells, which are added to the medium and stirred to prepare a cell suspension. Next, this cell suspension is injected onto the substrate 12 from the cell injection access port 111 using a syringe. Further, the medium is supplied from the medium supply container into the cell culture container 10 via the tube 16 and the medium transfer access port 112. Thereby, cell culture is started in the cell culture vessel 10.

Since the internal space of the cell culture container 10 is kept sealed, the cell culture can be continued aseptically even when the cell culture system is carried from the operating room to a non-sterile environment.
When the corneal epithelial cells are sufficiently proliferated, the cell culture system is again carried to the operating room in a sterile environment, and the easy peeling portion 141 of the cell culture container 10 is picked and the upper surface 14 is peeled off as shown in FIG. Then, the holding member 13 is taken out from the frame body 11. And the base material 12 is collect | recovered from the holding member 13, and the base material 12 is transplanted to a patient.

By using the cell culture vessel 10 in this way, the adherent cells can be cultured while maintaining a closed system without using a large-scale facility such as a cell processing center, and the substrate 12 to which the adherent cells are attached can be efficiently used. And can be quickly transplanted into a patient.
For this reason, according to the cell culture method of this embodiment and the usage method of a cell culture container, an adhesive cell can be cultured at low cost and it can be used suitably for regenerative medicine.

<Second embodiment>
[Cell culture vessel]
Next, the cell culture container of 2nd embodiment of this invention is demonstrated with reference to FIG. FIG. 4 is a perspective view showing the cell culture container of the present embodiment.
As shown in FIG. 4, the cell culture container 10 a of this embodiment has a lower surface 15 made of a gas permeable film, and a leg member 17 provided on the bottom of the frame body 11 so that gas can pass through the lower surface 15. This is different from the first embodiment. About another point, it can be set as the structure similar to 1st embodiment.

  That is, the cell culture container 10a is provided with leg members 17 at the four corners of the frame body 11, so that even when the cell culture container 10a is placed on a flat surface, the lower surface 15 is placed between the flat surface. A space can be provided. For this reason, according to the cell culture container 10a of this embodiment, it is possible to allow gas to permeate into the cell culture container 10a through this space and the lower surface 15.

The number, position, shape, and the like of the leg members 17 provided in the cell culture container 10a are not particularly limited as long as the cell culture container 10a can be stably placed.
Further, the material of the cell culture vessel 10a is not particularly limited, but the same material as that of the frame 11 can be suitably used.

According to the cell culture container 10a of this embodiment, by providing the leg member 17, the gas exchange efficiency in the cell culture container 10a can be improved, and the culture efficiency can be improved.
Moreover, the cell culture container 10a of this embodiment can be applied to the same cell culture method and the usage method of a cell culture container as 1st embodiment.

<Third embodiment>
[Cell culture vessel]
Next, the cell culture container of 3rd embodiment of this invention is demonstrated with reference to FIG. FIG. 5 is a perspective view showing the cell culture container of the present embodiment.
As shown in FIG. 5, the cell culture container 10b of the present embodiment is not provided with the holding member 13, but is provided with a base material 12a made of sponge as a scaffold for culturing adhesive cells. It is different from the form. About another point, it can be set as the structure similar to 1st embodiment.

The sponge is not particularly limited as long as it can be used as a scaffold for adhesive cells. For example, a sponge containing collagen can be preferably used.
Further, in the cell culture container 10b of the present embodiment, as a base material 12a, a gel containing collagen, a hydrogel using alginic acid or the like, a biological membrane such as amniotic membrane, or the like can be used.

According to such this embodiment, the base material 12 can be directly arrange | positioned without using the holding member 13 in the cell culture container 10b. For this reason, it can use suitably, when manufacturing a cell sheet etc. with a larger area compared with the cell culture container 10 of 1st embodiment.
Furthermore, the cell culture container 10b of the present embodiment can be applied to the same cell culture method as in the first embodiment and the method of using the cell culture container.

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 assumed that a plurality of cell culture vessels are provided, and a plurality of cultures can be performed simultaneously, or a sponge or a gel is used instead of the amniotic membrane in the holding member by combining the first embodiment and the third embodiment. It is possible to appropriately change the arrangement, for example, by providing a leg member in a cell culture container in which a sponge or gel is arranged without providing a holding member by combining the second embodiment and the third embodiment.

  The present invention can be suitably used when culturing adherent cells simply and at low cost.

DESCRIPTION OF SYMBOLS 10, 10a, 10b Cell culture container 11 Frame 111 Cell injection access port 112 Medium transfer access port 12, 12a Base material 13 Holding member 131 Hole 14 Upper surface film 141 Easy peeling part 15 Lower surface film 16 Tube 17 Leg member

Claims (9)

  An internal space is formed by a frame body and an upper surface and a lower surface bonded to the front and back of the frame body, and at least one of the upper surface and the lower surface is made of a gas permeable film, and cultures adhesive cells in the internal space. A cell culture container comprising: the base material of claim 1; and at least one access port provided in the frame.   The cell culture container according to claim 1, wherein at least one of the upper surface and the lower surface is adhered to the frame body so as to be peeled off and includes an easy peeling portion.   The cell culture container according to claim 1 or 2, wherein the frame is provided with at least one cell injection port and one medium transfer port.   The cell culture container according to any one of claims 1 to 3, wherein the lower surface is made of a gas permeable film, and a leg member is provided at the bottom of the frame body so as to allow gas permeation through the lower surface.   The cell culture container according to any one of claims 1 to 4, wherein the substrate is a biological membrane, a sponge, or a gel.   The cell culture container according to any one of claims 1 to 5, further comprising a holding member for holding the base material, wherein the holding member is a transwell, a petri dish, or a dish.   The cell culture container according to claim 1, wherein the gas permeable film is transparent.   A culture medium is sealed in the cell culture container according to any one of claims 1 to 7, and the adherent cells are seeded on the base material via the access port, and the adherent cells are cultured. Cell culture method.   In order to culture adhesive cells using the cell culture container according to any one of claims 1 to 7, and to transplant the adhesive cells together with the base material into a living body, the base material to which the adhesive cells are attached A method of using a cell culture container, wherein

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