JPH0677517B2 - Floating cell culture device - Google Patents

Description

TECHNICAL FIELD The present invention relates to a suspension cell culture device.

[Problems to be solved by conventional techniques and inventions]

In recent years, with the progress of biotechnology, various devices for culturing and utilizing microorganisms, cells, etc. have been proposed.

For example, as a device for culturing floating cells, a semi-batch reactor in which a reactor that requires batch operation is partially continuous, and a tank reactor such as a continuous tank reactor in which batch operation is improved to continuous operation, A tubular reactor suitable for continuous operation has been proposed and used.

The above-mentioned improvement has a certain effect in that the batch operation is semi-continuous and continuous, but in any case, suspension cells and medium and other necessary components (for example, physiologically active substances such as serum and lymphokine) Is a single system, and the desired cells and useful products are obtained by stirring the system.

Since cells and useful products obtained by culturing are present together with a large amount of medium, it is not easy to separate and purify the target substance, and how to increase the concentration of the cells and useful products, and An important issue is how to reduce the amount of medium that interferes with the operation of extracting useful products.

As a device for solving such a problem, there has been proposed a device for culturing cells or cells in a bag made of a membrane that does not permeate cells or cells provided inside the culture tank but allows the medium to permeate. (JP-A-62-122580).

By using this device, the effect of reducing the labor of separation and purification from a large amount of medium can be achieved by taking out the medium from the bag, and some results can be obtained, but the medium inside and outside the bag is obtained. There problem necessarily less likely to identical conditions, especially stability of important pH in the case of the culture of animal cells, HCO ₃ ^- levels maintain (depending on the cell type, usually 2
6 to 32 m ep / is preferable), and the concentration of cells that can be cultured is limited.

[Means for Solving the Problems]

In view of the problems as described above, the present invention has been made for the purpose of particularly increasing the concentration of floating cells to be cultured and reducing the ratio of the medium in the solution to be separated and purified as much as possible. A cell culture device in which a cell culture means and a gas exchange means are housed and arranged in a culture medium container to be provided, wherein the culture medium container is provided with a culture medium supply means and a culture medium extraction means, The flexible bag which is permeable to the culture medium but impermeable to the serum and the physiologically active substance, the frame body having a space through which the culture medium for supporting the bag can freely pass, and the bag body And a means for supplying cells and serum or a cell activator and collecting cells and products, wherein the gas exchange means has a hollow fiber bundle and a gas passage void for supporting the hollow fiber bundle. Is it a frame It made possible regarding suspension cell culture apparatus according to claim.

[Work]

In the device of the present invention, a specific cell culture means is arranged in the medium container, and cell culture is performed therein, so that cells, serum and cell activator and a large amount of medium do not form one system.

Further, since the gas exchange means using the hollow fiber bundle is arranged in the culture medium container, the gas exchange with the culture medium can be performed efficiently and easily, and the culture medium can be kept in a certain preferable state.

As a result, floating cells can be cultured at a high density of 10 ⁷ to 10 ⁸ cells / ml, and separation and purification of the cultured cells becomes easy. Further, the culture device can be made compact.

〔Example〕

The floating cell culture device of the present invention will be described based on FIG. 1, which is an explanatory view of one embodiment thereof.

The floating cell culture device (4) of the present invention has a medium container (1) for containing a medium, a cell culture means (2) and a gas exchange means (3) housed and arranged in the container.

The medium container (1) corresponds to a so-called culture bottle in a conventional floating cell culture device,
The size, shape, material, etc. are not particularly limited, and any conventional culture bottle having the same size, shape, material, etc. can be used.

As a preferred specific example of such a container (1), for example, in the case of a small one having a capacity of about 500 to 5000 ml, a bottom area of 50 to 50 formed of hard glass, synthetic resin, metal or the like is used.
Examples include a cylindrical container having a height of about 10 cm to 30 cm and a columnar container, a spherical container having a height of about 300 cm ² .

In such a small-sized device, the medium supply means (5), the medium extraction means (20), the cell culture means (2), the gas exchange means (3), etc. are usually attached to the mouth of the container, It is preferably broad. The remaining portion of the mouth portion provided with these means is sealed with a stopper, a lid, etc. to prevent contamination with germs and the like.

If the capacity is about 5 or more, medium to large size,
A cylindrical container with a diameter of about 0.15 to 2 m and a height of about 0.3 to 2 m formed from synthetic resin or metal, a prismatic container,
Examples include spherical containers.

In the case of such a large container, the medium supply means (5),
It is not necessary to provide the medium withdrawing means (20), the cell culture means (2), the gas exchange means (3), etc. on the stopper or lid of the container, but rather, when they are attached to these, opening and closing of the container When it becomes necessary, it is difficult to handle,
For the reason that it is not preferable to suspend the heavy cell culture means (2) or the gas exchange means (3), it is usually mounted on the bottom with a mounting base or attached on the side. It is preferable to provide and attach it.

The cell culture means (2) comprises a flexible bag (6) that allows the medium to permeate but not the suspended cells, but does not allow serum or a physiologically active substance to permeate, and a medium for supporting the bag (6). It comprises a means (8) for supplying floating cells and the like to a frame (7) and a bag (6) having freely permeable voids and recovering the floating cells and their products.

The number of cell culture means (2) housed and arranged in the medium container (1) does not have to be one, and may be two or more, depending on the size of the medium container (1). .

As described above, the bag body (6) does not allow suspended cells, serum or physiologically active substances to permeate therethrough, but the discharge of the medium or floating cells contained in the culture medium container (1), particularly the discharge to be discarded, is It is a bag-shaped container consisting of a membrane that permeates and supplies the nutrients necessary for the floating cells, oxygen and carbon dioxide gas, and removes unnecessary substances.

The size and shape of the bag (6) are not particularly limited, but it is preferably about 10 to 50% of the capacity of the culture medium container (1), and the contact area with the culture medium is preferably large. .
In addition, when two or more cell culture means (2) are provided, it is preferable that the total volume of the bag body (6) falls within the above range. Further, since the bag body (6) is supported inside the frame body (7) having a void through which the medium for supporting the bag body can freely pass, the shape of the inner side of the frame body (7) It is preferably about the same as the capacity.

Since the bag body (6) is permeable to the medium but impermeable to cells, serum and physiologically active substances, it usually has an average pore size of 30 to 80 Å depending on the types of the permeable substance and the impermeable substance. It is formed from a film having a degree of pores.

The material forming the membrane is not particularly limited, but it is necessary that the surface has hydrophilicity because it needs to allow the medium to permeate. Therefore, a preferable specific example of the material forming the membrane is a hydrophilic material such as regenerated cellulose. In the case of a membrane made of a non-hydrophilic material such as cellulose triacetate, cellulose diacetate, polypropylene, polyethylene, polytetrafluoroethylene, polysulfone, polyamide or polyester, for example, it is immersed in ethanol or a hydroxyl group is grafted onto the surface of the membrane. The membrane can be made hydrophilic, such as by polymerisation.

A frame (7) having a void through which the medium can freely pass
Since the bag body (6) does not have sufficient strength and is formed of a film that deforms with a slight force, it supports and supplements this and also supplies sufficient medium to the bag body (6). It was designed so as not to interfere with being played.

There is no particular limitation on the shape, size, material, etc. of the frame body (7), which surrounds and supports the bag body (6) and protects it, and the medium is freely supplied to the bag body (6). It can be used as long as it is volatile. A preferred specific example of such a frame body (7) is, for example, a bag body (6).
It has a shape such as a cylindrical shape, a prismatic shape, a spherical shape, or the like that can support the inside of the bag well, and is formed to have a size that is approximately the same as the size of the bag body (6) to a slightly larger size, a slit shape, It has voids in the shape of a lattice, a circle to an ellipse. It is preferable that the voids are substantially even, that is, the voids having the same shape and the same size are formed at equal intervals.

(8) in FIG. 1 is a means for supplying floating cells, serum or a cell activating substance to the bag to collect the proliferated cells and the physiologically active substance which is a product thereof. Although not particularly limited, for example, one using an injection plug can be cited, and floating cells and the like can be easily collected when the lower end thereof reaches near the lowermost portion of the bag (6). Further, in FIG. 1, the supply and recovery of the floating cells and the like are performed by one tube, but they may be continuously performed by using different tubes.

The gas exchange means (3) includes a gas supply line (19) and a gas discharge line (18), supplies a gas necessary for culture, for example, O _2, and produces a gas by the culture,
For example, as a means for discharging CO ₂ , it has a hollow fiber bundle (9) and a gas passage space (10) for supporting the hollow fiber bundle (9), as shown in detail in FIG. It consists of a frame (11).

The hollow fiber constituting the hollow fiber bundle (9) is not particularly limited as long as it has a gas exchange ability, but for example, the average pore size is about 30Å to 1 µm, and further 30Å to 0.
Hollow fibers of about 2 μm are preferably used.

The material forming the hollow fiber is not particularly limited, and examples thereof include regenerated cellulose, cellulose triacetate,
Cellulose diacetate, polypropylene, polyethylene, polytetrafluoroethylene, polysulfone, polyamide, polyester and the like are usually used.

The hollow fiber bundle (9) is used for gas exchange,
In some cases, it may be used not only for gas exchange but also for medium extraction. In this case, the medium needs to permeate the hollow fibers.Therefore, in the case of hollow fibers made of hydrophilic material or non-hydrophilic material, it is necessary to use hollow fibers having a hydrophilic surface. is there.

Although the hollow fibers differ depending on the volume of the medium, when the volume is 1000 ml, about 300 to 600 fibers are usually bundled and both ends are potted with, for example, urethane resin to form a hollow fiber bundle (9).

The cross-sectional shape, length, etc. when the hollow fibers are bundled may be appropriately selected, but it is preferable that the contact area with the medium is large, and a cross-sectional shape having a circular shape is usually employed.

The frame body (11) having the gas passage space (10) for supporting the hollow fiber bundle (9) does not have a constant shape by itself, and is intended to facilitate the handling of the difficult to handle hollow fiber bundle (9). Therefore, it is preferable that the gas exchange with the medium and the passage of the medium easily occur depending on the situation, and the shape and size are not particularly limited as long as such a purpose can be achieved. There is no. Therefore, the shape of the gas passage space (10) existing in the frame body (11) may be wide and long strip-like space, as shown in FIG. It may be a void having a circular shape, an elliptical shape, or the like.

It should be noted that the term "for gas passage" does not mean that only the gas passes therethrough, but the medium and the hollow fibers need to come into contact with each other.

For example, in the gas exchange means shown in FIG. 2, when a gas for gas exchange is supplied from above, the gas consumed by the culture has a high partial pressure in the hollow fiber, so that the gas dissolves into the medium. On the other hand, since the dissolved amount of the gas discharged by the culture is larger than that against the partial pressure of the gas in the hollow fiber, the gas is discharged from the medium into the hollow fiber and gas exchange is performed. At this time, if the surface of the hollow fiber is hydrophilic, the medium will penetrate into the hollow fiber if the pressure of the exchange gas is smaller than the water pressure, so when performing gas exchange, The pressure of the replacement gas needs to be greater than the water pressure. Further, when the gas pressure for exchange is made lower than the water pressure, the medium (drainage) permeates the hollow fibers and is discharged to the outside, so that the gas exchange means (3) also serves as the medium extraction means. Become.

The gas exchange means (3) provided in the culture medium container (1) is 1
The number may be one, but if necessary, two or more may be provided.

It goes without saying that the parts of the cell culture means (2) and the gas exchange means (3) where medium exchange and gas exchange occur must be present in the medium. Further, the supply of the medium from the medium supply means (5) is preferably performed from the upper part of the medium container (1).

The apparatus of the present invention does not necessarily need stirring means,
As shown in FIG. 1, when the agitating means (12) is provided to agitate the medium, gas exchange between the medium and the gas exchanging means is facilitated, and the medium is easily maintained in a constant state.
In addition, the medium easily permeates the bag (6). Further, since the medium is stirred, even if it is strongly stirred, the cells are not damaged. Furthermore, since the bag body (6) is constantly shaken to facilitate the floating of the cells, the cells are prevented from adhering to the surface of the bag body (6), and the cells are easily proliferated.

It should be noted that (13) in FIG. 1 is a degassing means for initially supplying the medium, and (14) in FIG. 2 is a potting portion.

Although FIG. 1 does not show a means for monitoring the state of the medium during culturing, it should be understood that it can be installed at any time.

The cells cultured by the device of the present invention include, for example, lymphocytes, bone marrow cells, macrophages, and the like, and cell-activating substances used in that case include lymphokineii and interferon, and the medium further includes, for example, Examples thereof include fetal bovine serum medium, serum-free medium and human serum medium.

Next, the device of the present invention will be described based on examples.

Example 1 The apparatus shown in FIG. 3 was used as the gas exchange means in the apparatus of the present invention as a gas exchange means having gas exchange and medium extraction functions.

The medium container (1) in FIG. 3 is made of hard glass and has a capacity of 33.
Cylindrical container of 00 ml, diameter of about 150 mm x height of about 190 mm, cell culture means (2) is a regenerated cellulose membrane with an average pore size of 40 Å, inner volume of 300 ml, height of 150 mm, diameter of 50 mm and surface area of 230 cm ²
(6), which is supported by a frame (7) having a substantially columnar shape, and has a pipe at the upper part of the bag body that reaches the lower part of the bag body. ) Is provided. Gas exchange and medium extraction means (1
5) is the average pore size made of cellulose triacetate
300 hollow fibers of 0.1 μm bundled and potted to an effective length of 120 mm (total surface area 250 cm ² ) has a cylindrical shape with an inner diameter of 20 mm and a side surface of approximately 80 mm × 10
It is supported by a frame with two wide strip-shaped voids of mm, with a gas supply port in the upper part and a gas exhaust / medium discharge port in the lower part (see Fig. 2). ). Then, the cell culture means (2), the gas exchange / medium extraction means (15), the medium supply means (5), and the 0.2 μm air filter (16) are provided on the lid that shuts off the medium container (1) from the outside. Degassing means (13) is arranged,
A stirring means (12) is provided. An air pump (17) connected to a gas supply line (19) having an air filter (0.2 μm) (16) is provided at the gas supply port, and an exhaust gas and a medium are discharged from the gas exhaust / medium exhaust port to the outside. A line (21) is provided for discharging the medium.

RPMI-1640 as a culture medium with the degassing means (13) open
After being supplied with 1000 ml (manufactured by Ajinomoto Co., Inc.), lymphocytes
About 110 ml of a cell suspension containing 10 ⁶ cells / ml × 100 ml, 10 ml of serum, and 200 units of lymphokine was supplied to the bag body (6).

Among them, the culture was carried out at 37 ° C. with stirring at a speed of 200 rpm. During this time, the medium is supplied from the medium supply means (5).
The cells were supplied at a rate of 1 ml / min and at the same time, air containing 5% by volume of CO ₂ was supplied at a flow rate of 10 ml / min using an air pump, and the cells were cultured for 20 days.

During the culture, the pH of the medium and the carbon dioxide partial pressure (PCO ₂ ) were measured every day, and it was confirmed that 7.2 ≦ pH ≦ 7.3 and 36 mmHg ≦ PCO ₂ ≦ 40 mmHg.

The culture results are shown in Table 1.

[Effects of the Invention] By using the device of the present invention, it is possible to stably maintain a constant medium condition even during cell culture. Further, since gas exchange is performed by a gas exchange means using a hollow fiber bundle in a medium in which no cells are present, cell damage caused by bubbling does not occur. In addition, cells, serum, and physiologically active substances are kept in the bag, which facilitates separation and purification of cultured cells and products. Further, the exchange of the medium and the collection of the cultured cells and the products thereof can be performed aseptically and continuously. And since the serum and the cell activating substance are retained in the bag, the consumption thereof is reduced.

As described above, by using the device of the present invention, high-concentration cultured cells and products can be efficiently obtained, so that the device can be made compact.

[Brief description of drawings]

FIG. 1 is an explanatory view relating to an embodiment of the device of the present invention, and FIG.
FIG. 3 is an illustration of an example of gas exchange means used in the device of the present invention, and FIG. 3 is an illustration of the device of the present invention used in the first embodiment. (Main symbols in the drawings) (1): Medium container (2): Cell culture means (3): Gas exchange means (4): Cell culture device (5): Medium supply means (6): Bag (7): Frame for supporting the bag (8): Supplying and collecting means for cells (9): Hollow fiber bundle (10): Gas passage void (11): Frame for supporting the hollow fiber bundle ( 15): Gas exchange and medium extraction means (20): Medium extraction means (21): Gas exhaust and medium extraction line

Claims (5)

[Claims] 1. A cell culture device in which a cell culture means and a gas exchange means are housed and arranged in a medium container for containing a medium, wherein the medium container is provided with a medium supply means and a medium extraction means. The cell culture means is provided with a flexible bag body which is permeable to a medium provided therein but is impermeable to serum and a physiologically active substance, and a space through which the medium for supporting the bag body can freely pass. A frame body, and means for supplying cells and serum or a cell activating substance to the bag body and for collecting cells and products, wherein the gas exchange means supports a hollow fiber bundle and the hollow fiber bundle. A floating cell culture device, comprising a frame having a gas passage space for 2. A flexible bag body in the cell culture means,
Cellulose triacetate with an average pore size of 30-80Å,
The device according to claim 1, which is a bag made of a membrane made of cellulose diacetate, regenerated cellulose, polypropylene, polyethylene, polytetrafluoroethylene, polysulfone, polyamide or polyester. 3. The hollow fiber bundle in the gas exchange means is a hollow fiber bundle made of cellulose triacetate, cellulose diacetate, regenerated cellulose, polypropylene, polyethylene, polytetrafluoroethylene, polysulfone, polyamide or polyester. Equipment. 4. The apparatus according to claim 1, wherein the gas exchange means also serves as the medium extraction means. 5. The flexible bag in the cell culture means comprises:
Cellulose triacetate with an average pore size of 30-80Å,
Cellulose diacetate, regenerated cellulose, polypropylene, polyethylene, polytetrafluoroethylene, polysulfone, a bag made of a membrane made of polyamide or polyester, the gas exchange means, cellulose triacetate, cellulose diacetate, regenerated cellulose, polypropylene, polyethylene 2. The device according to claim 1, which is a hollow fiber bundle made of polytetrafluoroethylene, polysulfone, polyamide or polyester, and which also serves as a medium extracting means.