JP2021013332A - Closed-system cell culture vessel - Google Patents

Abstract

To provide a closed-system cell culture vessel that can aseptically and easily perform cell culture, and can be stably supported, and can aseptically and stably perform various culture operations such as conveyance, culture medium replacement and observation.SOLUTION: A closed-system cell culture vessel 10 in which a culture base part 13 and a culture space 12 contacting the culture base part 13 are provided inside a hermetically sealed storage body 11, and cells are cultured on the culture base part 13 or inside the culture space 12. The hermetically sealed storage body 11 comprises: an annular frame body 15; and a flexible film 23 which fixes the annular frame body 15 immovably from an outer peripheral side and covers the body at least in a fluid-tight manner, and the culture space 12 and the culture base part 13 are provided in an inner space surrounded by the annular frame body 15.SELECTED DRAWING: Figure 2

Description

The present invention relates to a closed cell culture vessel for culturing cells inside a sealed container.

Conventionally, when cells are cultured inside an open system container such as a petri dish, a large-scale culture facility such as a cell processing center (CPC) is used to aseptically exchange the medium (culture solution) and take in and out the cultured cells. And large equipment such as cell processing isolators were needed.

In order to carry out cell culture without using a large-scale culture facility or device, a closed culture container provided with an access port for taking in and out of a medium and cells has been proposed. For example, Patent Document 1 below discloses a closed-type culture container comprising a closed container that can be opened and a cell culture body arranged in the closed container, and the cell culture body is provided so as to be removable from the closed container. There is.

Patent Document 1 describes that a petri dish-like airtight container or a part of the airtight container that can maintain its morphology by inserting a cell culture is heat-sealed with a film so that it can be peeled off. Further, a closed container in which a cell culture dish (cell culture body) is introduced into a flexible cell culture bag (cell culture container) and can be opened is also described.

International Publication No. WO2015 / 190090

However, in the conventional closed system culture container, the cell culture body and the base material for culturing the cells are not fixed to the sealed container, the frame, etc. in which these are housed and arranged in the closed system. Since the base material moves in a sealed container or frame, it is difficult to stably support or grip the closed culture container. For example, cell culture is performed in various culture operations such as transportation, medium exchange, and observation. There is a problem that the body, the base material, etc. may be displaced inside the sealed container, the frame, etc., which adversely affects the culture.

Therefore, in the present invention, cell culture can be aseptically performed, stable support (including grasping by an operator or the like) is easy, and various culture operations such as transportation, medium exchange, and observation are aseptic. An object of the present invention is to provide a closed cell culture vessel that can be stably performed.

In the closed cell culture vessel of the present invention that solves the above problems, the culture base and the culture space in contact with the culture base are provided inside the sealed container, and the cells are cultured on the culture base or in the culture space. In the culture vessel, the sealed container includes an annular frame and a flexible film that immovably fixes the annular frame from the outer peripheral side and covers it at least liquid-tightly, and the culture base and the culture space have an annular frame. It is provided inside surrounded by the body.

The closed cell culture vessel of the present invention preferably includes an inner case having an annular frame as a side peripheral wall and a culture base as a bottom wall.
Further, it is preferable that the flexible film covers at least a part of the outer peripheral surface of the annular frame in close contact and covers the entire outer circumference of the annular frame by covering the upper opening of the annular frame.

In the closed cell culture vessel of the present invention, the flexible film includes a lower film covering at least a part of the outer peripheral surface of the annular frame and an upper film covering the upper opening, and the lower film is the outer peripheral surface of the annular frame. It has a lower collar that protrudes more outward, and the upper film has an upper collar that protrudes outward from the outer peripheral surface of the annular frame, and the lower collar and the upper collar are at least liquid-welded. Is good. In that case, it is preferable that the lower film is provided with an annular constriction portion that covers the upper end surface of the upper opening between the side peripheral portion that is in close contact with the outer peripheral surface of the annular frame and the lower collar portion.

The closed cell culture vessel of the present invention is provided with a port portion capable of communicating the culture space with the outside, and the port portion has an inner protrusion protruding into the culture space and is flexible near the upper opening of the annular frame. It is preferably supported by the sex film. In that case, it is more preferable that the opening of the inner protrusion of the port portion can be displaced between the position of being immersed in the medium in the culture space and the position above the medium.
Further, it is preferable that the inner protrusion has a length corresponding to the height of the inner wall of the annular frame, and the opening of the inner protrusion can be immersed in the medium on the inner wall side of the annular frame.

In the closed cell culture vessel of the present invention, when the flexible film surrounds and covers the entire outer circumference of the annular frame, a guide portion is formed on the outer peripheral surface of the annular frame along the outer peripheral surface, and the guide portion is formed. It is preferable that it is covered with a flexible film together with the annular frame.

According to the closed-type cell culture vessel of the present invention, the annular frame is immovably fixed from the outer peripheral side with a flexible film and covered at least liquid-tightly, so that the culture group is immovable inside surrounded by the annular frame. A section and a culture space are provided. Therefore, the culture base and the culture space can be kept in a closed system, and cell culture can be performed aseptically.
At the same time, the culture base and the culture space can be fixedly arranged at a predetermined position with respect to the flexible film, and the culture space can be kept in a predetermined shape. Therefore, from the outside of the flexible film during or at the end of cell culture. The annular frame can be stably supported, and the culture base and the culture space can be suppressed from being displaced or deformed.
As a result, cell culture can be easily performed aseptically in a closed system, and it is easy to stably support the cells, and various culture operations such as transportation, medium exchange, and observation can be performed aseptically and stably. It is possible to provide a closed cell culture vessel that can be carried out.

In the closed cell culture vessel of the present invention, when the culture base is composed of the bottom wall of the inner case and the annular frame is composed of the lateral peripheral wall of the inner case, the inner case is integrated with the culture base and the annular frame. Therefore, the culture base and the culture space can be reliably maintained in a predetermined position and a predetermined shape, and cell culture can be reliably and stably performed in the culture base and the culture space.

In the closed cell culture vessel of the present invention, the flexible film closely covers at least the outer peripheral surface of the annular frame and covers the upper opening of the annular frame so that the flexible film can be used together with the annular frame in the culture space and the culture space. The culture base can be securely sealed to form a closed culture vessel. As a result, the inner surface of the annular frame is configured as the outer surface of the culture space, and the inner surface of the side peripheral wall of the inner case and the upper surface of the bottom wall composed of the annular frame and the culture base are outside the culture space, respectively. It is configured as a side surface and a bottom surface.
Therefore, regardless of the material of the flexible film, it is possible to freely select the outer surface and the lower surface of the culture space having desired properties according to the cells to be cultured. For example, the closed cell culture vessel of the present invention can be obtained by subjecting only the culture base of the inner case to a treatment such as hydrophilization and applying the above-mentioned invention to the inner case. In this case, a closed cell culture vessel having a hydrophilic culture base can be obtained without any treatment on the flexible film.

Further, since the flexible film is arranged on the outer peripheral surface of the annular frame or the outer side of the side peripheral wall of the inner case, the blade is pressed against the flexible film on the outer peripheral surface of the annular frame or the side peripheral wall of the inner case to be flexible. Only the film can be cut. Therefore, when the annular frame or inner case is taken out from the flexible film, the cutlery does not come into direct contact with the medium in the annular frame or inner case, and foreign substances generated during cutting do not get mixed in, and the cultured cells do not. And the medium can be prevented from being contaminated with foreign substances and germs.

In the closed cell culture vessel of the present invention, the flexible film comprises a lower film covering at least a part of the outer peripheral surface of the annular frame body and an upper film covering the upper opening, and the lower collar portion of the lower film is an annular frame. The lower collar portion and the upper collar portion are located outside the annular frame by being configured so as to project outward from the outer peripheral surface of the body and the upper collar portion of the upper film facing the outer peripheral surface. Therefore, the welding work becomes easy and the sealing can be surely performed.

In the closed cell culture vessel of the present invention, the lower film is provided by providing an annular constriction portion covering the upper end surface of the upper opening between the side peripheral portion and the lower collar portion of the lower film that is in close contact with the outer peripheral surface of the annular frame. Covers the upper end surface of the annular frame, so that the annular frame is sandwiched between the flexible films from above and below, and in addition to being able to stably fix the annular frame, the medium inside the annular frame is on the outside. It can be hard to overflow. In particular, by bringing the upper end of the opening of the annular frame into close contact with the lower film, more preferably the lower film and the upper end of the opening in close contact with the side peripheral portion of the lower film, the outer peripheral surface of the annular frame and the lower film It is possible to prevent the medium from entering between the two.
Further, since the annular constricted portion made of the flexible film can be easily deformed into a bellows shape, the upper film welded to the lower collar portion above the annular constricted portion is easily displaced or deformed. Therefore, while maintaining the closed system, the upper film can be deformed or displaced to change the volume, and operations such as accommodating and discharging the medium can be easily performed.

In the closed cell culture vessel of the present invention, the port portion that can communicate with the culture space and the outside is supported by the flexible film, so that the medium and cells can be opened and closed through the port portion that can be opened and closed without opening the flexible film. Can be easily discharged and introduced. At this time, since the annular frame body can be gripped and worked through the flexible film, the operation becomes easy. Further, since the port portion is provided on the flexible film, the degree of freedom of operation of the port portion is high even when a hard material is used for the annular frame.

Further, the opening of the inner protruding portion protruding into the culture space of the port portion can be displaced between the position of being immersed in the medium in the culture space and the upper position, so that the inner protruding portion of the port portion can be displaced. Cell culture can be performed by fixing the opening without immersing it in the medium, and it is possible to prevent vibration from being transmitted from the port to the medium and damage to the cell due to contact with the port, resulting in stable cells. Can be cultured. On the other hand, if the port portion is displaced, the opening of the medial protrusion can be immersed in the medium, for example, the medium can be gently discharged or introduced from the port portion, and the cells to be cultured are prevented from being excessively stimulated. it can.

Further, the inner protruding portion of the port portion has a length corresponding to the height of the side peripheral wall of the annular frame or the inner case, and the opening of the inner protruding portion is formed on the inner peripheral surface of the annular frame or the peripheral wall of the inner case. Since it can be immersed in the medium at a position along the route, even if the port portion is displaced and the opening of the medial protrusion is immersed in the medium, the medial protrusion is located on the central side of the cells cultured in the annular frame or the inner case. Can be prevented from damaging the cultured cells without contacting the cells.

In the closed cell culture vessel of the present invention, when the flexible film surrounds and covers the entire outer circumference of the annular frame or inner case, it is placed on the outer peripheral surface of the annular frame or inner case immovably fixed to the flexible film. When cutting the flexible film with a blade and taking out the annular frame or inner case by forming guide portions such as grooves, ridges or steps extending in the circumferential direction along the outer peripheral surface. By guiding the cutting tool to the guide portion, the flexible film can be easily and stably cut. Further, since the guide portion is formed on the outer peripheral surface of the annular frame or the inner case, the blade does not enter the culture space, and the contamination of the culture space by the blade can be suppressed.

It is a top view of the closed cell culture vessel which concerns on embodiment of this invention. It is a vertical sectional view of the closed cell culture vessel which concerns on embodiment of this invention. It is a partially enlarged view of the closed cell culture vessel which concerns on embodiment of this invention. It is a vertical cross-sectional view which shows the stationary state in the cell culture using the closed-type cell culture vessel which concerns on embodiment of this invention. It is a vertical sectional view which shows the state of the culture medium exchange in the cell culture using the closed-type cell culture vessel which concerns on embodiment of this invention. It is a vertical cross-sectional view which shows the state which the cell culture using the closed cell culture container which concerns on embodiment of this invention is completed, and the cell is taken out. (A) is a vertical sectional view showing a modification of the closed cell culture vessel according to the embodiment of the present invention, and (b) is a partial sectional view of the closed cell culture vessel according to the modification different from (a). Is. It is a partially enlarged top view of the closed cell culture vessel which concerns on the modification of embodiment of this invention. It is a top view of the closed-type cell culture vessel which concerns on another modification in embodiment of this invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
As shown in FIGS. 1 and 2, the closed cell culture container 10 of the present embodiment is a container provided with a culture base 13 and a culture space 12 in contact with the culture base 13 inside the sealed container 11, and has an upper opening. A hard inner case 21 having 21a, a flexible film 23 that immovably fixes the inner case 21 from the outer peripheral side and covers it airtightly, and a port portion 25 for communicating the culture space 12 and the outside are provided. ing. The inner case 21 is fixed to the flexible film 23 by being brought into close contact with the flexible film 23.

The sealed container 11 is a member that seals and houses cells, cell suspensions, media, and the like to be cultured, and is composed of an inner case 21 and a flexible film 23 in this embodiment. The cells to be cultured are not limited in any way, and may be adhesive cells or floating cells.

The inner case 21 airtightly closes one end of the circular annular frame 15 formed of the side peripheral wall 21b capable of securing the culture space 12 having a predetermined shape of the closed cell culture container 10 inside, and the side peripheral wall 21b. As described above, it has a flat culture base portion 13 composed of a bottom wall 21c provided integrally with the side peripheral wall 21b.
The inner case 21 is preferably made of a material having a hardness higher than that of the flexible film 23 and having transparency, and in that case, observation by a person or a machine (camera) can be easily performed from the outside. Flexibility It is more preferable that both the flume and the inner case are transparent because they can be observed from the outside even during the culturing operation. In the present embodiment, an existing petri dish-shaped culture container made of transparent resin such as glass or polystyrene is used as the inner case 21.
The inner case 21 is made of polystyrene resin, existing glass, for example, polyolefin resin such as polypropylene resin, polyethylene resin, ethylene-propylene copolymer, cycloolefin resin, polystyrene resin, polycarbonate resin, polyethylene. From terephthalate resin, methacrylic resin, vinyl chloride resin, polybutylene terephthalate resin, polyarylate resin, polysulfone resin, polyether sulfone resin, polyether ether ketone resin, polyetherimide resin, polymethylpentene resin, acrylic resin, etc. It is also possible to use plastics.
Among these, polystyrene resin is preferable from the viewpoint of moldability and sterilization required for the culture vessel. Further, when a resin culture container is used, it is easy to use a disposal type.

The flexible film 23 is made of a film capable of airtightly fixing the annular frame 15 made of the side peripheral wall 21b so as not to be movable from the outer peripheral side. The flexible film 23 is in close contact with at least the side peripheral wall 21b of the inner case 21 and covers the upper opening 21a of the inner case 21 so as to completely close the inner case 21, and the entire inner case 21 is hermetically surrounded by the flexible film 23. It has been.
Here, the term “adhesion” means that some of the films may be bonded to each other by adhesion, welding, or the like, but they may not be bonded to each other as long as the opposing contact surfaces are not separated from each other. The inner case 21 is in a state of being tensioned, that is, in close contact with the outer surface of the inner case 21 without sagging or stagnation, and in a state of being taut on the bottom wall side and the upper opening 21a side of the inner case 21. It is preferable to cover. For example, by using a heat-shrinkable film or the like as the flexible film 23, it is possible to form a close contact state between the inner case 21 and the flexible film 23. Further, the elasticity of the flexible film 23 can be utilized to form a close contact state between the inner case 21 and the flexible film 23. In this case, in addition to the side peripheral wall 21b, the flexible film 23 adheres more securely to the upper and lower ends of the side peripheral wall 21b, that is, the end on the upper opening 21a side and the end on the bottom wall 21c side. can do.

As shown in FIGS. 2 and 3, the flexible film 23 of the present embodiment includes a lower film 23a that covers the outer periphery of the side peripheral wall 21b and the bottom wall 21c, and an upper film 23b that covers the upper opening 21a so as to close it. The inner case 21 is airtightly covered by adhering them to each other. Here, it is preferable that the culture container is adhered by heat welding, which does not cause a problem of the volatile component of the adhesive.

The lower film 23a includes a flat lower end portion 23a1 that covers the bottom wall 21c of the inner case 21, and a side peripheral portion 23a2 that is formed in a tubular shape corresponding to the side peripheral wall 21b of the inner case 21 and closely covers the side peripheral wall 21b. , A lower collar portion 23a3 protruding radially outward from the side peripheral wall 21b over the entire circumference, and these have a three-dimensional shape and are integrally continuous.
The upper film 23b has an opening closing portion 23b1 that closes the upper opening 21a of the inner case 21, and an upper flange portion 23b2 that protrudes radially outward from the side peripheral wall 21b over the entire circumference, and these are flat and continuous. There is.

The lower film 23a and the upper film 23b are continuously welded in the circumferential direction with the lower collar portion 23a3 and the upper collar portion 23b2 in contact with each other. Here, since the flexible films 23 made of the same material are welded to each other, it is easy to set welding conditions and the like, and it is possible to reliably seal.
In the present embodiment, since the port portion 25 made of the resin molded body described later is arranged in a part of the lower collar portion 23a3 and the upper collar portion 23b2 in the circumferential direction, the lower collar portion 23a3 and the upper film of the lower film 23a are arranged. The upper flange portion 23b2 is welded to each other at a portion other than the port portion 25, and the remaining portion is welded to the outer peripheral surface of the port portion 25, respectively.
The lower collar portion 23a3, the upper collar portion 23b2, and the port portion 25 are welded all around in the circumferential direction, so that the lower film 23a and the upper film 23b are hermetically joined and sealed.

In the present embodiment, as further shown in FIG. 3, the upper end surface 21d around the upper opening 21a is closely and airtightly covered between the side peripheral portion 23a2 and the lower collar portion 23a3 of the lower film 23a. A continuous annular constriction portion 23a4 is provided on the peripheral portion 23a2.
The annular constricted portion 23a4 is provided by reducing the diameter from the tubular side peripheral portion 23a2 to the same as or smaller than the inner diameter of the inner case 21, and is continuous with the entire circumference in the circumferential direction.
Further, the distance between the lower end portion 23a1 of the lower film 23a and the annular constricted portion 23a4 corresponds to the height of the inner wall surface of the side peripheral wall 21b of the inner case 21, and the annular constricted portion 23a4 corresponds to the height of the upper opening 21a of the inner case 21. It is formed in close contact with the periphery of the.

Such a flexible film 23 may be made of, for example, a transparent and flexible resin film. For example, a heat-shrinkable film made of a thermoplastic resin can be easily brought into close contact with the inner case 21 by heating. Further, if it is a thermoplastic resin, it is suitable because it is easy to seal by welding. Further, it is preferable that the lower film 23a and the upper film 23b are made of the same material, but different materials may be used as long as they are made of compatible materials that can be welded to each other. For example, the upper film 23b may be made of the same material as the inner case 21 on the premise that the upper film 23b is made of a material having compatibility that can be welded to each other.
As the flexible film 23 of the present embodiment, as described above, an airtight film that does not allow air to pass through is used, but if only the liquid can be shielded, a liquidtight film may be used.
As the flexible film 23, a film suitable for the application and requirements can be used. For example, a gas-permeable flexible film of oxygen or a specific gas or a gas-impermeable film in which the permeation of oxygen or the like is suppressed can be selected according to the type of cultured cells and the culture environment such as high oxygen or low oxygen.
In accordance with the above applications and requirements, the flexible film 23 includes, for example, low-density or high-density polyethylene resin (including linear polyethylene), ethylene-vinyl acetate copolymer resin (EVA), polypropylene resin, and ethylene-propylene. A polymer resin, a polybutadiene resin, a styrene-butadiene copolymer resin and their hydrogenated resin, a polyurethane resin, a polyester resin, a polyamide resin (nylon resin), and a mixture of these resins may be used. In addition to using these as a single-layer or multi-layer film, they may be used as a laminated film in which different material films are combined. Further, a laminated film or the like using a gas barrier film such as a silica-deposited film, an alumina-deposited film or a polyvinyl alcohol film may be used for the surface layer or the intermediate layer of the film.
Further, the film to be used can be different depending on the part. For example, in cell culture, it is preferable to use an EVA film that allows oxygen to permeate, but by using an EVA film thinner than the lower film 23a for the upper film 23b, it is easier to supply more oxygen to the culture space. Can be done.

The port portion 25 is made of a resin molded body that extends in the axial direction corresponding to the radial direction of the inner case 21 and is provided with a through hole 25a in the axial direction. The port portion 25 is arranged in the vicinity of the upper opening 21a of the inner case 21 so as to communicate the culture space 12 with the outside and can be closed by a cap 29 or the like, and the lower film 23a and the upper film of the flexible film 23. It is airtightly joined with the 23b sandwiched between them.

The shape of the port portion 25 is not particularly limited, but is formed into a flat shape having a width w larger than the thickness t, and a cross-sectional shape in which the thickness t gradually decreases from the center side in the width w direction toward both side edges. have. Due to this shape, the lower film 23a and the upper film 23b can be easily and airtightly welded.

An inward projecting portion 25b projecting into the culture space 12 is provided on one end side of the port portion 25 in the mounted state in the axial direction, and an external device such as a pipette or a syringe projecting outward on the other end side in the axial direction. Is provided with an external connection portion 25c to which the is connected.
The inner protruding portion 25b has a length corresponding to the height of the side peripheral wall 21b of the inner case 21. The tip end side of the inner protrusion 25b is formed in a tapered shape, and the tip end is provided with an opening 25d of a through hole 25a.
On the other hand, although detailed illustration is omitted, the external connection portion 25c has a shape corresponding to the connection shape of the external device to be used, such as a lure taper port and a needleless rubber stopper, and does not connect an external device. In that case, it is closed with a cap 29 or the like.

The port portion 25 is supported between the lower film 23a and the upper film 23b of the flexible film 23, so that the port portion 25 can be displaced by the flexibility of the flexible film 23 as shown in FIGS. 4 and 5. ing. Due to this displacement, the inner protruding portion 25b is arranged along the opening closing portion 23b1 of the upper film 23b as shown in FIG. 4, and is located above the medium 27, and the opening 25d is the inner case 21 as shown in FIG. It can be displaced from the position of being immersed in the medium 27 on the side peripheral wall 21b side of the above. The port portion 25 is preferably made of a material having shape-retaining property so that it can be held at a position above the medium 27 and at a position where it is not immersed in the medium 27. The port portion 25 is preferably made of a material that can be bonded to the flexible film 23, and in this embodiment, a material that is weldable to the upper film 23b and the lower film 23a is used.

In order to produce such a closed cell culture vessel 10, first, an inner case 21 suitable for the cells to be cultured is prepared. If the culture base 13 composed of the surface of the bottom wall 21c of the inner case 21 is not suitable for the cells to be cultured as it is, the bottom wall 21c of the inner case 21 or the like is surface-treated with the inner case 21 alone. , The surface properties suitable for the cells to be cultured, for example, the hydrophilic surface is modified to prepare the culture base 13.

The inner case 21 is covered with a flexible film made of, for example, a flat thermoplastic resin from the bottom wall 21c side and heat-shrinked or evacuated to cause a lower end portion 23a1, a side peripheral portion 23a2, an annular constricted portion 23a4, and a lower collar. The lower film 23a having the portions 23a3 is formed in close contact with the outer periphery of the inner case 21.

Next, with the port portion 25 made of the resin molded body prepared in advance arranged at a predetermined position, the flat upper film 23b is arranged at a position that closes the upper opening 21a of the inner case 21, and the lower collar portion of the lower film 23a is arranged. The 23a3 and the upper flange portion 23b2 of the upper film 23b are brought into contact with each other.
In this state, the lower collar portion 23a3 of the lower film 23a and the upper collar portion 23b2 of the upper film 23b are welded, and the upper film 23b and the port portion 25 and the port portion 25 and the lower film 23a are welded together. As a result, the closed cell culture vessel 10 as shown in FIGS. 1 and 2 can be manufactured.

In order to cultivate cells using the closed cell culture vessel 10 produced in this manner, the closed cell culture vessel 10 as shown in FIG. 2 is sterilized and prepared in advance. In order to prepare the desired culture base 13 and culture space 12, various treatments may be applied to the inside of the sterilized closed cell culture vessel 10 as necessary. For example, in the case of coating such as collagen coating or fibronectin coating, a coating liquid is put into a syringe (not shown), the syringe is connected to the external connection portion 25c of the port portion 25 and injected, and the syringe is spread over the entire culture base portion 13 to be predetermined. Leave it for a while. Then, the coating liquid is sucked out from the port portion 25 with a syringe and extracted.

After these preparations are complete, cell seeding is performed. The cells to be cultured are suspended in the medium 27 to prepare a cell suspension, the cell suspension is placed in a syringe (not shown), connected to the external connection portion 25c of the port portion 25, and placed in the closed cell culture vessel 10. inject. Then, the cells to be cultured are allowed to stand in the incubator for a predetermined period to adhere to the culture base 13.
In order to remove the cells that did not adhere to the culture base 13, a predetermined amount to the entire amount of the cell suspension in the closed cell culture vessel 10 was removed by the same operation as the medium exchange described later, and a new medium 27 was injected. , Start culturing.

Culturing is performed by allowing the closed cell culture vessel 10 to stand in a predetermined culture atmosphere in which the temperature and humidity of an incubator or the like are adjusted. At this time, in a state where the port portion 25 is closed by the cap 29, the port portion 25 is arranged above the liquid level of the containing liquid composed of the medium 27, the cell suspension, or the like as shown in FIG. This prevents unnecessary vibrations, stimuli, and the like from being transmitted from the port portion 25 to the medium 27 in the culture space 12.

During such a culture period, the inside is regularly observed visually or with a microscope, and the medium is changed.
In order to observe the inside of the closed cell culture vessel 10, the closed cell culture vessel 10 arranged in the culture atmosphere as shown in FIG. 2 is supported by a manual or mechanical arm from the outside of the flexible film 23. Carry it in place and observe it visually or with a microscope. At this time, since the aseptic state of the culture space can be ensured by the flexible film 23, it can be performed in an external space where the aseptic state is not secured.

To exchange the medium, first, as shown in FIG. 4, the cap 29 of the port portion 25 is removed to expose the external connection portion 25c, and a syringe (not shown) is connected to the external connection portion 25c, as shown in FIG. Due to the flexibility of the flexible film 23, the inner case 1 is displaced with the vicinity of the upper opening 21a as a fulcrum. Since the length of the inner protruding portion 25b of the port portion 25 corresponds to the height of the side peripheral wall 21b of the inner case 21, the tip of the inner protruding portion 25b, that is, the opening 25d is mediumd on the side peripheral wall 21b side of the inner case 21 due to displacement. It can be immersed in 27 and can reach the corners of the bottom wall 21c.

In this state, the used medium 27 to be discharged by tilting the closed cell culture vessel 10 is collected, sucked from the opening 25d of the port 25 with a syringe (not shown), and after the suction operation is completed, the opening 25d of the port 25 is opened. By returning the medium to the vicinity of the upper opening 21a and removing the syringe (not shown) while taking care not to return the used medium 27, the used medium 27 is aseptically discharged easily and surely.
Next, a required amount of the sterilized new medium 27 was taken into the syringe, and in a state of being displaced as shown in FIG. 5, the syringe was connected to the external connection portion 25c of the port 25, and the new medium was transferred to the inner case 21. Gently pour into the corners of the bottom wall 21c on the side peripheral wall 21b side.
Then, the culture is continued by allowing it to stand in a culture atmosphere. The culture period can be appropriately set depending on the purpose and cell type.
In this way, the culture is terminated when the culture period elapses while aseptically performing one or more observations and medium exchange.

After completion of the culture, the cultured cells are collected from the closed cell culture vessel 10. First, the medium 27 in the closed cell culture vessel 10 is sufficiently removed in the same manner as in the medium exchange, and the phosphate buffer wash is performed in the same manner as in the medium exchange.
A predetermined amount of phosphate buffer is injected into the closed cell culture vessel 10 with a syringe and spread well in the inner case 21. Then, the phosphate buffer solution is sufficiently aspirated and sufficiently removed from the closed cell culture vessel 10. This phosphate buffer washing is repeated one or more times.

Then, the trypsin solution is used and treated in the same manner as the medium exchange. The trypsin solution is injected into the closed cell culture vessel 10 with a syringe and spread well in the inner case 21. Let it stand at room temperature or incubator for several tens of seconds to several minutes to allow trypsin to work.
Next, the trypsin solution is discharged, and a phosphate buffer solution containing serum, a medium 27, or the like is injected to make it recoverable in the state of a cell suspension or a cell sheet. At this time, pipetting or the like can be appropriately performed for the purpose of peeling the cultured cells from the culture base 13 or breaking the adhesion between the cells.

Then, the cell suspension of the cultured cells is sucked into a syringe to open the closed cell culture container 10 and taken out from the upper opening 21a of the inner case 21 for use.
When the closed cell culture vessel 10 is opened, the flexible film 23 can be cut to open the upper opening 21a of the inner case 21. In that case, as shown by a virtual line in FIG. 4, the blade 31 is applied to the portion corresponding to the side peripheral wall 21b of the inner case 21 to cut the flexible film 23 from the outside.

By moving the blade 31 once along the side peripheral wall 21b, as shown in FIG. 6, the upper film 23b side of the flexible film 23 is cut and separated, and the upper film 23b is removed from the upper opening 21a of the inner case 21. To do. At this time, since the upper film 23b is arranged in close contact with the inner case 21 without being adhered or welded, it can be easily removed from the upper opening 21a of the inner case 21 after cutting.
Then, the cells are taken out from the upper opening 21a of the inner case 21 and used.

According to the closed cell culture vessel 10 of the present embodiment as described above, first, the side peripheral wall 21b of the inner case 21 is fixed immovably from the outer peripheral side by the flexible film 23 and airtightly covered to cover the annular frame. The culture space 12 and the culture base 13 are immovably provided inside the body 15 surrounded by the lateral peripheral wall. Therefore, the culture space 12 and the culture base 13 can be kept in a closed system, and cell culture can be performed aseptically.

At the same time, the culture space 12 and the culture base 13 can be fixedly arranged at a predetermined position with respect to the flexible film 23, the culture space 12 can be kept in a predetermined shape, and the outer surface of the inner case 21 and the flexible film 23 Since the cells are in close contact with each other, the inner case 21 having a higher hardness than the flexible film 23 can be reliably gripped from the outside through the flexible film 23 during or at the end of cell culture, and the culture space 12 And it is possible to prevent the culture base 13 from being displaced or deformed.

As a result, cell culture can be easily performed aseptically in a closed system, and stable support can be achieved by either a human or a mechanical arm. Therefore, various culture operations such as stable transportation, medium exchange, and observation can be performed aseptically and stably, and automation by a machine is also possible.

In the closed cell culture vessel 10 of the present embodiment, since the culture base 13 is composed of the bottom wall 21c of the inner case 21 and the annular frame 15 is composed of the side peripheral wall 21b of the inner case 21, the culture base 13 is formed in the inner case 21. And the annular frame 15 are integrally provided. Therefore, the culture base 13 and the culture space 12 can be reliably maintained in a predetermined position and a predetermined shape, and the cells can be reliably and stably and aseptically cultured in the culture base 13 and the culture space 12.

In the closed cell culture vessel 10 of the present embodiment, since the flexible film 23 covers the entire outer periphery of the inner case 21, the flexible film 23 ensures the culture space 12 and the culture base 13 regardless of the inner case 21. Can be sealed in. Therefore, the material of the inner case 21 can be freely selected regardless of the material of the flexible film 23, and for example, even a combination in which the inner case 21 and the flexible film 23 have low compatibility and cannot be welded can be used.

Further, the inner case 21 can be easily sealed by the flexible film 23 by applying various treatments. For example, in the case of the inner case 21 for culturing adhesive cells, the inner case 21 is hydrophilized in the culture base 13 as a single body. It is possible to perform various treatments on the inner case 21 depending on the cells to be cultured, the environment, and the like, such as treatment. That is, it is possible to easily obtain a closed cell culture vessel 10 in which a required site is subjected to a desired treatment.

Further, since the flexible film 23 is arranged on the outside of the side peripheral wall 21b, the flexible film 23 can be cut by the cutting tool 31 on the outer peripheral side of the side peripheral wall 21b. Therefore, when the inner case 21 is taken out from the flexible film 23, the cutting tool 31 does not come into direct contact with the medium 27 in the inner case 21, and foreign matter generated during cutting does not fall into the inner case 21 and is cultured. It is possible to prevent the cells and the medium 27 from being contaminated with foreign substances, germs, and the like.

In the closed cell culture vessel 10 of the present embodiment, the flexible film 23 includes a lower film 23a covering the side peripheral wall 21b and an upper film 23b covering the upper opening 21a, and has a diameter larger than that of the side peripheral wall 21b of the inner case 21. The lower collar portion 23a3 of the lower film 23a protruding outward in the direction and the upper collar portion 23b2 of the upper film 23b are airtightly welded. Even if the inner case 21 is present, the lower collar portion 23a3 and the upper collar portion 23b2 are located on the outside of the inner case 21, which facilitates the welding work and enables reliable sealing. Therefore, the culture space 12 and the culture base 13 in the inner case 21 can be easily and surely sealed.

In the closed cell culture vessel 10 of the present embodiment, an annular constriction portion 23a4 covering the upper end surface 21d of the upper opening 21a is provided between the side peripheral portion 23a2 and the lower collar portion 23a3 in close contact with the side peripheral wall 21b of the lower film 23a. It is provided. Therefore, by covering the upper end surface 21d of the inner case 21 with the lower film 23a, the inner case 21 is sandwiched from above and below, the inner case 21 can be stably fixed, and the medium 27 in the inner case 21 is outside. It can be hard to overflow. In particular, by bringing the lower film 23a into close contact with the upper end surface 21d of the upper opening 21a, it is possible to prevent the medium 27 from entering between the inner case 21 and the lower film 23a.

Moreover, since the annular constriction portion 23a4 is easily deformed into a bellows shape with respect to the side peripheral portion 23a2 of the lower film 23a that is closely fixed to the side peripheral wall 21b, the upper film 23b welded to the lower collar portion is displaced. Or easily deformed. Therefore, even if the inner case 21 is hard, the volume can be changed by deforming or displacing the upper film 23b while maintaining the closed system formed by welding the upper film 23b and the lower film 23a. For example, the medium 27. Operations such as accommodating and discharging the film can be easily performed.

In the closed cell culture container 10 of the present embodiment, the port portion 25 that communicates the culture space 12 and the outside is displaceably supported by the flexible film 23, so that the flexible film can be opened and closed without opening. The medium and cells can be easily discharged and introduced through the port portion 25. At this time, since the port portion 25 is provided on the flexible film 23, the degree of freedom in operation of the port portion 25 is high.

In the closed cell culture vessel 10 of the present embodiment, the opening 25d of the inner protruding portion 25b protruding into the culture space 12 of the port portion 25 is located at a position where it is immersed in the medium 27 in the culture space 12 and at an upper position. It can be displaced between them. Therefore, the opening 25d of the inner protruding portion 25b of the port portion 25 can be arranged at a position above the medium 27 to perform cell culture, and vibration is transmitted from the port portion 25 to the medium 27 or contact with the port portion 25. Therefore, it is possible to prevent the cells from being damaged and to perform stable cell culture. On the other hand, if the port portion 25 is displaced, the opening 25d of the inner protruding portion 25b can be immersed in the medium 27, for example, the medium 27 can be gently discharged or introduced from the port portion 25, and the cells to be cultured are excessive. Can be prevented from stimulating the culture medium.

In the closed cell culture vessel 10 of the present embodiment, the inner protruding portion 25b has a length corresponding to the height of the side peripheral wall 21b, and the opening 25d of the inner protruding portion 25b is located along the peripheral wall portion of the inner case 21. Since the cells can be immersed in the medium 27 in the medium 27, even if the port portion 25 is displaced and the opening 25d of the inner protruding portion 25b is immersed in the medium 27, the cells cultured in a film shape in the inner case 21 are inside the center side. It is possible to prevent the protrusion 25b from coming into contact with the cultured cells and causing damage to the cultured cells.
Further, the medium 27 can be taken in and out by displacing the port 25 or tilting the inner case 21 according to the discharge or introduction of the medium 27, so that the medium 27 touches the inner surface of the upper film 23b or the like. Opportunities can be reduced. The inner surface of the upper film 23b is naturally sterilized, but the risk of contamination of the medium 27 can be further reduced by reducing the chance that the medium 27 comes into contact with a portion that does not need to be touched.

[Modification example]
Hereinafter, a modified example of the present embodiment will be described.
7 (a) and 7 (b) show a closed cell culture vessel 10 of a modified example of the above embodiment. In the closed cell culture vessel 10 of FIG. 7A, a guide member 33 is arranged on the entire circumference of the outer peripheral side of the side peripheral wall 21b of the inner case 21, and the guide member 33 is provided together with the side peripheral wall 21b of the inner case 21. , The lower film 23a of the flexible film 23 continuously adheres and covers the flexible film 23.

Here, the guide member 33 is made of a tubular hard ring, and is loosely fitted to the outer peripheral side of the bottom wall 21c side end of the side peripheral wall 21b of the inner case 21 so as not to shift. As a result, a stepped portion 35 as a continuous guide portion is formed on the outer periphery of the side peripheral wall 21b between the side peripheral wall 21b and the guide member 33. The guide member 33 and the side peripheral wall 21b are continuously and tightly covered with the flexible film 23 via the stepped portion 35.
An annular groove may be provided on the outer circumference of the guide member 33, and in that case, the outer circumference of the groove is also covered with the flexible film 23.
Others are the same as those in the above embodiment.

Even with such a closed cell culture vessel 10, the same effects as those of the above embodiment can be obtained. Moreover, in this modification, the guide member 33 is arranged on the outer peripheral side of the side peripheral wall 21b, and the step portion 35 is formed on the entire circumference and is covered with the flexible film 23.
Therefore, when the flexible film 23 is cut by the blade 31 on the outer peripheral side of the side peripheral wall 21b, the blade 31 is moved along the side peripheral wall 21b with the blade 31 in contact with the stepped portion 35 of the guide member 33. , The flexible film 23 can be easily cut. That is, the step portion 35 guides the blade 31 as a guide portion when cutting the flexible film 23.

In this modification, the guide member 33 is arranged on the outer peripheral side of the side peripheral wall 21b to form the stepped portion 33 as the guide portion, but the guide portion may have a different configuration. For example, as shown in FIG. 7B, by providing one or more ridges 36 as guide portions on the outer peripheral surface of the side peripheral wall 21b along the assumed cutting line, the cutting tool 31 to be pressed is provided by the ridges 36. You may want to guide you.
Since the guide portion only needs to be able to guide the blade 31 to be pressed on the outer peripheral surface of the side peripheral wall 21b, it can be formed by a groove or a plurality of convex portions in addition to the ridge 36.

The above embodiments and modifications can be appropriately changed within the scope of the present invention.
In the above embodiment, an example of a closed cell culture vessel 10 in which an inner case 21 having a bottom wall 21c and a side peripheral wall 21b is at least liquid-tightly covered with a flexible film 23 has been described, but is not particularly limited. ..
For example, as shown in FIG. 8, a closed cell culture vessel 10 can be obtained by covering the annular frame 15 having a ring shape with both ends open at least in a liquid-tight manner with a flexible film 23. In that case, the inner surface of the flexible film 23 in the inner space surrounded by the annular frame 15 becomes the culture base 13, and the space inside the annular frame 15 becomes the culture space 12. Even with such a configuration, it can be used as a closed cell culture vessel 10 capable of aseptically and stably performing various culture operations such as transportation, medium exchange, and observation, as in the above embodiment. it can.

Further, the culture base 13 does not have to be the surface of the sealed container itself such as the bottom wall 21c of the inner case 21 or the flexible film 23, and the inside of the sealed container 11 may be separated from a plate material, a film, a mesh, or the like. The culture base 13 made of the members may be accommodated and configured.

Further, in the above embodiment, the annular frame 15 is circular in the top view, but it may be any shape that can hold the culture space 12 inside, such as a polygon such as an ellipse, a quadrangle, or a hexagon in the top view.
Further, it is also possible to use an annular frame body 15 whose inside is divided into a plurality of sections. For example, as shown in FIG. 9, the annular frame 15 having a plurality of partitions inside may be covered with a flexible film 23 at least in a liquid-tight manner to provide a culture space 12 or a culture base 13 partitioned into a plurality of cells. .. As a result, the cultured cell group (cell sheet) cultured in the form of a sheet can be taken out for each section, and the operability is good.
Further, by arranging one or a plurality of displaceable port portions 25 corresponding to each compartment, it is possible to supply and discharge the medium so as to be suitable for the compartmentalized culture base portion 13. In some cases, an operation of peeling the cultured cell group (cell sheet) from the bottom wall can be performed by jetting the medium from the port portion 25.

Further, in the above embodiment, an example in which the flexible film 23 is configured to cover the entire inner case 21 has been described, but as long as the space inside the inner case 21 can be hermetically sealed, one of the inner cases 21 It can also be configured to cover the portion. That is, if the flexible film 23 covers only the outer peripheral surface of the side peripheral wall 21b of the inner case 21 so as to be in close contact with the inner case 21, and the upper opening 21a can be closed to at least liquid-tightly seal the inner case 21, the rest of the inner case 21 is flexible. It does not have to be covered with the sex film 23.

Further, in the above embodiment, an example in which the flexible film 23 has a two-divided structure of the lower film 23a and the upper film 23b has been described, but the flexible film 23 is integrated or the flexible film 23 is divided into three or more. , The inner case 21 and the annular frame 15 may be covered.

In the above embodiment, an example in which the flexible film 23 is sealed by welding the lower collar portion 23a3 and the upper collar portion 23b2 by heating has been described, but it is also possible to join and seal the flexible film 23 by another method such as adhesion. is there. When an adhesive or the like is used, it is preferable to bond at a site that does not come into direct contact with the cells to be cultured, the cell suspension, the medium 27, or the like, and for example, the adhesive may be bonded on the outer peripheral side of the annular frame 15.
Further, when the lower collar portion 23a3 and the upper collar portion 23b2 of the flexible film 23 are sealed by welding, a weak seal that can at least ensure liquidtightness, a so-called easy peel, may be adopted. In that case, in order to facilitate the peeling of the upper film when taking out the cultured cells or the like in the culture space, it is desirable to form, for example, a tongue piece-shaped peeling knob portion.
Further, in the above embodiment, an example in which the opening 25d of the inner protrusion 25b is displaceable by being displaceably supported by the flexible film 23 has been described, but the present invention is not limited thereto. For example, the opening 25d of the inner protrusion 25b may be displaceable by expanding or contracting the flexible film 23.

10 Closed cell culture container 11 Sealed medium 12 Culture space 13 Culture base 15 Circular frame 21 Inner case 21a Upper opening 21b Side peripheral wall 21c Bottom wall 21d Upper end surface 23 Flexible film 23a Lower film 23b Upper film 23a1 Lower end 23a2 side Peripheral part 23a3 Lower collar part 23a4 Circular constriction part 23b1 Opening closure part 23b2 Upper collar part 25 Port part 25a Through hole 25b Inner protrusion 25c External connection part 25d Opening 27 Medium 29 Cap 31 Blade 33 Guide member 35 Step part (guide part)
36 ridge (guide part)
t thickness w width

Claims (9)

A closed cell culture vessel in which a culture base and a culture space in contact with the culture base are provided inside a sealed container, and cells are cultured on or in the culture base.
The sealed container includes an annular frame and a flexible film that immovably fixes the annular frame from the outer peripheral side and covers it at least liquid-tightly.
The culture base and the culture space are closed cell culture vessels provided in an inner space surrounded by the annular frame. The closed cell culture vessel according to claim 1, further comprising an inner case having the annular frame as a side peripheral wall and the culture base as a bottom wall. The second aspect of the present invention, wherein the flexible film covers at least the outer peripheral surface of the annular frame in close contact and covers the entire outer periphery of the annular frame by covering the upper opening of the annular frame. Closed system cell culture vessel. The flexible film includes a lower film that covers at least a part of the outer peripheral surface of the annular frame, and an upper film that covers the upper opening.
The lower film has a lower collar portion protruding outward from the outer peripheral surface of the annular frame body.
The upper film has an upper flange portion protruding outward from the outer peripheral surface of the annular frame body.
The closed cell culture vessel according to any one of claims 1 to 3, wherein the lower collar portion and the upper collar portion are at least liquid-welded. The fourth aspect of the present invention, wherein the lower film is provided with an annular constriction portion that covers the upper end surface of the upper opening between a side peripheral portion that is in close contact with the outer peripheral surface of the annular frame body and the lower collar portion. Closed cell culture vessel. A port portion capable of communicating the culture space with the outside is provided.
The closed system according to any one of claims 2 to 5, wherein the port portion has an inward protruding portion protruding into the culture space and is supported by the flexible film near the upper opening of the annular frame. Cell culture vessel. The closed cell culture vessel according to claim 6, wherein the opening of the medial protrusion of the port portion can be displaced between a position where the opening is immersed in the medium in the culture space and a position above the medium. The seventh aspect of claim 7, wherein the inner protruding portion has a length corresponding to the height of the inner wall of the annular frame, and the opening of the inner protruding portion can be immersed in the medium on the inner wall side of the annular frame. Closed system cell culture vessel. The method according to any one of claims 1 to 8, wherein a guide portion is formed on the outer peripheral surface of the annular frame body along the outer peripheral surface, and the guide portion is covered with the flexible film together with the tubular frame body. Closed system cell culture vessel.

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