JPS61280270A - Method for cell culture and apparatus therefor - Google Patents

Abstract

PURPOSE:To enable the efficient proliferation of cells and to maintain and culture the cell in high density, by using a membrane permeable to the nutrient necessary for the proliferation of the cell and impermeable to the cell and culturing the cell while circulating the suspension of the cell or the culture liquid. CONSTITUTION:The apparatus is provided with hollow fibers having wall membrane permeable to the nutrient necessary for the proliferation of the cell and impermeable to the cell. The culture liquid is sent from the culture liquid tank 8 with the pump 9 to the cell culture apparatus 10 in which the cells are cultured in the space 6 between the above hollow fibers and/or outside of the hollow fibers. The cell suspension or culture liquid between the fibers is circulated with the pumps 11, 12 through the circuits 13, 14 and the space 6.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method and apparatus for culturing animal cells. For more information, see Hollow! The present invention relates to a method and an apparatus for efficiently culturing cells at high density using a cell culture vessel made of ll-like membranes.

[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, large quantities of animal cells have been developed. Culture technology has become an important development theme9.

Various methods are known to date for culturing animal ichor cells. On a laboratory scale, there are methods such as petri dishes or culture bottles. 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, the 1967 Van
Wezel et al. developed the so-called microcarrier method using DEAE-Sephadex (trade name) as a carrier to increase the cell adhesion area when culturing adherent 111 cells, and agitation culture of adherent cells became possible. It's here. On the other hand, with regard to floating cells, attempts to obtain 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 is being considered. has been done. However, in conventional aerated agitation culture, the number of cells is at most 2 x 10' cells/
ml, and the concentration of the physiologically active substance produced was also insufficient, posing many industrial difficulties. Recently, we have recently developed a method to solve these problems using planktonic cells by increasing the number of cells to approximately 5x10G.
Cultivation methods that increase the density of cells/d 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
Publication No.-9482, Tolbert et al.: Invi
tro 17 885 (1981)) However, a common problem with these methods is that the continuous separation of cells in culture and the culture medium is not always technically solvable, especially in continuous culture for several weeks. It's not that I'm doing it. 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, K
Hollow fiber M housed in a cylindrical case by Nazek et al.
Since the reporting of a culture method in which cells are attached to the membrane surface, culture medium is circulated through the hollow part of the hollow fiber, and nutrients are supplied to the cells through the wall membrane, culture methods using hollow m-fibers have been developed. Some proposals have been made, such as the Publication of Special Publication No. 54-6634,
Japanese Patent Publication No. 57-21978, Japanese Patent Publication No. 56-4258
Publication No. 4). For example, in JP-A No. 56'-42584, a cell culture vessel in which hollow fibers are covered with a shell and both ends of the hollow fibers are opened to the outside of the shell is used, and a culture solution is poured into the hollow portion between the shell and the hollow fibers. A method of culturing planktonic cells 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 the cells are cultured through the wall membrane of hollow fibers, the cells and the culture medium must be separated in advance, and even if the cells are cultured in an attached or suspended state, it is difficult to apply any shearing force 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 lIi cells, 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 require dialysis through the wall membrane. Alternatively, since it is carried out based on the principle of filtration, the microenvironment for cell growth differs depending on the cell's positional relationship with the membrane, such as the inlet and outlet of the culture medium container, or the distance from the membrane. It is difficult to maintain the environment, and conventional methods are insufficient to maintain and culture cells at high density.
At present, the advantages of N-type incubators are not fully utilized.

[Objective of the invention] In view of the above situation, 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. Although intensive research was carried out with the aim of producing it in large quantities, the present invention was not completed.

[Structure of the Invention] That is, the present invention provides a method for forming a hollow All-in-one bundle at both ends, which has a wall membrane that is permeable to nutrients necessary for cell proliferation but not permeable to cells. is fixed to both ends of the container by partition walls so as to open to the outside of the container,
In addition, a cell culture vessel is provided with a conduit that communicates with the hollow part opened at both ends of the hollow fiber, and at least two conduits that communicate with the hollow fiber dispersion gap, and culture is carried out in the hollow part of the hollow fiber. The liquid is poured into the hollow fiber dispersion gap and/or
Alternatively, a method for culturing cells on the outer surface of hollow fibers, characterized in that the cell suspension or culture solution in the hollow fiber dispersion gap is cultured while being circulated by a circulation means provided outside the culture container. and equipment.

Furthermore, when circulating the cell suspension or culture solution in the hollow fiber dispersion gap, the present invention prevents the circulating solution from containing cells as much as possible, and furthermore, the circulating solution contains more cells necessary for cell proliferation. This is a method and device for culturing cells while supplying essential components and removing part of the circulating fluid from the system as necessary.

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. Both ends of the hollow fiber dispersed bundle have a frontage to the outside of the container. fixed to both ends of the container by partition walls so as to
There is no particular limitation as long as a conduit communicating with the hollow portions of both ends of the hollow fibers and at least two conduits communicating with the hollow fiber dispersion gap are provided.

A specific example is shown in FIG. Hollow fiber 1 consisting of multiple fibers
Both ends of the dispersed bundle are connected to the partition walls 3a and 3b at both ends of the container 2.
is fixed. Furthermore, both ends of the hollow fibers are open on the outer surface of the partition and communicate with the end chambers 4a, 4b and into the end conduits 5a, 5b. A culture solution can be taken in and out from the end conduits 5a and 5b. Also, at least two conduits 7 communicating with the hollow fiber dispersion gap 6
a.

7b is attached to the container 1, and is used as an inlet/outlet for inoculating cells and for circulating a cell suspension or a culture solution being cultured in the hollow fiber dispersion gap 6.

Any hollow fiber may be used as long as the membrane itself is not cytotoxic and is not altered or degraded by sterilization or culture medium. Examples of materials made from polymeric materials include cellulose esters, acrylic polymers, polysulfones, polyethersulfones, fluoropolymers, polyoffine polymers, polycarbonates, and cellulose. Furthermore, 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, a culture solution tank 8 contains a culture solution for growing cells, and this culture solution is pumped into a cell culture device 10 by a pump 9.
The liquid is sent to the end conduit 5a of the hollow fiber, exits from the end conduit 5b through the hollow portion of the hollow fiber, returns to the culture solution tank 8, and can be circulated. Cells are inoculated into the hollow fiber dispersion gap 6, and the cells are inoculated into the hollow fiber dispersion gap 6, and
The cells are supplied with nutrients from the culture solution flowing through the hollow part of the hollow fiber through the wall membrane of the hollow fiber, and grow. 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 make the microenvironment in which cells grow uniform, hollow m
The cell suspension or culture solution in the fiber dispersion gap 6 is supplied to the pump 11.
．． 12 allows circulation through the circuit 13, 14 and the hollow fiber distribution gap 6. A cell sedimentation tube 15 is provided between the hollow am dispersion gap 6 and the circuit 13 to prevent cells from being included in the circuit 13 as much as possible. Furthermore, a floating cell outlet 16 is provided at the bottom of the cell sedimentation tube 15. Between circuit 13 and circuit 14,
A circulating fluid storage tank 17 is provided, and a conduit 18 for supplying oxygen or nutrients and a circulating fluid outlet 19 are attached. When necessary components such as nutrients and oxygen necessary for the growth of cells in the hollow fiber dispersion gap 6 are insufficient to be supplied from the culture solution in the culture solution tank 8, or when DH adjustment is required, they are supplied through the conduit 18. Oxygen etc. are supplied.

Further, the circulating fluid can be taken out from the circulating fluid outlet 19 as needed, and metabolic products such as useful physiologically active substances produced by the cells can be recovered. The culture solution tank 8 is provided with a tank 20 for supplying a new culture solution and a tank 21 for taking out the old culture solution and useful physiologically active substances contained therein from the culture solution tank 8. By continuously supplying a culture solution from the tank 20, cells can be continuously grown.

Commonly used pumps such as tube pumps, plunger pumps, diaphragm pumps, and spiral pumps can be used as a means for circulating the cell suspension or culture solution in the hollow fiber dispersion gap, but they are easy to sterilize. 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 method. When using a pump, cells passing through the pump section are damaged to some extent, so it is desirable to prevent cells from being included in the circulating fluid passing through the pump section as much as possible. 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.

When culturing adherent cells, cells proliferate on the hollow fiber surface.1. Since a three-dimensional structure is constructed, it is not necessarily necessary to provide cell separation means in the circulation circuit. In addition, even when culturing planktonic cells (2), if the circulation is carried out by the drop-down method without using a pump, there is no cell damage caused by the pump, so it is not necessarily necessary to provide cell separation means, but the circulation Since it is subjected to shearing force, it is generally preferable to circulate it 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. Further, it is preferable to provide means for taking out the circulating fluid.

Generally, when the number of cells reaches a high density of approximately 5 x 10 Gcells/-, the rate of nutrient uptake by the cells increases rapidly, and the supply of oxygen becomes an especially 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 is true not only for oxygen, but also for hollow TA fibers used in cell culture vessels that are selected to be relatively impermeable to nutritional components in the polymeric matrix, and components that are difficult to pass through. Alternatively, it is preferable to supply a culture solution. Furthermore, in order to recover metabolites produced by the cells, particularly useful physiologically active substances, if necessary, it is also possible to remove the circulating fluid and add an appropriate amount of culture medium.

In order to maintain cell growth over a long period of time and obtain large amounts of useful physiologically active substances produced by cells, hollow lI
i. Timely expel some of the cells proliferating in the fiber dispersion gap from the culture system, maintain the number of cells in the hollow fiber dispersion gap as constant as possible, and maintain useful substance production activity of the cells. is preferred. 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, renal cell 2 epithelial cells, and the like. Examples of floating cells include lymphocytes, myeloma cells, white blood cells, and cells obtained by cell fusion of myeloma cells and other cells (hybridoma).
Examples include.

[Effects of the Invention] According to the culture method and culture device of the present invention, it is possible to efficiently proliferate cells and maintain a high cell density. At the same time, it is possible to efficiently produce useful physiologically active substances produced by cells.
By using @ or a plurality of them, it can be applied to mass culture.

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 fraction of the membrane is 50,000, and the container in the hollow fiber dispersion gap is 70d. Using this cell culture device, the cell culture device shown in FIG. 2 was assembled and steam sterilized. Culture solution tank 8 contains 3 types of culture solution (GIBCO's RPM
A medium (consisting of 90% I-1640 medium and 10% fetal bovine serum) was added, and the culture solution was circulated through the hollow part of the hollow fiber.

Further, in the hollow fiber dispersion gap 6, mouse myeloma cells P
2 x 10 mouse-mouse fusion cells using 3U1 as the parent strain
The suspension in the above culture solution was inoculated at 5 seeds/day. The circulating fluid storage tank was filled with 60 volumes 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. In the culture liquid tank 8 and circulating liquid storage tank 17: GLIW22.
18. Vent a mixed gas of 95% air and 25% CO or a mixed gas of 95% oxygen and 25% CO into the tank,
The dissolved oxygen concentration in the culture solution was maintained at 5 ppm. The circulation speed of the culture solution in the hollow portion of the hollow fiber was 30 m/min, and the circulation speed of circuit 13.14 was 5.47 m1n. 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 day, the 12th day, and 1st on the 16th day, the culture solution in the culture solution tank 8 was replaced with fresh culture solution.

Table 1 Unit: cells/Kai (blank below) As a comparative example, culture was carried out in the same manner as in Example 1 without circulating the cell suspension in the hollow IIi fiber dispersion gap. Compared to the invention, cell proliferation was slow and the number of cells was small.

Example 2 A cell culture vessel was assembled in the same manner as in Example 1 by bundling 2000 cellulose acetate hollow m-fibers with an inner diameter of 320 μm and a film thickness of 70 μm. The molecular weight fraction of the membrane is about 1 million, and the capacity of the hollow fiber dispersion gap is 70 m. The cell culture vessel was sterilized with ethylene oxide gas, the other parts were sterilized with steam, 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 speed in the hollow part of the hollow fiber was 60 d/min. The cell suspension was collected over time and the number of cells was measured, and the results shown in Table 2 were 19.

Table 2 (Margin below)

[Brief explanation of drawings]

FIG. 1 is an example of a cross-sectional view of a cell culture vessel used in the present invention. FIG. 2 shows an example of the cell culture device of the present invention. 1... Hollow fiber, 2... Container, 3... Partition wall,
4... End chamber, 5... End conduit, 6... Middle-
Empty fiber dispersion gap, 7... Side conduit, 8... Culture solution tank, 10... Cell culture device, 11, 12...
Pump, 13.14... Circulation circuit, 15... Cell sedimentation tube, 16... Cell suspension outlet, 17... Circulating fluid storage tank, 18.22... Conduit, 19... Circulation Liquid outlet

Claims (10)

[Claims] (1) A partition wall with a wall membrane that is permeable to nutrients necessary for cell growth but not permeable to cells so that both ends of the hollow fiber dispersed bundle open to the outside of the container. A cell culture vessel is provided with a conduit fixed to both ends of the container and communicating with a hollow part opened at both ends of the hollow fibers, and at least two conduits communicating with the hollow fiber dispersion gap. , a method in which a culture solution is poured into the hollow part of the hollow fiber and cells are cultured in the hollow fiber dispersion gap and/or on the outer surface of the hollow fiber, the cell suspension or culture solution in the hollow fiber dispersion gap is poured into a culture vessel. A cell culture method characterized by culturing while circulating using an external circulation means. (2) When circulating the cell suspension in the hollow fiber dispersion gap, the culture is carried out so that the circulating solution passing through the external circulation means does not contain cells as much as possible. The method described in paragraph 1. (3) When circulating the cell suspension or culture solution in the hollow fiber dispersion gap, while supplying nutrients, oxygen, and other components necessary for cell proliferation to the circulating solution, The method according to claim 1, characterized in that the culture is carried out while being taken out of the system. (4) The method according to claim 1, wherein the floating cells in the hollow fiber dispersion gap are cultured while being removed from the system as necessary. (5) The method according to claim 1, wherein the cells are floating animal cells. (6) The method according to claim 1, wherein the cells are adherent cells of an animal. (7) A partition with a wall membrane that is permeable to nutrients necessary for cell growth but not permeable to cells so that both ends of the hollow fiber dispersed bundle open to the outside of the container. a cell culture vessel, which is fixed to both ends of the container and connected to a hollow portion opened at both ends of the hollow fibers, and at least two conduits connected to the hollow fiber dispersion gap; A cell culture device comprising means for supplying a culture solution into the hollow portion of the hollow fibers and external circulation means for circulating the cell suspension or culture solution in the hollow fiber dispersion gap. (8) The device according to claim 7, wherein the external circulation means includes cell separation means. (9) The external circulation means includes means for supplying components necessary for cell proliferation such as nutrients and oxygen, and means for taking out the circulating fluid outside the system. Device. (10) The device according to claim 7, further comprising means for removing floating cells in the hollow fiber dispersion gap to the outside of the system.