JP6412369B2 - Method for culturing adherent cells - Google Patents

Description

  The present invention relates to a method for culturing adherent cells using a bag-like container.

Conventionally, a tissue culture petri dish or tissue culture flask made of styrene is generally used as a culture vessel for adhesive cells.
The styrene resin itself that forms the culture surface of these containers is hydrophobic, and adherent cells are difficult to adhere, and as a result, the cells are extremely difficult to grow and often die.
However, petri dishes or flasks for tissue culture make the culture surface hydrophilic by ultraviolet irradiation treatment or plasma treatment, etc., thereby making it easy for adhesive cells to adhere to the culture surface.

Moreover, although the container which culture | cultivates a cell using a bag-shaped container is marketed, most of these culture | cultivate a floating cell. These bag-like containers are generally formed of a flexible olefin-based resin, the inner surface is hydrophobic, and the contact angle thereof is near 90 ° or more than 90 °. It is considered unsuitable and is generally not used for adherent cell culture.
However, the bag-like container has an advantage that it can be cultured in a closed system, and some attempts have been made to use it for culturing adhesive cells.

  For example, like a styrene petri dish or flask, the inner surface of the bag-like container, that is, the culture surface is hydrophilized by surface treatment by ultraviolet irradiation treatment or corona discharge treatment, etc. There is something to offer. For example, in Patent Document 1, after irradiating a film surface with ultraviolet rays to make it hydrophilic, two films are overlapped so that the hydrophilic surface becomes the inner surface, and the periphery is sealed to form a bag for adhesive cell culture The container is formed. Further, in Patent Documents 2 and 3, after corona discharge treatment of the film surface, two films are overlapped so that the hydrophilic surface becomes the inner surface, and the periphery is sealed to form a bag shape for adhesive cell culture. A container is formed.

  However, in any of these methods, the inner surface of the bag-shaped container, that is, the surface to be the culture surface must be surface-treated in the state exposed to the outside air, and at that time, foreign matter and bacteria adhere to the culture surface. It was difficult to avoid. For example, inflation molding can easily form a film free of foreign substances and bacteria on the inner surface. However, even in the case of corner inflation molding, the surface to be cultured in the future must be exposed to the outside air in order to perform surface treatment. It is difficult to avoid foreign substances and bacteria from adhering to the same surface.

  Another problem is that in the case of highly flexible olefin-based resins such as polyethylene, there is a drawback that surface treatment is likely to deteriorate over time, making it difficult to implement as a product. It is thought that this is one of the reasons why the product is not generalized.

  In addition, the bag-like container is deformed so as to follow the shape of the contained liquid due to its flexibility, and thus a wave that repeats even with a small inertia force generated during movement is generated, which is generated in the liquid container in the container. It causes a flow to inhibit the cells from adhering or to help the adhered cells detach. Since this tendency is strong when the amount of the liquid container is small relative to the capacity of the bag-like container, in the culture of adhesive cells, the amount of the liquid container is significantly smaller than the capacity of the bag-like container. The tendency to inhibit the cells from adhering or to peel off the adhered cells becomes stronger. This also made it more difficult to culture adherent cells in bag-like containers.

  On the other hand, Patent Literature 4 describes a culture tray for improving the culture performance of a bag-like container by suppressing the fluidity of the liquid container previously invented and patented by the present inventors.

  According to Patent Document 4, a culture tray that holds a bag-like container in which a liquid material containing cells and a medium is accommodated, the floor surface on which the bag-like container is placed in a state of being laid in a horizontal direction. A tray body, a pressing plate that presses the bag-shaped container between the floor surface of the tray main body, and a spring member that presses the pressing plate in the floor surface direction. The bag-like container is set on a culture tray, wherein the bag-like container is held in a state in which the internal pressure of the bag-like container is increased by pressing to generate tension on the container wall of the bag-like container. It is described that if cells are cultured, the flow of the liquid substance is suppressed and cell clusters are not destroyed, so that information transmission between cells is promoted and cell culture performance is improved.

  However, Patent Document 4 describes a method for culturing adherent cells by increasing the adherence of adherent cells in a bag-like container having a culture surface on which adherent cells are difficult to adhere. Not.

JP 2009-27944 A Japanese Patent Laid-Open No. 3-160984 JP-A-6-9756 Japanese Patent No. 4806671

The first problem to be solved by the present invention is to provide a new culture method capable of easily culturing adhesive cells in a bag-like container.
A second problem is to provide a culture method capable of easily culturing adherent cells in an environment that is not affected by foreign substances and bacteria.

A third problem is to provide a culture method capable of easily culturing adherent cells without worrying about changes over time in the culture surface.
Moreover, the 4th subject is providing the culture | cultivation method which can culture | cultivate an adhesive cell easily and cheaply by using an inexpensive member.

  In order to solve the above-mentioned problems, the present inventors have intensively studied and cultured in the same culture method as the culture method in which the flow of the liquid material in the culture tray described in Patent Document 4 is suppressed for the culture of adhesive cells. As a result, at least, in general, a container having a culture surface formed of a material that has been said to be unable to culture adhesive cells without treatment, that is, having a culture surface to which adhesive cells are difficult to adhere, and Even in the case of a bag-like container, it has been found that the adhesion of adherent cells can be enhanced and cultured, and the adhered cells are not easily detached, leading to the following invention.

The method for culturing adhesive cells according to claim 1 is a method for culturing adhesive cells by enclosing a liquid container containing adhesive cells and a liquid medium in a bag-like container having a container wall serving as a cell culture surface. A method for culturing, wherein the cell culture surface is formed of a low density polyethylene resin, a low density linear polyethylene, an ethylene / vinyl acetate copolymer, or a mixture thereof. A first member having a mounting surface for mounting the container wall and a pressing member for pressing the container wall facing the container wall, without using a surface treatment for improving cell adhesion on the surface. The bag-like container is set in a shape holding means provided with the above, and the internal pressure of the container is increased by pressing the bag-shaped container with the placement surface and the pressing surface, and the liquid storage is performed by increasing the internal pressure. This suppresses the flow of things By culturing while maintaining the state, prompting that the adhesive cells adhere to the cell culture surface, or to suppress that the adhered cells detached, characterized in that to improve the culture efficiency.

  When the internal pressure in the container rises by pressing the bag-like container, tension is generated on the container wall of the bag-like container, and the liquid container is as if the surroundings were covered with a rigid body, and the fluidity in the vicinity of the container wall Is lost. Adhesive cell culture generally has a small amount of medium, so that the liquid container is pushed into a narrow gap, and the vicinity of all of the liquid container is occupied by a container wall, so that the liquid The flow of the entire contents is suppressed.

  Adhesive cells mentioned here include cells that cannot grow or die if they are not attached to the carrier, but they can grow without being attached to the carrier, depending on the generation or environment. Or even if it is a cell which can proliferate, the cell culture | cultivated in anticipation of an effect is also included by making it adhere | attach and culture | cultivate to a support | carrier.

The cell culture surface referred to here is a surface on which cells adhere and grow or proliferate, and in the case of a bag-like container, it is located on the inner surface of the container, that is, the inner container wall surface.
The culture performance here includes not only the ability of cells to grow by adhering to the culture surface, but also the ability to grow into a form suitable for the purpose of culture, even if they do not grow.

  Further, the bag-like container here is an envelope-like form in which the peripheral edges of two overlapped films or sheets are sealed or sealed, and the outer shape is easily deformed depending on the shape of the contents. Say. Furthermore, in the present invention, the container is flexible enough to deform according to the shape of the liquid container. For example, the liquid container can be deformed together with the container wall with a small inertia force generated when the container is moved. It is a container.

  In addition, the present invention generally enhances the adhesion of adhesive cells, even in a container having a culture surface formed of a material that has been said to be unable to culture adhesive cells without treatment, It was cultivated, and it was discovered that the adhered cells were not easily detached and reached the present invention, but this effect is obtained by suppressing the flow of the liquid container, It is clear that this effect also occurs in the cultivation of adherent cells in a general bag-like container, that is, a container that has been surface-treated so that the adherent cells can easily adhere.

  For example, this effect also occurs when cells with poor cell adhesion are cultured on a culture surface generally called a cell-adhesive surface, and the conventional surface treatment for improving cell adhesion deteriorates. However, even with such materials, there is a synergistic effect that culture can be performed more reliably without considering deterioration of the surface treatment.

In the method for culturing adhesive cells of the present invention, the cell culture surface is a surface to which adhesive cells are difficult to adhere.
Here, the surface to which adherent cells are difficult to adhere refers to a cell that grows or proliferates only when cultured by the method for culturing live adherent cells according to claim 1 and must be cultured by the same culture method. That is, when cultured without using the shape-retaining means, the adhesion of the cells is poor, and the cells do not adhere to the cell culture surface, or peel off even if they grow halfway, so that the cells form aggregates. It is formed and does not proliferate or die, and the container alone cannot be used as an adhesive cell culture container.

That is, a container formed with a surface to which adherent cells are difficult to adhere cannot achieve the purpose of adherent cell culture unless it is cultured by the adherent cell culture method of the present invention.
In addition, that it does not hold as an adhesive cell culture container means that there is no merit of commercialization and an industrial merit.

In the method for culturing adhesive cells of the present invention, the cell culture surface is formed of a hydrophobic material having flexibility, and is not subjected to a surface treatment for improving cell adhesion.

The adhesion cell culture method according to claim 2 is characterized in that, in addition to the description of claim 1 , the contact angle of the cell culture surface with water is in the vicinity of 90 ° or 90 ° or more. .

  According to the method for culturing adhesive cells of the present invention, the inner surface of the bag-like container is not surface-treated only by pressing the bag-like container to increase the internal pressure of the container and suppressing the flow of the liquid container. Since the adherent cells can be cultured, a new culture method capable of easily culturing the adherent cells using the bag-like container can be provided.

  Moreover, according to the culture | cultivation method of the adhesive cell of this invention, the process of surface-treating the inner surface of a bag-shaped container can also be omitted. Therefore, from the manufacturing process of the bag-like container, it is possible to eliminate the process that has a risk of foreign matter and bacteria adhering to the culture surface, that is, the step where the culture surface is exposed to the outside air. Adherent cells can be cultured.

  Moreover, according to the culture | cultivation method of the adhesive cell of this invention, the process of surface-treating the inner surface of a bag-shaped container can also be omitted. Therefore, adherent cells can be stably cultured without worrying about changes in the culture surface over time.

  Further, according to the method for culturing adhesive cells of the present invention, the inner surface of the bag-like container is surface-treated only by pressing the bag-like container to increase the internal pressure of the container and suppressing the flow of the liquid container. Adhesive cells can be cultivated without any problems, and the process of using expensive equipment and the complicated and laborious process can be largely eliminated in the manufacturing process of the bag-like container. Therefore, according to the method for culturing adherent cells of the present invention, adherent cells can be cultured easily and inexpensively.

It is a perspective view which shows the state which set the bag-shaped container which accommodated the liquid container in the shape holding means used for the culture method of embodiment of this invention. It is sectional drawing which shows the state which set the bag-shaped container which accommodated the liquid container in the shape holding means used for the culture method of embodiment of this invention. It is a disassembled perspective view of the shape holding means shown in FIG. It is a microscope picture which shows the state of the cell in Example 1, (a) shows the state 24 hours after cell seeding, (b) shows the state 4 days after cell seeding. It is a microscope picture which shows the state of the cell in the comparative example 1, (a) shows the state 24 hours after cell seeding, (b) shows the state 4 days after cell seeding.

Hereinafter, embodiments of the present invention will be described.
In the culturing method of the embodiment of the present invention, as shown in FIGS. 1 to 3, a liquid container containing adhesive cells and a liquid medium in a bag-like container 2 having a container wall 21 to be a cell culture surface 22. 6, a shape including a first member 3 having a placement surface 31 on which the container wall 21 is placed and a second member 4 having a pressing surface 42 that holds the container wall 21 opposite to the container wall 21. The bag-like container 2 is set on the holding means 1 and the bag-like container 2 is pressed by the mounting surface 31 and the holding surface 42 to increase the internal pressure of the container, and the increase in the internal pressure causes the liquid container 6 to flow. By suppressing and incubating while maintaining this state, the adherent cells are encouraged to adhere to the cell culture surface 22, or the adhered cells are prevented from peeling off, thereby improving the culture performance.

[Liquid container]
The liquid container 6 of the present embodiment is a liquid having a fluidity mainly composed of adhesive cells and a liquid medium, contained in the bag-like container 2, and includes serum, other additives, indicators, and the like. May be included.

[Bag-like container]
The bag-like container 2 of the present embodiment is a flat resin-made bag-like container in which an accommodation space is formed between a pair of container walls 21 and 21 facing each other. Around the periphery, a port 23 and a tube 24 are provided for communicating the accommodation space with the outside. The end portion of the tube 24 serves as a connection portion 25 that can be connected to a syringe or another tube and can be sealed when the liquid container 6 is accommodated or discharged from the accommodation space.

  Here, having flexibility means that the container wall 21 is flexible enough to be deformed by its own weight when, for example, a part of the container is supported while the liquid container 6 is accommodated. By the deformation | transformation of the wall 21, it means the softness | flexibility of the grade which can discharge | emit substantially the whole quantity of the liquid container 6 accommodated, without the gas going in and out of a container.

  Each container wall 21 of the bag-like container 2 is made of resin, and the inner surface of the container wall 21 that contacts the placement surface 31 during culture is the cell culture surface 22. The cell culture surface 22 may be a surface to which adherent cells easily adhere during culture, but in this embodiment, the surface treatment for improving cell adhesion is not performed, and the adherent cells adhere. It is a difficult aspect.

  Here, the surface treatment for improving cell adhesion is, for example, ultraviolet irradiation, corona discharge, plasma discharge, electron beam, etc. on the surface of the resin film or resin molded product constituting the container wall 21 of the bag-like container 2. This is a treatment for generating a hydrophilic functional group or polar group on the resin surface by irradiating a high energy electromagnetic wave or particle beam.

  In addition, at least a part, preferably all, of the container wall 21 of the bag-like container 2 has gas permeability capable of supplying oxygen necessary for cell growth and discharging carbon dioxide as a cell metabolite. It is good. The gas permeability required for the container wall 21 varies greatly depending on the type of cells accommodated in the container, the target number of cells grown, the purpose of culture, and the size of the culture container. The container wall material, the container wall area, and the container wall thickness may be selected and adjusted according to the culture purpose. Moreover, it should have transparency that allows the shape of the cells in the accommodation space, the state of the cells, the progress of the culture, and the like to be observed with a microscope or the like.

  In the container wall 21 of this embodiment, at least the culture surface is formed of a material having high hydrophobicity so that adherent cells are less likely to adhere in a general culture method, and surface treatment is performed to improve cell adhesion. Absent. Here, examples of highly hydrophobic materials that are difficult to adhere to adherent cells by a general culture method include low density polyethylene resin, low density linear polyethylene resin, ethylene / vinyl acetate copolymer, vinylidene fluoride. Examples thereof include a film or sheet formed from a resin, a tetrafluoroethylene / hexafluoropropylene copolymer, and a mixture thereof, and a multilayer film or sheet using the material as at least the cell culture surface 22.

[Shape holding means]
The shape holding means 1 of the present embodiment presses the bag-like container 2 in which the liquid container 6 is sealed with the mounting surface 31 and the pressing surface 42 to increase the internal pressure of the bag-like container 2 and increase the internal pressure. This is means for suppressing the flow of the liquid container 6. This shape holding means 1 has a structure that not only holds the bag-like container 2 continuously during incubation but also allows each operation during cell culture as it is.

  As shown in FIG. 3, the shape holding means 1 includes a tray-shaped first member 3, a second member 4 provided with a biasing member 43 that can press the bag-like container 2, a first member 3, and a second member 2. It is comprised from the cover body 5 for maintaining the distance of the member 4 and maintaining a press state. The first member 3 and the lid 5 are fixed to the first member 3 by a projection 52 provided on the first member 3 and a projection receiver 51 provided on the lid 5.

  The first member 3 has a placement surface 31 on which the bag-like container 2 is placed, and an outlet 32 such as a tube 24 connected to the bag-like container 2. The placement surface 31 is provided in a flat shape and is maintained substantially horizontal during culture, and the bag-like container 2 is placed with one container wall 21 in contact with the placement surface 31.

The second member 4 includes a pressing plate 41 and an urging member 43, and the pressing plate 4 has a pressing surface 42 disposed to face the placement surface 31, and an urging member 43 such as a spring is incorporated therein. The pin 5 is movably supported by the lid 5 and is biased in a direction away from the lid 5.
The bag-like container 2 was placed on the first member 3, the lid 5 was closed, and the first member 3 and the lid 5 were fixed by the projection 52 and the projection receiver 51, so that the bag was placed on the placement surface 31. The other container wall 21 facing the one container wall 21 is pressed by the pressing surface 42.

  The shape of the pressing plate 41 and the pressing surface 42 is not particularly limited as long as the bag-like container 2 can be pressed, but in order to ensure gas permeability of the container wall 21 in contact with the pressing plate 41, the pressing plate 41 has a plurality of vent holes. 45 may be provided, or may be formed of a material having air permeability.

  The first member 3, the lid 5, and the pressing plate 41 of the shape holding means 1 have a strength that can prevent the bag-like container 2 from being deformed during holding. For example, these may be formed of a hard material. Moreover, in order to make it easy to observe the inside of the bag-like container 2 held during the incubation period, it is preferably formed of a transparent material.

  In such a shape holding means 1, the bag-like container 2 containing the liquid container 6 is disposed between the mounting surface 31 of the first member 3 and the pressing surface 42 of the pressing plate 41, thereby providing a biasing member. The bag-like container 2 can be pressed by the placement surface 31 and the pressing surface 42 according to the urging force 43, and can be held in a state where the internal pressure of the container is increased.

[Culture method]
Next, a method for culturing adhesive cells using such a bag-like container 2 and shape holding means 1 will be described.

  First, a bag-like container 2 that contains a liquid medium in advance and a syringe that is filled with a cell suspension of adhesive cells in advance are prepared. The syringe is connected to the connection portion 25 of the tube 24 of the bag-like container 2, and a cell suspension of adhesive cells is injected into the bag-like container 2 via the tube 24, and the tube 24 is sealed and adhered. A bag-like container 2 in which a liquid container 6 containing sex cells and a liquid medium is enclosed is prepared. At this time, it is better that no gas enters the accommodation space of the bag-like container 2. This is because the movement of the gas in the container causes a flow in the liquid container 5.

  One container wall 21 of the bag-like container 2 is placed on the placement surface 31 of the first member 3, and the pressing surface 42 of the pressing plate 41 is brought into contact with the other container wall 21 to close the lid 5. That is, the bag-like container 2 is set in a state where it is laid sideways on the shape holding means 1.

  In the set state, the bag-like container 2 is pressed by the mounting surface 31 and the pressing surface 42 according to the urging force of the urging member 43, and the internal pressure of the container increases. When the internal pressure in the container rises, tension in the container wall 21 is generated in a direction parallel to the liquid contact surface, and the bag-like container 2 is held in a certain shape as if the container wall 21 is a rigid body. . The liquid container 5 in the bag-like container 2 is not deformed by the inertia force because the container wall 21 is maintained like a rigid body, that is, the liquid container 5 moves. Since there is no room, flow is regulated.

The internal pressure of the bag-like container 2 needs to be appropriately set according to the magnitude of the inertia force. For example, as the mass of the liquid container 6, the acceleration during movement of the bag-like container 2, or the strength of vibration received by the bag-like container 2, the larger the inner pressure, the higher the internal pressure needs to be set. That is, it is necessary to give the bag-like container 2 an internal pressure enough to prevent deformation of the bag-like container 2 due to the inertial force of the liquid container 6.
Further, when the bag-like container 2 is tilted or inverted during the culture operation, the internal pressure is increased to such an extent that the container wall is not substantially deformed with respect to the change in hydraulic pressure caused by the height difference of the storage space. There is a need.

  Adhesive cells are produced by themselves and adhere to the cell culture surface 22 via an adhesive component attached to the cell culture surface 22 of the container wall 21 and grow or proliferate for the first time. Therefore, not only the positional relationship between the adherent cells and the cell culture surface 22 is maintained, but the adhesion components produced by the cells are concentrated around the adherent cells and the cell culture surface 22 in the vicinity thereof without diffusing to the surroundings. It is desirable to hold it.

  According to the culture method of the present embodiment, by regulating the fluidity of the medium, the positional relationship between the adhesive cells and the cell culture surface 22 is maintained, and without spreading the adhesive components produced by the cells to the surroundings, By concentrating and holding the adherent cells and the vicinity of the cell culture surface 22 in the vicinity thereof, the adherent cells can be adhered and cultured even on the culture surface to which the adherent cells are difficult to adhere.

  On the culture surface where the adherent cells are difficult to adhere, even in the process where the adherent cells adhere and proliferate, the adhesive force is weak, and if the adherent cells adhere to the cell culture surface 22, the cell culture surface 22 Therefore, if the liquid container 6 in the vicinity of the cell culture surface 22 flows, the cells adhered to the cell culture surface 22 will receive a large frictional force or shearing force from the liquid container 6. Adhered cells are easy to peel off. In addition to this, when the culture container is a bag-like container, the deformation of the container wall 21 accompanying the flow of the liquid container 6 further promotes cell detachment.

In particular, in the later stage of culture, cells proliferate and gather, cell adhesion progresses, and a film-like cell aggregate is formed on the cell culture surface 22. On the culture surface to which the adherent cells are difficult to adhere, the adhesive force between the adherent cells and the cell culture surface 22 is weaker than the cell-cell adhesive force. When subjected to a force or a shearing force, the force concentrates on the end of the membrane-like cell aggregate, and falls off from the end while being engulfed to form an unusable cell mass.
According to the culturing method of the present embodiment, by regulating the fluidity of the medium, it is possible to perform culturing while maintaining an available cell form without peeling until the late stage of culturing.

In addition, since the adhesiveness of the adherent cells to the cell culture surface 22 varies depending on the cell type and the material of the cell culture surface 22, the degree of flow of the liquid container 6 affected by the adhesive cells varies. Therefore, in the culture method according to the present embodiment, the flow of the liquid container 6 in the vicinity of the cell culture surface 22 is prevented if the following two points are clear as compared with the culture method that does not prevent or limit the deformation of the container wall 21. Or it can be considered restricted.
The first point is that the fluidity of the liquid container 6 is lowered, and the second point is that the adherence or proliferation of cells to the cell culture surface 22 rather than the fluidity of the liquid container 6. Or the peel resistance of the cells adhered to the cell culture surface 22 may be superior.

  In the present embodiment, the incubation is performed while maintaining the state in which the internal pressure of the bag-like container 2 is increased as described above. Here, the shape holding means 1 in which the bag-like container 2 is set is left in an incubator so that the placement surface 31 is substantially horizontal, and is held at a predetermined temperature for a predetermined time. As a result, the adherent cells are adhered and cultured on the cell culture surface 22 which is the inner surface side of one container wall 21 brought into contact with the placement surface 31.

During the incubation period, various operations may be performed by moving the bag-like container 2 such as observing the shape of the cell, the state of the cell, the progress of the culture, etc. with a microscope. Even in such a case, it is preferable that various operations can be performed while the state in which the bag-like container 2 is set in the shape holding means 1 is maintained.
Here, the incubation means that the culture vessel is left in the incubator together with the cells and the culture solution, and the incubation period refers to the period from the seeding of the cells to the completion of the series of incubations. This means that the period in which the temporary incubation is interrupted by observation of cells, addition or replacement of a medium or an additive, etc. is also included.

  As a normal culture method in this embodiment, after the adherent cells adhere to or grow on the cell culture surface 22 of the bag-like container 2 after incubation, the grown or proliferated adherent cells are taken out, or after a lapse of a predetermined period. The liquid medium is added or changed, and the continuous incubation is repeated one or more times and then removed. The liquid medium is added by connecting a syringe containing a new liquid container 6 containing a liquid medium or the like to the connection portion 25 of the tube 24 of the bag-like container 2, and placing the liquid container 6 in the bag-like container 2. Slowly infuse.

  In addition, the liquid medium is exchanged by connecting an empty syringe to the connection portion 25 of the tube 24 of the bag-like container 2, slowly extracting the liquid container 6 of the bag-like container 2 through the tube 24, and then the liquid medium. The syringe containing the new liquid container 6 containing etc. may be connected to the connection part 25 of the tube 24 of the bag-like container 2 and the liquid container 6 may be slowly injected into the bag-like container 2. .

  In addition, the adherent cells are taken out by connecting an empty syringe to the connecting portion 25 of the tube 24 of the bag-like container 2, slowly extracting the liquid container 6 of the bag-like container 2 through the tube 24, and then storing the liquid. The syringe containing the cell detachment liquid as the object 6 is connected to the connection portion 25 of the tube 24 of the bag-like container 2 and slowly injected into the bag-like container 2 so that the cell detachment liquid spreads uniformly on the cell surface. Then, after waiting for the cells to detach from the cell culture surface 22 and the connection between the cells to break, that is, when the cells are divided into individual cells or have a uniform spheroid shape, The syringe containing the liquid is connected to the connection portion 25 of the tube 24 of the bag-like container 2, and the cell-dispersing liquid is injected into the bag-like container 2 through the tube 24 to disperse the cells and then the bag-like shape. Container 2 contains liquid 6 may be Nukidase the syringe.

In addition, when cells are detached unintentionally during the addition or exchange of liquid medium by the above method, or when an unintentional cell mass is formed during cell removal, it is more reliable. As a method for preventing separation, the bag-like container 2 is inverted together with the shape holding means 1 so that the cell culture surface 22 faces up, and then a syringe containing a certain amount of sterile air is connected to the connection portion of the tube 24 of the bag-like container 2. 25, injecting air into the bag-like container 2 to separate the liquid surface of the liquid container 6 of the bag-like container 2 from the cells adhered to the cell culture surface 22, and again reversing the cell culture surface After performing the operation of returning so that 22 is at the bottom, an operation of extracting an air from the bag-like container 2 by connecting an empty syringe to the connecting portion 25 of the tube 24 of the bag-like container 2 may be added.
The adherent cells recovered in a dispersed state in this way are then used after thoroughly washed with, for example, a washing solution.

[Function and effect]
According to the adhesive cell culture method of the present embodiment as described above, the flow of the liquid container 6 in the vicinity of the cell culture surface 22 is regulated by pressing the bag-like container 2 to increase the internal pressure of the container. Since various culture operations can be performed while maintaining this state, even in a bag-like container having a cell culture surface 22 to which adherent cells are difficult to adhere, adherent cells can be easily cultured.

  For example, in the initial stage of culture, by controlling the fluidity of the medium, the positional relationship between the adherent cells and the cell culture surface 22 is maintained, and the adhesive components produced by the cells are not diffused to the surroundings. By concentrating and holding around the cell culture surface 22 in the vicinity thereof, the adherent cells can be adhered and cultured even on a culture surface to which the adherent cells are difficult to adhere.

  During the incubation period, various operations are performed by moving the bag-like container 2 such as observing the shape of the cells, the state of the cells, the progress of the culture, etc. with a microscope. Various operations can be performed while maintaining the state in which the bag-like container 2 is set in the state, that is, while maintaining the state in which the cells are not easily detached.

  Also, in the later stage of culture, by controlling the fluidity of the culture medium, it is possible to suppress detachment while curling up from the end of the membrane-like cell aggregate and maintaining the usable cell morphology. Can do. In addition, the frictional force and shearing force applied to the cells by the discharge of the liquid container 6 and the flow of the liquid container 6 by the injection operation can be minimized in the operation of adding the medium, exchanging the medium, and taking out the cells.

In this culturing method, the cell culture surface 22 may be a surface to which adherent cells are difficult to adhere, or may be a surface made of an unmodified resin that has not been treated to improve cell adhesion. Since it is good, manufacture of the bag-shaped container 2 is easy.
Further, the deformation of the cell culture surface 22 is restricted and the flow of the liquid container 6 in the vicinity of the cell culture surface 22 is restricted only by pressing the bag-like container 2 between the pressing surface 42 and the mounting surface 31. Therefore, the configuration of the shape holding means 1 can be simplified. For this reason, it is possible to easily cultivate adherent cells without taking time and effort for preparation of adherent cells.

In addition, the said embodiment can be suitably changed within the scope of the present invention.
For example, the configuration of the shape holding means 1 can be appropriately changed as long as it includes a mounting surface 31 on which the container wall 21 is mounted and a pressing surface 42 that presses the container wall 21 and raises the internal pressure.
In the above, the mounting surface 31 is fixedly provided on the first member 3 and the pressing plate 41 having the pressing surface 42 is provided so as to be displaceable. However, the pressing surface 42 is fixed and provided. The mounting surface 31 and the pressing surface 42 may be both urged by being provided so as to be displaceable.

In the above description, the mounting surface 31 and the pressing surface 42 are provided on separate members. However, the part that functions as the mounting surface 31 and the part that functions as the pressing surface 42 may be provided on an integral member. .
In the above embodiment, an example has been described in which one shape holding means 1 holds one bag-like container 2 and culture is performed, but the number of shape holding means and the number of bag-like containers can be arbitrarily set. It is. For example, a plurality of shape holding means may each hold one bag-like container. In that case, the shape holding means may be arranged in any way.

It is also possible to hold a plurality of bag-like containers on one shape holding means. In that case, a plurality of placement surfaces may be provided on the shape holding means, a bag-like container may be placed on each placement surface, and a pressing surface may be provided on each of the placement surfaces to press the bag-like container.
Further, when a plurality of shape holding means are provided, or when a plurality of placement surfaces are provided on the shape holding means, a plurality of shape holding means and placement surfaces within a range that does not hinder gas supply and discharge of each bag-like container. It is also possible to arrange them in a stacked manner.

  In the said embodiment, although what has a flat inner surface shape was illustrated as one container wall 21 which comprises the cell culture surface 22 of the bag-shaped container 2, a some recessed part is provided in the inner surface of the container wall 21, and each The bottom surface of the recess may constitute the cell culture surface 22.

Examples of the present invention will be described below.
[Example 1]
Adhesive cells were cultured using the bag-like container 2 and the shape holding means 1 as shown in FIGS.

(Culture preparation)
Normal human neonatal foreskin fibroblasts (Kurabo Co., Ltd.) were used as the adherent cells.
The liquid medium was prepared by adding fetal bovine serum to 5 wt% using a fibroblast synthetic medium EIDF (+) (trade name, manufactured by Nipro Co., Ltd.).

The bag-like container 2 has a pair of container walls 21 made of a 0.14 mm-thick sheet formed by inflation molding of an ethylene vinyl acetate copolymer (Ultrasen 630 (registered trademark) manufactured by Tosoh Corporation). The maximum width and maximum length of the accommodation space formed between 21 and 21 was 5 cm × 10 cm, and the area of the cell culture surface 22 was about 50 cm ² . The cell culture surface 22 was not surface-treated, and the water contact angle of the cell culture surface 22 was 93 degrees.

(Cell seeding)
In the bag-like container 2, 25 ml of the liquid container 6 is sealed in the liquid medium in advance, and the cell suspension is injected with a syringe, so that the adherent cells are adjusted so that the cell seeding concentration is 5000 cells / cm ^2. At the same time, the mixed air was removed so that no air remained in the bag-like container 2. At this time, the dimension between the two container walls 21 and 21 of the bag-shaped container 2 was an outer dimension of about 5 mm.

  The bag-like container 2 was set on the shape holding means 1 while being laid sideways, and the pair of container walls 21 were pressed by the placement surface 31 and the pressing surface 42. In the set state, the internal pressure of the bag-like container 2 was a 300 mm water column.

(Culture process)
The bag-like container 2 was placed in the incubator while being set in the shape holding means 1, and incubated for 4 days under humidification at 37 ° C., a carbon dioxide concentration of 5%, and a humidity of 95% or more.
During the incubation period, the bag-like container 2 was set in the shape holding means 1 and was taken out of the incubator and observed for cell adhesion using a microscope.
During the incubation period, the medium was changed once by the normal culture method in the present embodiment described above. On the third day from the start of culture, 20 ml of the liquid container was extracted and discarded, and 20 ml of fresh medium was newly injected.

After the incubation period, cells were taken out by the method of inverting the bag-like container 2 among the methods described above.
The bag-like container 2 is slowly inverted together with the shape holding means 1 so that the cell culture surface 22 faces up, and then a syringe containing 10 ml of sterile air is connected to the connection portion 25 of the tube 24 of the bag-like container 2. After injecting air into the bag-shaped container 2, the liquid surface of the liquid container 6 in the bag-shaped container 2 is separated from the cells adhered to the cell culture surface 22, and then the liquid in the bag-shaped container 2 is passed through the tube 24. After slowly removing the contents 6, a syringe containing 3 ml of trypsin (0.25% Trypsin-EDTA Gibco) diluted 10-fold with PBS as a cell detachment solution is connected to the connection portion 25 of the tube 24 of the bag-like container 2. The cell detachment solution was slowly injected into the bag-like container 2. Next, the cell detachment liquid and the cells were brought into contact with each other so that the cell culture surface 22 was returned to the lower side, that is, the cell detachment solution spread slowly and uniformly on the cell surface.

  After 1 minute, a syringe containing 10 ml of PBS as a cell dispersion liquid is connected to the connection portion 25 of the tube 24 of the bag-like container 2, and PBS is slowly injected into the bag-like container 2 to disperse the cells. Then, it was extracted into a syringe and collected.

(Culture result)
FIGS. 4 (a) and 4 (b) show micrographs taken 24 hours after cell seeding and immediately before 4 days after cell seeding. Table 1 shows the evaluation of cell growth state and cell concentration.
From FIG. 4 (a), 24 hours after cell seeding, most of the cells adhered to the cell culture surface, and no cell aggregation was observed. Moreover, although it became confluent immediately before the cell collection | recovery 4 days later from FIG.4 (b), what peeled while curling and which peeled and became a cell lump was not seen. For this reason, the cells could be collected in a dispersed state by the trypsin recovery operation, and the number of cells could be counted. From Table 1, the total number of collected cells is sufficiently increased from the time of cell seeding, and it can be confirmed that the cells proliferated smoothly.

[Comparative Example 1]
Without using the shape holding means 1, the bag-like container 2 in which the liquid container 6 was enclosed was placed on a flat acrylic plate without being pressed and cultured. Therefore, the adherent cells were cultured in the same manner as in Example 1 except that the medium exchange on the third day and the recovery operation after 4 days were not performed.

Micrographs taken 24 hours after cell seeding and 4 days after the cell recovery are shown in FIGS. 5 (a) and 5 (b). Table 1 shows the evaluation of cell growth state and cell concentration.
From FIG. 5 (a), some cells adhere to the cell culture surface 22 24 hours after cell seeding, but the number is small, and they float without adhering to the cell culture surface 22. Some also formed. Further, from FIG. 5 (b), after 4 days, no adherent cells were seen, and agglomerates of slightly larger cells were sparsely seen. From a quantitative point of view, this was thought to be a result of agglomeration of the seeded cells rather than a result of cell mass growth.
In addition, since this cell mass was not disperse | distributed to each cell even if it processed with a trypsin, it did not reach to specification of a cell number.

DESCRIPTION OF SYMBOLS 1 Shape holding means 2 Bag-shaped container 3 1st member 4 2nd member 5 Lid body 6 Liquid container 21 Container wall 22 Cell culture surface 23 Port 24 Tube 25 Connection part 31 Placement surface 32 Draw-out port 41 Holding plate 42 Holding surface 43 Biasing member 44 Pin 45 Ventilation hole 51 Protrusion receiver 52 Protrusion

Claims (3)

A culture method for culturing adhesive cells by enclosing a liquid container containing adhesive cells and a liquid medium in a bag-like container having a container wall to be a cell culture surface,
The cell culture surface has a low density polyethylene resin, a low density linear polyethylene, an ethylene / vinyl acetate copolymer, or a mixture thereof. Use without surface treatment to improve,
The bag-like container is set in a shape holding means comprising a first member having a placement surface for placing the container wall and a second member having a pressing surface for holding the container wall opposite to the container wall,
The bag-like container is pressed by the placement surface and the pressing surface to increase the internal pressure of the container, and by increasing the internal pressure, the flow of the liquid container is suppressed, and the culture is performed while maintaining this state. Thus, the method for culturing adhesive cells is characterized in that the adherent cells are promoted to adhere to the cell culture surface, or the adhered cells are prevented from peeling off, thereby improving the culture performance. 2. The method for culturing adhesive cells according to claim 1 , wherein a contact angle of the cell culture surface with water is in the vicinity of 90 ° or 90 ° or more. By maintaining the cell culture surface in contact with the plane-shaped mounting surface and maintaining substantially horizontal, the adhesive component produced by the adhesive cells is concentrated and held around the cell culture surface in the vicinity of the adherent cells. The method for culturing adherent cells according to claim 1 or 2, wherein the culture is performed while the cells are cultured.