JPH0360477B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method and a culture vessel for culturing cells. More specifically, the present invention relates to a hollow fiber cell culture device suitable for efficiently culturing cells at high density. [Prior Art] In recent years, in order to obtain on an industrial scale physiologically active substances produced by animal cells, such as interferon, urokinase, monoclonal antibodies, lymphokines, and other high value-added proteins and peptides, bioactive substances have been developed. Mass culture technology has become an important development theme. Various methods have been known to date for culturing animal cells. On a laboratory scale, there are methods such as Schare or culture solution. on the other hand,
As methods for culturing cells in large quantities, microcarrier methods and methods using hollow fiber culture vessels have been developed, which basically aim to increase the adhesion area of cells. In particular, a method by Knazek et al. in which cells are attached to the surface of a hollow fiber membrane and a culture solution is circulated through the hollow part of the hollow fiber to supply nutrients to the cells through the wall membrane (Japanese Patent Application Laid-Open No. 49-41579) is This method has attracted attention as a method for culturing cells efficiently. Furthermore, nutrients
Several improved methods have been proposed for efficiently exchanging waste products and gaseous substances (Japanese Patent Application Laid-open No. 1973-
Publication No. 36684, Japanese Patent Application Laid-Open No. 1983-98382, Japanese Patent Application Publication No. 1983
-42584, JP-A-59-175878). For example, Japanese Patent Application Laid-open No. 56-42584 describes a cell culture device in which a hollow fiber is covered with a shell and both ends of the hollow fiber are opened to objects outside the shell, and a culture solution is injected into the hollow part for a long time. A method of culturing between a shell and a hollow fiber is disclosed. Also, JP-A-59
-175878 discloses a method in which cells are confined on one side of a semipermeable membrane that allows the components in the culture solution to pass through but does not allow the desired products produced by the cells to pass through, and the culture solution is continuously supplied to the other side. Disclosed. The advantage of hollow fiber culture vessels is that cells can be cultured at high density because cells are cultured through the wall membrane of the hollow fibers. In other words, when the purpose is to culture cells and produce a large amount of physiologically active substances, culturing the cells as densely as possible allows for miniaturization of the equipment, and also reduces the concentration of the product. This is an extremely effective method in terms of increasing the chemical content and facilitating purification. For this purpose, it is necessary to constantly maintain a physiological environment suitable for cell growth in the culture system, and to do so, it is necessary to supply the nutritional components necessary for cell growth, and on the other hand, to increase the amount of cells produced during the growth process. It is considered necessary to continuously remove factors that inhibit this. In this respect, the method of culturing cells through hollow fiber membranes is extremely useful as a method for mass culturing cells, as it has conditions suitable for high-density culture and is not subject to the same shearing force as in agitated culture. It is hoped that this method can be used as a method. However, even with the prior art available up to now, it is difficult to say that mass culture technology for cells has been sufficiently established. In other words, in the case of hollow fibers, cells are usually cultured by leaving them on one side of the hollow fiber, so it is difficult to achieve a uniform dispersion like in agitation culture, and the supply of nutrients and waste products produced by cells are difficult. Because growth-inhibiting components such as The microenvironment for proliferation of cells is different, and it is difficult to maintain a uniform microenvironment for all cells. In order to make this microenvironment uniform, it is desirable that each hollow fiber is dispersed as uniformly as possible. Generally, when a hollow fiber bundle is fixed to a container, it is unavoidable that the hollow fibers come into close contact with each other along the length direction. If the hollow fibers come into close contact with each other, for example, when cells are cultured in the hollow fiber dispersion gap, uniform dispersion of cells will be hindered. On the other hand, if the number of hollow fibers is reduced in order to prevent adhesion, it not only results in a decrease in membrane area but also creates an excessively large hollow fiber dispersion gap, which is not good. In addition, when culturing cells in the hollow part of the hollow fiber,
If the hollow fibers are in close contact with each other, it becomes difficult for the culture solution to flow evenly around the hollow fibers, resulting in uneven distribution of nutrients to the cells. Conventional methods are insufficient to maintain and culture cells at a high density, for example, 1 x 10 ⁷ cells/ml or higher, because they do not create a sufficient microenvironment for cell proliferation. The current situation is that it is not fully utilized. [Purpose of the invention] In view of the above circumstances, it is possible to utilize the characteristics of hollow fiber cell culture vessels to provide cells with a better microenvironment and to proliferate them efficiently and at high density, thereby contributing to mass culture. With this in mind, we have conducted extensive research and have completed the present invention. [Structure of the Invention] That is, the present invention provides a cell culture method using a hollow fiber dispersed bundle having a wall membrane that is permeable to nutrients necessary for cell proliferation but not permeable to cells. In this method, a modified hollow fiber having fins extending in the longitudinal direction on the outer periphery is used for most of the hollow fiber, and cells are cultured on one side of two regions separated by the wall membrane of the hollow fiber. A cell culture method characterized in that both ends of a hollow fiber dispersed bundle having a wall membrane that is permeable to nutrients necessary for cell proliferation but not permeable to cells are placed outside the container. A conduit is fixed to both ends of the container by a partition wall so as to open at both ends of the hollow fiber, and is provided with a conduit that communicates with the hollow portion opened at both ends of the hollow fiber, and at least two conduits that communicate with the hollow fiber dispersion gap. In a cell culture vessel, most of the hollow fibers are deformed hollow fibers with fins extending in the longitudinal direction on the outer periphery, and cells are grown on one side of two regions separated by the wall membrane of the hollow fibers. A hollow fiber type cell culture device characterized by the following is provided. FIG. 1 shows the basic configuration of an embodiment of a hollow fiber cell culture device according to the present invention. Both ends of a dispersed bundle of hollow fibers 1 consisting of a large number of fibers are connected to partition walls 3 at both ends of the container 2.
It is fixed at a and 3b. Further, both ends of the hollow fibers are open to the outer surface of the partition wall, and the end chambers 4a, 4
b and to the end conduits 5a, 5b. Further, at least two conduits 7a and 7b communicating with the hollow fiber dispersion gap 6 are attached to the container 1. Hollow fibers have a wall membrane that is permeable to nutrients necessary for cells but not permeable to cells, and has fins extending in the longitudinal direction on the outer periphery. It is a uniquely shaped hollow fiber. Cells are seeded in the hollow dispersion gap 6, and a culture solution is flowed into the hollow fibers through the conduits 5a and 5b. Alternatively, cells are seeded in the hollow part of the hollow fiber, and a culture solution is poured into the hollow fiber dispersion gap through the conduits 7a and 7b. A major feature of the present invention is the use of irregularly shaped hollow fibers having fins extending in the longitudinal direction at the outer periphery of the hollow fibers. Therefore, the entire hollow fiber membrane surface can function effectively without causing the hollow fibers to come into close contact with each other, and the microenvironment for cell proliferation can be made uniform. FIG. 2 shows an example of an enlarged cross section of a modified hollow fiber useful in the present invention. The number of fins is at least one, but if the number of fins is 15 or more, the effective membrane area will be significantly reduced due to the base of the fins, the culture medium supply performance will be significantly reduced, and the effective hollow fiber dispersion gap will be reduced, making it impractical. Generally, the number of fins is preferably 1 to 14, more preferably 2 to 10, and a particularly effective range is 3 to 7. Other shapes and dimensions of the irregularly shaped hollow fibers in the present invention are not particularly limited, but the outer diameter (d) is 100 to 1000μ, preferably 200 to 800μ, and the film thickness (h) of the portion without fins is is 5 to 100μ, preferably 10 to 80μ, and the height (H) of the fin is 5 to 100μ.
The height/outer diameter (H/d) ratio of the fins is preferably 0.01 to 0.8, more preferably 0.03 to 0.3. In addition, the width (W) of the base of the fin is 15 to 80μ, preferably
A thickness of 20 to 60μ is suitable. The cross-sectional shape of the hollow portion is not limited to a circular shape, but may be an elliptical shape. Furthermore, the fins on the outer periphery of the hollow fiber may be formed in a spiral shape. Note that when one hollow fiber has multiple fins, the height (H) and width (W) of each fin may be the same or different. Further, two or more hollow fibers may be connected via fins, but in that case, two hollow fibers are preferred. Furthermore, not only a single irregularly shaped hollow fiber but also a mixture of several different irregularly shaped hollow fibers may be used. Further, the irregularly shaped hollow fibers may be crimped. Note that the hollow fiber bundle of the present invention may partially include hollow fibers without fins. When filling a container with hollow fibers, it is desirable to fill the container with the hollow fibers as uniformly as possible so that there are no spaces where no hollow fibers exist or excessive gaps for dispersing the hollow fibers. In this regard, in the case of the irregularly shaped hollow fiber with fins of the present invention, it can be filled extremely well, but when expressed in hollow fiber filling rate, it is 30
~60% is desirable. Here, the hollow fiber filling rate indicates the occupancy rate of the outer periphery standard cross section of the hollow fibers excluding the fin portion of the entire irregularly shaped hollow fiber bundle with respect to the cross section perpendicular to the axial direction of the hollow fiber bundle on the inner wall of the container. If the filling rate is too low, not only will the effective membrane area decrease, but also an excessive space will be created, making it difficult to maintain cells in the hollow fiber dispersion gap, which is unfavorable from the viewpoint of the microenvironment for cell proliferation. Furthermore, if the filling rate is too high, it will not only be difficult to insert and fill the hollow fibers into the container, but also, especially when culturing cells in the hollow fiber dispersion gap, the volume will be too small, which is not preferable from the point of view of mass culture. do not have. The finned irregularly shaped hollow fiber used in the present invention may have a plurality of crimps, if necessary. The preferred range of crimp amplitude is 1 to 500% of the outer diameter (d) of the hollow fiber;
The preferred wavelength range is 5 to 1000 times the outer diameter (d). Materials for the hollow fiber membrane used in the present invention include:
Any membrane may be used as long as the membrane itself is not cytotoxic and is not altered or degraded by sterilization or culture medium. For example, those made from polymeric materials include cellulose, cellulose ester, acrylic polymer, polysulfone, polyethersulfone, fluorine polymer, polyolefin polymer, polycarbonate, and copolymers thereof;
This includes mixtures with other substances. Examples of materials made from inorganic materials include glass and ceramics. In the cell culture device of the present invention, cells may be seeded in the hollow fiber dispersion gap 6 and cultured by pouring a culture solution into the hollow fiber, or conversely, cells may be seeded in the hollow fiber dispersion gap 6. The seeds may be seeded and cultured by pouring a culture solution into the hollow fiber dispersion gap. That is, the cells are cultured by supplying nutrients from one side to the cells on the other side through the wall membrane of the hollow fiber. In addition, when cells are cultured on the side of the hollow fiber dispersion gap, there is less close contact between the hollow fibers, making it easier to maintain uniform culture conditions, and especially when culturing adherent cells, there is an advantage that the adhesion area can be increased. The hollow fiber used in the present invention has a wall membrane that allows nutrients necessary for cell proliferation to pass through, but does not allow cells to pass through. The nutrients necessary for cell growth include inorganic salts, amino acids, sugars,
fatty acids, vitamins, coenzymes, nucleobases,
This refers to hormones, albumin, transferrin, various other cell growth factors, components in serum, etc., and it is inherently desirable for hollow fiber membranes to permeate all of these necessary components, ranging from low-molecular weight substances to high-molecular weight substances. However, this does not necessarily mean that all components must be transmitted. That is, a membrane having any molecular weight cutoff can be selected as required, but it is sufficient that it can at least pass most of the low molecular weight components necessary for cell proliferation, such as glycose and amino acids. In this case, if there are components that cannot pass through the membrane, they can be supplemented by directly supplying them to the cell side. For example, when attempting to obtain a physiologically active substance produced by a cell, it is preferable to concentrate the physiologically active substance while retaining it on the cell side. In this case, a hollow fiber membrane is used that has a molecular weight cut-off that does not allow physiologically active substances to pass through, so some of the components necessary for cell proliferation may not be able to pass through, but this component is directly transferred to the cell side. It is sufficient to supply and supplement it. One example is the case where a monoclonal antibody is obtained by culturing a hybridoma; in this case, a dialysis membrane used as a so-called artificial kidney may be sufficient. The cell culture vessel of the present invention uses irregularly shaped hollow fibers with fins, so it provides an extremely favorable microenvironment for cell proliferation. In addition to creating microflows near cells, we also actively move the cell suspension or culture solution near the cells, such as by applying vibrations, rotating them, or circulating the solution. It can also be used for culture. The cells cultured according to the present invention are not limited to animal cells but also include plant cells, but animal cells are preferably applied. Alternatively, the cells may be cells that have been modified artificially or by genetic manipulation. Examples of adherent cells include fibroblasts, kidney cells, hamster ovary cells, epithelial cells, and endothelial cells. Examples of floating cells include lymphocytes, myeloma cells, white blood cells, and hybrid cells (hybridomas) obtained by cell fusion of myeloma cells and other cells. [Effects of the Invention] According to the cell culture device of the present invention, it is possible to homogenize the microenvironment for cell proliferation, to efficiently proliferate cells, and to maintain the number of cells at a high density. It is. At the same time, it is possible to efficiently produce useful physiologically active substances produced by cells. The present invention will be explained below using Examples, but the present invention is not limited to these Examples. Examples 1 to 3, Comparative Example 1 100 parts of cellulose diastate was mixed with 50 parts of polyethylene glycol (molecular weight 200), the mixture was melted at 230°C, and a finned hollow fiber cap and It was spun from a regular finless circular tube spinneret. Next, the polyethylene glycol was eluted by immersion in hot water, followed by a saponification reaction with an aqueous solution of caustic soda, followed by immersion in an aqueous glycerin solution and drying with hot air to obtain circular hollow fibers and fins having the shapes shown in Table 1. A circular hollow fiber having no shape was obtained. The circular diameter of each of these hollow fibers was 200μ, and the thickness of the portion without fins was 25μ. Also, the height of the fins is
It was 35μ and the width was 30μ. The molecular weight cutoff of the membrane is approximately 1.5
It was ten thousand. 6,000 hollow fibers obtained as described above were bundled and filled into a polycarbonate cylindrical container, and partition walls were molded with urethane resin to assemble a cell culture vessel. The cell culture device shown in FIG. 3 was assembled using this incubator. Culture solution tank 8 contains culture solution 3 (GIBCO's RPMI-1640 medium 90%, fetal bovine serum 10%
%), and using pump 9, the culture solution was circulated through the hollow part of the hollow fiber at a rate of 50 ml/mm. In addition, in the hollow fiber dispersion gap 6, hybrid cells obtained by fusing mouse myeloma cells P3U1 with mouse antibody-producing cells are suspended in the above culture medium at a density of about 5 x 10 ⁵ cells/ml. Approximately 40 ml of the solution was inoculated through conduit 7. The entire apparatus is installed in a thermostat at 37°C, and the culture tank 8 is filled with 95% air through the conduit 11.
A gas mixture of 5% _CO2 and 95% oxygen if necessary.
A mixed gas containing 5% CO ₂ was vented into the tank to maintain the dissolved oxygen concentration in the culture solution at 5 ppm. The number of proliferated cells was measured on the 8th day after the start of culture, and the results shown in Table 1 were obtained. Cell proliferation using the irregularly shaped hollow fibers with fins of the present invention was extremely good compared to hollow fibers without fins.

[Table] Examples 4 to 5, Comparative Example 2 A spinning solution prepared by dissolving 20 parts of polyether sulfone in 80 parts of dimethyl sulfoxide was coagulated with water through a hollow fiber spinneret with fins and an ordinary annular spinneret without fins. The mixture was spun into a bath to obtain circular hollow fibers having fins and no fins as shown in Table 2. The circular diameter of each of these hollow fibers was 230μ, and the thickness of the portion without fins was 50μ. Also, the height of the fins is
It was 35μ and the width was 30μ. The molecular weight cutoff of the membrane is approximately 5
It was ten thousand. 4,000 hollow fibers obtained as described above were bundled and filled into a polycarbonate cylindrical container, and partition walls were molded with urethane resin to assemble a cell culture vessel.
Using this incubator, the number of proliferated cells on the 8th day was measured by the same method as in Examples 1 to 3, and the results shown in Table 2 were obtained.

[Table] Example 6 Using the cell culture device made of hollow fibers with six fins of Example 2, the cell culture device shown in FIG. 4 was assembled and cultured for a long period of time. Culture tank 8 is filled with the culture solution from step 3 (consisting of 90% GIBCO's RPMI1640 medium and 10% fetal bovine serum), and the hollow part of the hollow fiber is 50
The culture medium was circulated at a rate of ml/mm. In addition, in the hollow fiber dispersion gap 6, mouse-human hybrid cells obtained by fusing mouse myeloma cells P3U1 and human antibody-producing cells were suspended in the above culture medium at a concentration of 6 x 10 ⁵ cells/ml. 50 ml of the cloudy solution was inoculated. The culture solution in the hollow fiber dispersion gap was circulated by pumps 12 and 13 at a rate of 5 ml/mm. The entire apparatus was placed in a constant temperature bath at 37°C. 95% air is supplied to the culture solution tank 8 and circulating fluid storage tank 14 through conduits 11 and 15.
A mixed gas of 5% _CO2 , 95% oxygen, 5% _CO2 , and _CO2 gas were aerated to maintain the dissolved oxygen concentration in the culture solution at 5 ppm. Additionally, the pH was maintained below 7.4. A cell suspension in the hollow fiber dispersion gap was collected over time, and the number of proliferated cells was measured. When replacing the culture solution, replace the entire culture solution in the culture solution tank 8 with the storage tank 17.
The fresh culture solution 3 was then supplied from the storage tank 16 to the culture solution tank 8. 10% serum medium on the 6th and 9th day after the start of culture, 5% serum medium on the 11th day, and then RPMI- without adding serum as a general rule.
Only 1640 medium was exchanged daily, but some serum was also added as needed. Table 3 shows the results of cell number measurements. It was possible to maintain and proliferate cells at high density for a long period of time.

【table】 [Brief explanation of drawings]

FIG. 1 is an example of a cross-sectional view of the cell culture device of the present invention. FIG. 2 is an example of a cross-sectional view of a modified hollow fiber with fins used in the present invention. Figures 3 and 4 are flowcharts of cell culture. 1... Hollow fiber, 2... Container, 3... Partition wall, 4...
...End chamber, 5...End conduit, 6...Hollow fiber dispersion gap, 7...Side conduit, 8...Culture liquid tank, 9,1
2,13...Pump, 10...Cell culture device, 1
1, 15... Conduit, 14... Circulating fluid storage tank, 16,
17...Storage tank.

Claims (1)

[Scope of Claims] 1. A cell culture method using a dispersed hollow fiber bundle having a wall membrane that is permeable to nutrients necessary for cell proliferation but not permeable to cells. It is characterized by using irregularly shaped hollow fibers having longitudinally extending fins in the outer periphery of most of the hollow fibers, and culturing cells on one side of two regions separated by the wall membrane of the hollow fibers. cell culture method. 2. The cell culture method according to claim 1, wherein the irregularly shaped hollow fiber has 1 to 14 fins. 3. The container is provided with a partition wall so that both ends of the hollow fiber dispersed bundle, which has a wall membrane that is permeable to nutrients necessary for cell growth but not permeable to cells, are open to the outside of the container. fixed at both ends,
and a cell culture device that is provided with a conduit that communicates with a hollow portion opened at both ends of the hollow fiber, and at least two conduits that communicate with the hollow fiber dispersion gap, wherein most of the hollow fiber is located at its outer periphery. A hollow fiber type cell culture characterized in that the hollow fiber is an irregularly shaped hollow fiber having fins extending in the longitudinal direction, and cells are cultured on one side of two regions separated by a wall membrane of the hollow fiber. vessel.