JPH0352954B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to an apparatus for culturing animal cells. More specifically, the present invention relates to a method and apparatus for efficiently culturing cells at high density using a cell culture vessel made of a hollow fiber membrane. [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, the use of animal cells has been developed. Mass culture technology has become an important development theme. Various methods are known to date for culturing animal cells. On a laboratory scale, there are methods such as a sialet or a culture bottle. On the other hand, as a method for culturing in large quantities, attempts have been made to use agitation culture as an extension of conventional microbial fermentation technology. For example, in 1967, Van Wezel et al. developed the so-called microcarrier method that uses DEAE-Sephadex (trade name) as a carrier to increase the cell attachment area when culturing adherent cells. It has become possible. On the other hand, with regard to floating cells, attempts to obtain large quantities of monoclonal antibodies from hybridomas obtained by cell fusion of mouse bone marrow tumor cells and immunized mouse spleen cells have rapidly increased, and a method using agitation culture has been investigated. ing. However, conventional aerated agitation culture can only reach a cell count of about 2×10 ⁶ cells/ml at most, and the concentration of physiologically active substances produced is also insufficient, posing many industrial difficulties. . Therefore, recently, as a method to solve these problems especially in planktonic cells, the number of cells has been increased to approximately 5 × 10 ⁶ cells/
Cultivation methods that increase the density to ml or higher are attracting attention. That is, it is an extremely effective method in that it is possible to downsize the apparatus by culturing cells at high density, and it also facilitates purification by increasing the concentration of the product. The conditions for culturing cells at high density are not necessarily clear, but in general, it is necessary to constantly maintain a physiological environment suitable for cell proliferation in the culture system, It has been proposed to supply necessary nutrients while continuously removing cell-inhibiting factors produced during the growth process.
As a method for this purpose, for example, in agitation culture, several methods have been proposed in which a new culture solution is supplied to the culture tank and at the same time a large amount of culture solution is taken out from the culture tank (for example, JP-A-60-9482, Tolbert et al.; In
vitro 17 885 (1981)). However, a common problem with these methods is that continuous separation of cultured cells and culture medium has not always been technically solved, especially in continuous culture for several weeks. Furthermore, unlike microorganisms, animal cells do not have resistance to shearing forces such as those caused by stirring, and although efforts have been made to overcome this problem, they are not always sufficient. On the other hand, as a method for efficiently culturing adherent cells, Knazek et al. reported that cells were allowed to adhere to the surface of a hollow fiber membrane housed in a cylindrical case, and a culture solution was circulated through the hollow part of the hollow fibers through the wall membrane. Since the first report of a culture method that supplies nutrients to cells, several culture methods using hollow fibers have been proposed (Japanese Patent Publication No. 54-6634, Japanese Patent Publication No. 57-21978, Japanese Patent Publication No. 56-42584). For example, JP-A-56
-42584 uses a cell culture vessel in which a hollow fiber is covered with a shell and both ends of the hollow fiber are opened to the outside of the shell, and a culture solution is poured into the hollow part to culture floating cells between the shell and the hollow fiber. A method is disclosed. These methods are worthy of attention because they have the potential to solve the problems of high-density culture using agitation culture described above. In other words, since cells are cultured through the wall membrane of hollow fibers, the cells and culture medium are separated in advance, and even if cells are cultured in an attached or suspended state, shearing force is not as strong as in the case of agitation culture. This is not acceptable. However, even with these methods, it is currently difficult to say that high-density culture has been sufficiently established. In other words, in the case of hollow fibers, the cells are not uniformly dispersed as in agitation culture, and the supply of nutrients and the removal of inhibiting components such as waste products produced by the cells are carried out through the wall membrane by dialysis or Because this is carried out based on the principle of filtration, the microenvironment for cell growth differs depending on the positional relationship between the cell and the membrane, such as the inlet and outlet of the culture solution container, or the distance from the membrane. It is difficult to maintain a uniform microenvironment, and conventional methods are insufficient to maintain and culture cells at high density.
At present, the advantages of hollow fiber culture vessels are not fully utilized. [Objective of the invention] In view of the above circumstances, it is an object of the present invention to utilize the characteristics of hollow fiber cell culture vessels and the advantages of agitation culture to efficiently proliferate cells, maintain and culture them at high density, and thereby extract useful physiologically active substances from cells. With the aim of producing large quantities of , we conducted extensive research and completed the present invention. [Structure of the Invention] The present invention provides 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 located 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 container, and 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. a cell culture device, a first external circulation means for circulating the culture solution in the hollow part of the hollow fibers, and a second external circulation means for circulating the cell suspension or culture solution in the hollow fiber dispersion gap, The first external circulation means is equipped with a first culture solution storage tank and a first pump means, each having a culture solution discharge means and a fresh culture solution supply means containing components necessary for cell proliferation; A cell characterized in that the means is equipped with a second culture solution storage tank equipped with a means for supplying components necessary for cell proliferation, a second pump means, and a cell separation means upstream of the second pump means. Regarding a culture device. Furthermore, the present invention includes a device characterized in that the cell culture device includes a means for removing floating cells in the hollow fiber dispersion gap to the outside of the system. The cell culture vessel used in the present invention has a wall membrane that is permeable to nutrients necessary for cells but not permeable to cells, and both ends of the hollow fiber dispersed bundle are open to the outside of the container. Particularly limited if it is fixed to both ends of the container by a partition wall so as to be attached, and a conduit communicating with the hollow portion of both ends of the hollow fibers and at least two conduits communicating with the hollow fiber dispersion gap are attached. It is not something that will be done. A specific example is shown in FIG. Both ends of a dispersed bundle of multiple hollow fibers 1 are fixed to partition walls 3a and 3b at both ends of the container 2. Furthermore, both ends of the hollow fibers are open to the outer surface of the partition wall, and the end chamber 4
a, 4b and to end conduits 5a, 5b. A culture solution can be taken in and out from the end conduits 5a and 5b. Further, at least two conduits 7 communicating with the hollow fiber dispersion gap 6
a and 7b are attached to the container 1, and are used as an inlet and an inlet for inoculating cells and for circulating a cell suspension or a culture solution during cultivation in the hollow fiber dispersion gap 6. As a hollow fiber, the membrane itself is not cytotoxic;
Any material may be used as long as it is not altered or decomposed by sterilization or culture medium. Examples of polymer materials include cellulose esters, acrylic polymers, polysulfones, polyethersulfones, fluorine polymers, polyoffine polymers, polycarbonates, and cellulose. Examples of materials made from inorganic materials include glass and ceramics. The present invention uses the above-mentioned cell culture device, and cultures cells in the hollow fiber dispersion gap or the outer surface of the hollow fiber by pouring a culture solution into the hollow fiber. Another major feature is that the culture solution is circulated. Furthermore, when circulating, it is preferable to provide a cell separation means in the circulation system to prevent cells from being included in the circulation fluid as much as possible. In addition, by providing a means for supplying oxygen and nutrients to the circulatory system or a means for taking circulating fluid and cell suspension out of the system, cells can be efficiently grown and useful physiologically active substances can be continuously produced. I can do it. FIG. 2 shows a specific example of the cell culture device of the present invention. That is, the first culture solution storage tank 8 contains a culture solution for growing cells, and this culture solution is pumped into the end conduit 5a of the cell culture device 10 by the first pump means 9.
It passes through the hollow portion of the hollow fiber, exits from the end conduit 5b, returns to the first culture solution storage tank 8, and can be circulated. Cells are inoculated into the hollow fiber dispersion gap 6 and proliferate on the outer surface of the hollow fiber or in the hollow fiber dispersion gap 6 by being supplied with nutrients from the culture solution flowing through the hollow fibers through the wall membrane of the hollow fibers. Note that inhibiting components such as waste products produced by cells are removed into the culture solution flowing through the hollow portion of the hollow fiber through the wall membrane of the hollow fiber.
In order to homogenize the microenvironment in which the cells grow, the cell suspension or culture medium in the hollow fiber dispersion gap 6 can be circulated through the circuits 13, 14 and the hollow fiber dispersion gap 6 by means of second pump means 11, 12. It's becoming like that. Hollow fiber dispersion gap 6 and circuit 1
Between 3 and 3 is a cell sedimentation tube 15 as a cell separation means.
is provided so that cells are not included in the possible circuit 13. Furthermore, a floating cell outlet 16 is provided at the bottom of the cell sedimentation tube 15.
A second culture solution storage tank 1 is provided between the circuit 13 and the circuit 14.
7 is provided, and a conduit 18 for supplying oxygen or nutrients and a circulating fluid outlet 19 are attached. Necessary components such as nutrients and oxygen necessary for cell proliferation in the hollow fiber dispersion gap 6 are
When the supply from the culture solution in the culture solution storage tank 8 is insufficient or when the pH needs to be adjusted, oxygen or the like is supplied through the conduit 18. Further, the circulating fluid can be taken out from the circulating fluid outlet 19 as needed, and metabolites such as useful physiologically active substances produced by the cells can be recovered. The first culture solution storage tank 8 includes a tank 20 for supplying new culture solution and a tank 20 for taking out the old culture solution and useful physiologically active substances contained therein from the first culture solution storage tank 8 via a pump means. A tank 21 is also provided, and cells can be continuously grown by continuously supplying a culture solution from the tank 20 via pump means. In this way, the first pump means, the first culture solution storage tank, etc. constitute the first external circulation means, and the second pump means, the second culture solution storage tank, etc. constitute the second external circulation means. As a means for circulating the cell suspension or culture solution in the hollow fiber dispersion gap, commonly used pumps such as tube pumps, plunger pumps, diaphragm pumps, and spiral pumps can be used, but sterilization is easy. A tube pump is preferred. In addition, it is also suitable to use a method in which negative pressure is applied intermittently to the circuit without using a direct pump and circulated using a head system. When using a pump, the cells passing through the pump will be damaged to some extent, so
It is desirable to prevent cells from being included in the circulating fluid passing through the pump section. For this purpose, it is preferable to provide cell separation means in the circulation circuit. Methods for separating cells include methods using various filters or methods using sedimentation tubes that take advantage of the fact that the specific gravity of cells is greater than the specific gravity of the culture medium, but these methods do not separate cells. If so, it is not particularly limited. In the case of culturing adherent cells, the cells proliferate on the hollow fiber surface and construct a three-dimensional structure, so it is not necessarily necessary to provide cell separation means in the circulation circuit. Furthermore, even when culturing planktonic cells, if the circulation is carried out by a drop-down method without using a pump, there will be no cell damage caused by the pump, so it is not necessarily necessary to provide cell separation means, but there will be some shear caused by the circulation. Since the cells are subjected to force, it is generally preferable to circulate them in a manner that contains as few cells as possible, whether it is circulated by a pump or by a head method. Furthermore, it is preferable to provide a storage tank for storing circulating fluid in the circulation circuit for the cell suspension or culture fluid in the hollow fiber dispersion gap, and to provide means for supplying oxygen and nutrients necessary for cell proliferation as necessary. Moreover, it is preferable to provide means for taking out the circulating fluid. Generally, when the number of cells reaches a high density of about 5 x 10 ⁶ cells/ml, the rate of nutrient uptake by the cells increases rapidly, and the supply of oxygen becomes a particularly important issue. In order to maintain the dissolved oxygen in the culture solution that circulates through the hollow part of the hollow fiber, methods such as directly blowing oxygen into the culture solution in the culture solution tank have been used, but problems such as bubbling have not always been technically resolved. .
In order to maintain a high density of cells, this problem can be solved by supplying oxygen to the cell suspension in the hollow fiber dispersion gap or to the circulating culture solution. This applies not only to oxygen, but especially to
The same holds true when the hollow fibers used in the cell culture vessel are selected to prevent passage of nutrient components in a relatively high molecular weight range, and it is preferable to supply components or culture fluid that are difficult to pass through. In addition, in order to recover metabolites produced by cells, especially useful physiologically active substances, as needed, circulating fluid is removed.
It is also possible to add a corresponding amount of culture medium. In order to maintain cell proliferation over a long period of time and obtain a large amount of useful physiologically active substances produced by the cells, a portion of the cells proliferating in the hollow fiber dispersion gap should be expelled from the culture system in a timely manner. It is preferable to maintain the number of cells in the hollow fiber dispersion gap as constant as possible and maintain the useful substance producing activity of the cells. This method is particularly preferred in the case of floating cells. The cells cultured according to the present invention are not limited to animal cells but also include plant cells, but animal cells are preferably used. Examples of adherent cells include fibroblasts, kidney cells, and epithelial cells. Also,
Examples of floating cells include lymphocytes, myeloma cells, white blood cells, and cells obtained by cell fusion of myeloma cells and other cells (hybridoma). [Effects of the Invention] According to the culture device of the present invention, it is possible to efficiently proliferate cells and maintain the cell number at a high density. It is possible to efficiently produce useful physiologically active substances produced by cells at the same time, and it can be applied to mass culture by using one or more cell culture vessels. The present invention will be explained below using Examples, but the present invention is not limited to these Examples. Example 1 4000 polyethersulfone hollow fibers with an inner diameter of 230 μm and a film thickness of 50 μm were bundled and filled into a polycarbonate cylindrical container, and partition walls were molded with urethane resin to assemble a cell culture vessel as shown in FIG. 1. The molecular weight cutoff of the membrane is 50,000, and the volume of the hollow fiber dispersion gap is 70 ml. Using this cell culture device, the cell culture device shown in FIG. 2 was assembled and steam sterilized. 3 for culture solution tank 8
culture solution (GIBCO's RPMI-1640 medium 90%,
(consisting of 10% fetal bovine serum) was added, and the culture solution was circulated through the hollow part of the hollow fiber. Further, the hollow fiber dispersion gap 6 was inoculated with mouse-mouse fusion cells whose parent strain was mouse myeloma cell P3U1 suspended in the above culture solution at a concentration of 2×10 ⁵ cells/ml. The circulating fluid storage tank was filled with 60 ml of the above culture solution, and the cell sedimentation tube 15 and circuits 13 and 14 were similarly filled with the above culture solution. The entire apparatus was placed in a constant temperature bath at 37°C. 95% air is passed through the conduits 22 and 18 to the first culture solution storage tank 8 and the second culture solution storage tank 17.
A mixed gas of 5% CO ₂ or a mixed gas of 95% oxygen and 5% CO ₂ was vented into the tank to maintain the dissolved oxygen concentration in the culture solution at 5 ppm. The circulation rate of the culture solution in the hollow portion of the hollow fiber was 30 ml/min, and the circulation rate in circuits 13 and 14 was 5 ml/min. A cell suspension in the hollow fiber dispersion gap was collected over time, and the number of proliferated cells was measured. The results are shown in Table 1. However, on the 6th, 12th, and 16th days, the entire amount of the culture solution in the first culture solution storage tank 8 was replaced with a new culture solution.

[Table] As a comparative example, culture was performed in the same manner as in Example 1 without circulating the cell suspension in the hollow fiber dispersion gap. As shown in Table 1, the cell proliferation was slower than in the present invention. The number of cells was also small. Example 2 A cell culture vessel was assembled in the same manner as in Example 1 by bundling 2000 cellulose acetate hollow fibers with an inner diameter of 320 μm and a membrane thickness of 70 μm. The molecular weight cutoff of the membrane is approximately 1 million,
The capacity of the hollow fiber dispersion gap is 70ml. The cell culture vessel was sterilized with ethylene oxide gas, the other parts were steam sterilized, and the cell culture apparatus shown in FIG. 2 was assembled. The cell culture vessel was circulated and washed with saline followed by culture medium. Next, the fused cells were cultured in the same manner as in Example 1. However, the culture solution circulation rate in the hollow part of the hollow fiber was 60 ml/min. A cell suspension was collected over time, the number of cells was measured, and the results shown in Table 2 were obtained.

【table】 [Brief explanation of drawings]

FIG. 1 is an example of a cross-sectional view of a cell culture vessel used in the present invention. Figure 2 shows one of the cell culture apparatuses of the present invention.
This is an example. 1... Hollow fiber, 2... Container, 3... Partition wall, 4
... End chamber, 5 ... End conduit, 6 ... Hollow fiber dispersion gap, 7 ... Side conduit, 8 ... First culture solution storage tank, 10 ... Cell culture vessel, 11, 12 ... No. 2 pump means, 13, 14... circulation circuit, 15... cell sedimentation tube, 16... cell suspension outlet, 17...
Second culture solution storage tank, 18, 22... Conduit, 19...
Circulating fluid outlet.

Claims (1)

[Claims] 1. Both ends of a hollow fiber dispersed bundle having 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. A cell is fixed to both ends of the container by a partition wall so as to be connected to the container, and 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. It consists of a culture vessel, a first external circulation means for circulating the culture solution in the hollow part of the hollow fibers, and a second external circulation means for circulating the cell suspension or culture solution in the hollow fiber dispersion gap, The external circulation means is equipped with a first culture solution storage tank and a first pump means, which are equipped with a culture solution discharge means and a fresh culture solution supply means containing components necessary for cell proliferation; A cell culture device comprising a second culture solution storage tank equipped with means for supplying components necessary for cell proliferation, a second pump means, and a cell separation means upstream of the second pump means. . 2. The device according to claim 1, further comprising means for removing floating cells in the hollow fiber dispersion gap to the outside of the system.