JPH0257174A - Device for culturing suspended cell - Google Patents

Abstract

PURPOSE:To obtain a device for culturing suspended cells, equipped with a special cell culturing means and gas exchanging means in a vessel for containing a culture medium and capable of readily separating and collecting cultured cells or products without causing bubbling. CONSTITUTION:A device constituted of a cell culturing means and a gas exchanging means in a vessel 1 for containing a culture medium. The cell culturing means is constituted of a flexible bag body 6 capable of permeating the culture medium without permeating a physiologically active substance, a frame body 7 supporting the bag body and a means 8 for feeding cells, blood serum or cell activator, and collecting the cells or products. The gas exchanging means is constituted of a hollow yarn bundle 9 and a frame body 11 for supporting the hollow yarns in a state of formed gaps for passing gases therethrough between the hollow yarns.

Description

[Detailed description of the invention] [Industrial application 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 devices for culturing suspended cells, there are semi-batch reactors, which are partially continuous reactors that require batch operations, and seed reactors, such as continuous tank reactors, which have improved batch operations into continuous operations. Tubular reactors suitable for continuous operation have been proposed and used.

The above improvements have some effect in making batch operations semi-continuous or continuous, but both of them have the effect of converting suspended cells, culture medium, and other necessary components (e.g., serum, physiologically active substances such as lymphokines, etc.) This is a method in which desired cells and useful products are harvested by forming a single system and stirring the system.

Since cells and useful products obtained through culture exist together with a large amount of medium, it is not easy to separate and purify the target product, and it is difficult to increase the concentration of the cells and useful products. An important issue is how to reduce the amount of media and other materials that interfere with the removal of useful products.

As a device to solve these problems, a device has been proposed that cultivates bacteria or cells in a membrane bag that is provided inside a culture tank and does not allow the bacteria or cells to pass through, but allows the culture medium to pass through. (Japanese Unexamined Patent Publication No. 82-122580).

By using this device, the labor of separation and purification from a large amount of culture medium can be reduced by taking out the contents of the bag, and the results may be improved, or the culture medium inside the bag and outside the bag can be separated. The problem is that it is difficult to maintain the same conditions, especially in the case of culturing animal cells, the stability of pl and the maintenance of II 003-level (depending on the cell type, usually 36 to 40 m eq/ρ) There is a problem that it is difficult to measure (preferably), and therefore there are restrictions on the concentration of cells that can be cultured.

[Means to solve the problem]

In view of the above-mentioned problems, the present invention was made with the aim of increasing the concentration of suspended cells to be cultured and minimizing the proportion of the medium in the solution to be separated and purified. A cell culture bag device in which a cell culture means and a gas exchange means are housed in a culture medium container containing a medium, wherein the culture medium container is provided with a medium supply means and a medium removal means, and the cell culture means However, a flexible bag that allows the medium provided inside to pass through but not serum or physiologically active substances, a frame that supports the bag and has a gap through which the medium can freely pass, and the above-mentioned The gas exchange means includes means for supplying cells, serum, and cell activating substances to the bag and recovering cells and products, and the gas exchange means includes a hollow fiber bundle and a gas passage for supporting the hollow fiber bundle. The present invention relates to a floating cell culture device characterized by comprising a frame having voids.

[For production]

In the apparatus of the present invention, a specific cell culture means is placed in a medium container and cell culture is carried out therein, so that cells, serum, cell activating substance, and a large amount of medium are not combined into one system.

Furthermore, since a gas exchange means using a hollow fiber bundle is disposed in the culture medium container, gas exchange with the culture medium can be performed efficiently and easily, and the culture medium can be maintained in a certain desirable state.

As a result, floating cells can be cultured at a high density of 107 to 108 cells/ml, and the cultured cells can be easily separated and purified. Furthermore, the culture apparatus can be made more compact.

〔Example〕

The suspension cell culture device of the present invention will be explained based on FIG. 1, which is an explanatory diagram of one embodiment thereof.

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

The medium container (1) corresponds to a so-called culture bottle in a conventional suspension cell culture device, and there are no particular limitations on its size, shape, material, etc.
If it is the same size, shape, and material as a traditional culture bottle, you can use it.

Preferred concrete examples of such a container (1) include, for example, a small container with a capacity of about 500 to 5000 ml, a container made of hard glass, synthetic resin, metal, etc., with a bottom area of about 50 to 300 c# and a high Examples include cylindrical containers, prismatic containers, and spherical containers with a length of about 10 to 30 cm.

In such small devices, the medium supply means (5)
Since the cell culture means (2), gas exchange means (3), etc. of the medium extraction means are attached to the mouth of the container, it is preferable that the medium have a wide mouth. The remaining part of the mouth where these means are provided is sealed with a stopper, lid, etc., to prevent contamination by germs and the like.

In addition, for medium to large items with a capacity of about 5g or more,
Diameter 0.15-2 made from synthetic resin or metal
Examples include cylindrical containers, prismatic containers, and spherical containers with a height of approximately 0.3 to 2 m.

A space for such a large container, a medium supply means (5),
The cell culture means (2) and the gas exchange means (
3) etc. are not necessarily required to be attached to the stopper or lid of the container, but if they are attached to these, it will be difficult to handle when the container needs to be opened and closed, and the cell culture will be heavy. Because it is undesirable to hang the means (2) and gas exchange means (3), etc., they are usually mounted on a rack with a stand on the bottom, or with mounting means on the side. It is preferable to do so.

The cell culture means (2) includes a flexible bag (6) that is permeable to a medium or not permeable to suspended cells, serum or physiologically active substances, and a medium for supporting the bag (6). A frame body (sword and bag body) with a void that can freely pass through.
It consists of a means (8) for supplying floating cells and the like to 6) and collecting the floating cells and their products.

Note that the cell culture means (
2) does not need to be one, and may be two or more, depending on the size of the culture medium container (1).

The bag (6) does not allow floating cells, serum, or physiologically active substances to pass through, as described above, but it does not allow the medium or suspended cells contained in the medium container (1) to pass therethrough, especially the waste that should be disposed of. It is a bag-shaped container made of a membrane that permeates through the cell, supplying sufficient nutrients, oxygen, carbon dioxide, etc. necessary for floating cells, and removing unnecessary substances.

There are no particular limitations on the size, shape, etc. of the bag (6), but it is preferably about 10 to 50% of the capacity of the culture medium container (1), and it is preferable that the contact area with the culture medium is large. . In addition, when two or more cell culture means (2) are provided, it is preferable that the total capacity of the bag (6) falls within the above range.

In addition, the bag (6) is a frame with a gap through which a culture medium can freely pass through to support the bag (6). Preferably, they are substantially the same.

The bag (6) needs to be permeable to the medium or impermeable to cells, serum, and physiologically active substances, so it usually has an average pore size of 30 to 80, depending on the type of the permeable substance or impermeable substance. It is formed from a membrane with human-sized pores.

There are no particular limitations on the material constituting the membrane, but since it is necessary to allow the medium to permeate, the surface must be hydrophilic.

Therefore, preferred specific examples of the material constituting the membrane include hydrophilic materials such as regenerated cellulose. In the case of membranes made of non-hydrophilic materials, such as cellulose triacetate, cellulose diacetate, polypropylene, polyethylene, polytetrafluoroethylene, polysulfone, polyamide or polyester, for example, immersion in ethanol or grafting of hydroxyl groups onto the membrane surface is recommended. The membrane can be made hydrophilic by methods such as polymerization.

A frame (7) provided with a gap through which the medium freely passes.
Since the bag body (6) does not have sufficient strength and is made of a membrane that deforms with a slight force, it is necessary to support and compensate for this and to supply a sufficient amount of culture medium to the bag body (6). This was done so that it would not prevent it from being carried out.

There are no particular limitations on the shape, size, material, etc. of the frame (7), and it surrounds, supports, and protects the bag (6), and allows the medium to be freely supplied to the bag (6). It can be used as long as it is moist. A frame like this (7
) is a cylindrical, prismatic, or spherical shape that can effectively support the bag (6) inside, and has a shape that is about the same size as the bag (6). ~It is formed in a slightly thick size, with a slit shape,
It has voids in the shape of a lattice, circular to elliptical, etc. Preferably, the voids are substantially uniform, that is, have the same shape and size, or are equally spaced.

(8) in Figure 1 is a means for supplying floating cells, serum, and cell activating substances to the bag body, and collecting proliferated cells and physiologically active substances that are their products. There is no particular limitation, for example, it may be possible to use an injection plug, and it is easier to collect floating cells etc. if it reaches the bottom end of the injection plug or near the bottom of the bag body (6). Further, in FIG. 1, the supply and recovery of floating cells, etc. are carried out in one tube, but they may be carried out continuously using separate tubes.

The gas exchange means (3) is equipped with a gas supply line 091 and a gas exhaust line 08), and supplies gas necessary for culture, such as 02, and discharges gas generated by culture, such as CO2. For example, as shown in FIG. 2 in detail, it consists of a hollow fiber bundle (9) and a frame body 01) having a gas passage gap 00) for supporting the hollow fiber bundle (9).

The hollow fibers constituting the hollow fiber bundle (9) are not particularly limited as long as they have gas exchange ability; for example, the average pore size is about 30 to 1 μm, and more preferably 30 to 0.2 μm. Hollow fibers of about am are preferably used.

There are no particular limitations on the material constituting the hollow fibers; for example, regenerated cellulose, cellulose triacetate, cellulose diacetate-1, polypropylene, polyethylene, polytetrafluoroethylene, polysulfone, polyamide, polyester, etc. are usually used. Ru.

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 culture medium removal. In this case, it is necessary for the medium to pass through the hollow fibers, so in the case of hollow fibers made of hydrophilic material or hollow fibers made of non-hydrophilic material, it is necessary to use hollow fibers whose surfaces are made hydrophilic. be.

The hollow fiber has a capacity of 1010, although it varies depending on the capacity of the medium.
In the case of 0O, the hollow fiber bundle (9) is usually made by bundling around 300 to 600 fibers and potting both ends with, for example, urethane resin.

The cross-sectional shape, length, etc. of the bundled hollow fibers may be selected as appropriate, but it is preferable to increase the contact area with the culture medium, and a circular cross-sectional shape is usually adopted.

The frame body 01) having a gas passage gap (IQ) that supports the hollow fiber bundle (9) has a certain shape,
Since the purpose is to make the hollow fiber bundle (9), which is difficult to handle, easy to handle, it is preferable to use a material that facilitates gas exchange with the culture medium, and in some cases facilitates passage of the culture medium. As long as it's Uruchino,
There are no particular limitations on shape, size, etc. Therefore, the shape of the gas permeation gap 00) existing in the frame body 01) may be wide and long strip-shaped, for example as shown in FIG. 2, or may be slit-shaped, grid-shaped, or circular. - It may be a void having a shape such as an ellipse.

Note that the above-mentioned "for gas passage" does not mean that only gas passes through, but since the culture medium and the hollow fibers need to be in contact with each other, it is a matter of course that the culture medium also passes through.

For example, in the gas exchange means shown in Fig. 2, when gas for gas exchange is supplied from one side, the gas consumed during cultivation has a high partial pressure inside the hollow fibers, so the gas is dissolved into the culture medium. On the other hand, since the dissolved amount of gas discharged by culture is greater than the partial pressure of the gas in the hollow fibers, the gas is discharged from the culture medium into the hollow fibers, and gas exchange takes place. At this time, if the surface of the hollow fiber is hydrophilic, if the pressure of the exchange gas is lower than the water pressure, the culture medium will penetrate into the inside of the hollow fiber, so when performing gas exchange, Is it necessary to make the pressure of the replacement gas higher than the water pressure? Furthermore, when the exchange gas pressure is lower than the water pressure, the culture medium (drained liquid) passes through the hollow fibers and is discharged to the outside, so the gas exchange means (3) also serves as the culture medium removal means. Become.

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

It goes without saying that parts of the cell culture means (2) and gas exchange means (3) where medium exchange or gas exchange occurs must be present in the medium. Further, it is preferable that the medium is supplied from the medium supply means (5) from the top of the medium container (1).

Although the device of the present invention does not necessarily require stirring means,
As shown in FIG. 1, when the stirring means 02) is provided to stir the culture medium, gas exchange between the culture medium and the gas exchange means is facilitated, and the culture medium is easily maintained in a constant state.
In addition, the culture medium easily permeates through the bag body (6). In addition, since the medium is stirred, the cells will not be damaged even if strongly stirred.Furthermore, the bag (6) is constantly shaken to make it easier for the cells to float, so that the cells adhere to the surface of the bag (6). This prevents this and makes it easier for them to proliferate.

Note that 03) in FIG. 1 is a degassing means when initially supplying the culture medium, and 04) in FIG. 2 is a potting part.

Although FIG. 1 does not show any means for monitoring the condition of the medium during culture, it is a matter of course that the scale should be installed from time to time.

Examples of cells cultured by the apparatus of the present invention include lymphocytes, bone marrow cells, and macrophages; examples of cell activating substances used at this time include lymphokines and interferon;
Examples include fetal bovine serum medium, serum-free medium, and human serum medium.

Next, the apparatus of the present invention will be explained based on examples.

Example 1 An apparatus shown in FIG. 3 was used in which the gas exchange means in the apparatus of the present invention was a gas exchange means having gas exchange and culture medium removal functions.

The culture medium container (1) in Figure 3 is made of hard glass and has a capacity of 3
300 ml, diameter approx. 150 x height approx. 190 +++
+n cylindrical container, cell culture means (2) is a regenerated cellulose membrane with an average pore size of 40, and a content volume of 300 ml.
It is a bag (6) with a height of 150 mm, a diameter of 50 mm, and a surface area of 230 at, and a frame having an approximately cylindrical shape (supported by force, and a pipe reaching the bottom of the bag at the top of the bag). The gas exchange and medium removal means (8) are provided with a cell supply and collection means (8) having an average pore size of 0.1 made of cellulose triacetate.
00 pieces are bundled and potted to have an effective length of 120 mm (total surface area: 250 cIIl'), which has a cylindrical shape with an inner diameter of 20 +++n and a lJ wide strip shape of approximately 80 mm x 10+++n+ on its side. It is supported by a frame with two gaps, and has a gas supply port at the top and a gas exhaust/medium discharge port at the bottom (see Figure 2). The cell culture means (2), the gas exchange/medium removal means, and the medium supply means (5
) and 0.2. A degassing means (131 is arranged and stirring means 02) having an air filter 00 of am is provided. Note that an air pump 07) connected to a gas supply line 09 having an air filter (0,2ρ) 00 is connected to the gas supply port, and exhaust gas and culture medium are discharged to the outside from the gas exhaust and culture medium discharge port. A line (21) is provided for this purpose.

With the gas venting means 03) open, RP old-16 was used as a culture medium.
40 (manufactured by Ajinomoto ■) was supplied, and then 110 ml of cell suspension consisting of 106 lymphocytes/ml x room ml, 10 ml of serum, and 200 units of lymphokine was supplied to the bag (6).

After that, stir at a speed of 200 rpm.
Culture was performed at 7°C. During this time, the medium supply means (5)
Culture medium was supplied from the culture medium at a rate of 1 ml/min, and air containing 5% by volume of CO2 was supplied using an air pump at a flow rate of 10 ml/min for 20 days.

During culture, the pll of the medium and the partial pressure of carbon dioxide (PC
O2) was measured and 7.2≦IIH≦7.3.3[in+
It was confirmed that +nHg≦PCO2≦40mm11g.

The culture results are shown in Table 1.

[Margin below]

Table 1 makes it easy to separate and purify cultured cells and products. Furthermore, exchange of the medium and collection of cultured cells and their products can be performed continuously in an aseptic manner. And since serum and cell activation substances are retained within the bag,
Its consumption will also be reduced.

As described above, by using the apparatus of the present invention, highly concentrated cultured cells and products can be obtained efficiently, so that the apparatus can be made more compact.

[Effects of the Invention] By using the apparatus of the present invention, constant culture medium conditions can be stably maintained even during cell culture. Furthermore, since gas exchange is performed in a medium without cells using a gas exchange means using a hollow fiber bundle, damage to cells caused by bubbling does not occur. In addition, cells, serum, and physiologically active substances are stored inside the bag.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram of one embodiment of the apparatus of the present invention, and FIG.
The figure is an explanatory diagram of an example of the gas exchange means used in the apparatus of the present invention, and FIG. 3 is an explanatory diagram of the apparatus of the present invention used in Example 1. (Main symbols in the drawing) (1): Medium container (2): Cell culture means (3): Gas exchange means (4): Cell culture device (5): (6): (7): (8): ( 9) 2002 old version: Culture medium supply means bag Frame for supporting the body bag Supply/recovery means for cells etc. Hollow fiber bundle Gaps for gas passage Receptacle frame for supporting the hollow fiber bundle Gas exchange and culture medium Removal means Culture medium removal means Gas disposal and culture medium removal line Applicant: Nissho Co., Ltd. Procedural amendment (Salmon) 5. Subject of amendment (1) "Claims" column of the specification (2) "Details of the invention" of the specification Column 3: “Brief Description of Drawings” Column 1: Indication of the case 1988 Patent Application No. 208862 2: Name of the invention Suspension cell culture device 3: Person making the amendment Relationship with the case Patent Applicant address: 3-9-3 Honjonishi, Oyodo-ku, Osaka-shi 6 Contents of amendment (1) The "Claims" of the specification will be amended as shown in the attached "Amended Scope of Patents." (2) r 3B~40+n eq/ on page 5, line 11 of the specification
i) Correct J to "26-32meq/, Ill". (3) Correct "confirmed" in line 8 of page 20 to "confirmed". (4) Correct "disposal" in line 10 of page 23 to "exhaust". Address: 2-37 Tanimachi, Higashi-ku, Osaka NS Bill et al. Amended claims [A cell culture device in which a cell culture means and a gas exchange means are housed in a culture medium container containing one culture medium] , the culture medium container is provided with a culture medium supply means and a culture medium removal means, and the cell culture means includes a flexible bag through which the medium provided inside is permeable but not through which serum or physiologically active substances are permeable; It consists of a frame provided with a gap through which a culture medium can freely pass to support the bag, and a means for supplying cells, serum, and cell activating substances to the bag and collecting cells and products. . A floating cell culture device, wherein the gas exchange means comprises a frame having a hollow fiber bundle and a gas passage gap for supporting the hollow fiber bundle. 2. The flexible bag in the cell culture means is a bag made of a membrane made of cellulose triacetate, cellulose diacetate, regenerated cellulose, polypropylene, polyethylene, polytetrafluoroethylene, polysulfone, polyamide, or polyester with an average pore size of 30 to 80. The apparatus according to claim 1. 3. The apparatus according to claim 1, wherein 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. 4. The apparatus according to claim 1, wherein the gas exchange means also serves as the medium removal means. 5 A flexible bag in the cell culture means, or a bag made of a membrane made of cellulose triacetate, cellulose diacetate, regenerated cellulose, polypropylene, polyethylene, polytetrafluoroethylene, polysulfone, polyamide, or polyester with an average pore size of 30 to 80. the gas exchange means or a hollow fiber bundle made of cellulose triacetate, cellulose diacetate, regenerated cellulose, polypropylene, polyethylene, polytetrafluoroethylene, polysulfone, polyamide or polyester; and a culture medium removal means. The device according to claim 1, which also serves as a device. "that's all

Claims (1)

[Scope of Claims] 1. A cell culture device in which a cell culture means and a gas exchange means are housed in a culture medium container containing a culture medium, wherein the culture medium container is provided with a culture medium supply means and a culture medium removal means. The cell culture means includes a flexible bag that allows a culture medium to pass through but does not allow serum or physiologically active substances to pass through, and a gap that allows the culture medium to freely pass through to support the bag. a frame provided with the bag, and means for supplying cells, serum, and cell activating substances to the bag and recovering cells and products, and the gas exchange means connects the hollow fiber bundle to the bag. 1. A floating cell culture device comprising a frame having a gas passage gap for support. 2. The flexible bag in the cell culture means is a bag made of a membrane made of cellulose triacetate, cellulose diacetate, regenerated cellulose, polypropylene, polyethylene, polytetrafluoroethylene, polysulfone, polyamide, or polyester with an average pore size of 30 to 80 Å. 2. The apparatus of claim 1. 3. The apparatus according to claim 1, wherein 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. 4. The apparatus according to claim 1, wherein the gas exchange means also serves as the medium removal means. 5 The flexible bag in the cell culture means is made of cellulose triacetate, cellulose diacetate, regenerated cellulose, polypropylene, with an average pore size of 30 to 80 Å,
The bag is made of a membrane made of polyethylene, polytetrafluoroethylene, polysulfone, polyamide, or polyester, and the gas exchange means is made of cellulose triacetate, cellulose diacetate, regenerated cellulose, polypropylene, polyethylene, polytetrafluoroethylene, polysulfone, or polyamide. 2. The device according to claim 1, wherein the device uses a bundle of hollow fibers made of polyester and also serves as a medium removal means.