JPS6233878B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial Application Field The present invention relates to a culture method for culturing and propagating cells. More specifically, it relates to the method used for large-scale cell culture, and is a suspension-type cell culture system designed to efficiently provide nutrients to cells while removing by-product growth inhibitors and substances. This invention relates to a cell culture method. (b) Prior art For example, large-scale cell culture using viruses,
It is essential for the production of vaccines, antiviral agents such as interferon, and biological drugs such as hormones. Particularly in recent years, the production of monoclonal antibodies targeting specific proteins has been based on mass culturing of hybridomas using antibody-producing cells and myeloma, and solving this technology is an industrially important theme. Conventionally, cell culture has generally been carried out on a laboratory scale using Schare test tubes, culture bottles, and the like. On the other hand, in recent years, several proposals have been made regarding mass culture methods for cells and devices for the same. These proposals can be broadly divided into attached culture (anchorage culture).
Cell culture is classified into two types: dependent culture and suspension culture, and one of these methods is determined depending on the characteristics of the cells to be cultured. The present invention relates to an improved method for suspension-type cell culture. There have been several recent proposals on how to culture cells by suspension culture, for example by means of a magnetic stirrer or an impeller on a mechanically driven shaft, arranged in a spinner flask. Culture methods equipped with a stirring function have been proposed (for example, see Japanese Patent Application Laid-open No. 1983-65180). However, in the above method, since the cells are cultured in a fixed amount of nutrients, the growth and proliferation of cells stops at a relatively low density. (c) Structure of the Invention Therefore, the present inventors overcame the drawbacks of the conventional methods as described above and conducted research on a culture method that enables large-scale and high-density culture using the suspension culture method, and as a result, the present invention was developed. invention has been achieved. That is, the present invention provides a suspension liquid in a suspension-type cell culture tank with a hollow space formed by a wall membrane that substantially permeates nutrients and growth-inhibiting substances necessary for cell culture, but does not substantially permeate cells. A capillary bundle consisting of a large number of threads, in which at least one unit is fixed by a fixing layer is housed at both ends of the capillary bundle, and (i) the culture solution in the suspension solution is transferred to the walls of the hollow fibers of the unit. (ii) After that, an aqueous liquid medium that does not adversely affect the cell culture is supplied from outside the culture tank to both ends or one end of the unit through the conduit. This cell culture method is characterized in that the liquid medium is then allowed to flow into the suspension liquid through the wall membrane of the hollow fiber, and the above-mentioned discharge and inflow are repeated alternately. Furthermore, in the method of the present invention, at least two of the units are housed in the suspension liquid of a suspension type cell culture tank, and each unit is operated so that the discharge and inflow are alternately repeated; More advantageous cultivation can be achieved by mutually controlling the intervals between discharge and inflow of each unit so that the discharge of the culture solution in the suspension solution in the culture tank does not stop. Thus, according to the culture method of the present invention, a unit consisting of at least one capillary bundle is submerged in a suspension liquid in a suspension-type culture tank, and waste products, metabolites, and The old culture solution containing other growth-inhibiting substances flows from the suspension solution into the hollow fiber through the wall membrane of the hollow fiber, and is discharged from both ends or one end of the unit to the outside of the culture tank via a conduit, and after a certain period of time has passed. This discharge is stopped, and then an aqueous liquid medium that does not adversely affect the cell culture is supplied from outside the culture tank to both ends or one end of the unit through the conduit, and the liquid medium flows into the suspension liquid through the hollow fiber wall membrane. I am forced to do it. Therefore, in the method of the present invention, the unit submerged in the suspension liquid supplies an aqueous liquid medium into the suspension liquid through the wall membrane of a large number of hollow fibers provided therein, and Since the drainage from the John's solution to the old culture solution is repeated alternately, the pores of the hollow fiber wall membrane will not be clogged with cells or other solids, which will result in a substantial decrease in wall area efficiency, which is advantageous. It is possible to smoothly supply an aqueous liquid medium and to drain old culture medium, making it possible to culture cells at an extremely high density. In particular, in the method of the present invention, two or more units can be immersed into the suspension liquid separately or in a suitable combination, and two or more units can be easily connected in series or in parallel for use. , it is possible to easily adapt the wall area of the hollow fiber according to changes in cell type and density, culture conditions, and culture scale. In addition, in the present invention, at least two units are used in combination, and each unit is operated so that the suspension liquid of the culture tank is repeatedly supplied with the liquid and the old culture liquid is discharged. In both units, the intervals between the draining and inflowing of the liquid in each unit are mutually controlled so that the old culture medium is constantly drained, that is, the draining of the culture medium in the suspension liquid in the culture tank does not stop. can do. The cell culture method of the present invention is applied to suspension type cell culture.
It refers to various types of suspension culture in which cells are grown in an aqueous medium while floating themselves, while floating on microcarriers, or in microcapsules. In particular, the present invention is advantageously used in a system in which the cells themselves are cultured while being suspended. The cells cultured in the cell culture method of the present invention may be plant cells, animal cells, microbial cells, etc., or cells that have been modified artificially or by genetic manipulation. In particular, the culture method of the present invention is suitable for culturing animal cells. In the suspension-type cell culture tank of the present invention, cells to be cultured are cultured in a suspended state in a culture solution. The culture solution is an aqueous medium consisting essentially of water, to which are added additives commonly used in cell culture, such as various inorganic salts, vitamins, enzyme traps, glucose, amino acids, and antibiotics. Further, serum can be added to the culture solution, or a so-called serum-free medium that does not use serum can also be used as the culture solution. In the method of the present invention, the "aqueous medium that does not adversely affect cell culture" that is supplied from outside the culture tank to the unit in the suspension liquid through a conduit is mainly sterile water, in which one of the above-mentioned additives is added. or more can be used. This aqueous medium may be a fresh culture medium containing all the additional components necessary for the culture, and need not contain all of these components, some of which may be added to the suspension solution by other means of supply. It can also be introduced. Furthermore, as the aqueous medium, it is also possible to use part of the culture solution (hereinafter sometimes referred to as "old culture solution") discharged from the unit to the outside of the culture tank through the conduit according to the method of the present invention. . The hollow fiber capillary constituting the unit used in the method of the present invention may be made of various polymers, and preferred examples of such polymers include cellulose acetate, polysulfone, polyacrylonitrile, and fluorine-based polymers. Examples include: The wall of the hollow fiber capillary has many micropores, and the wall of the capillary is permeable to nutrients, cell waste products, metabolic products, etc. Microcapsules are provided with many pores that are sized to prevent penetration. When the cells themselves are cultured in suspension, the size of the pores depends on the size of the cells, but generally an average pore size of 10 μm or less, preferably 8 μm or less is suitable. On the other hand, when cells are cultured by adhering to the surface of microcarriers (microcarriers) or when cells are cultured using microcapsules, the pores need to be of a size that does not allow them to pass through. It is also desirable that the capillary wall membrane has the ability to permeate water to some extent. That is, it is advantageous for the wall membrane to have a water permeability coefficient (ml/m ² ·hr · mmHg) of 10 or more, preferably 100 or more.
On the other hand, there is no particular upper limit, but 20,000 or less, preferably
Desirably 10000 or less. Furthermore, as the wall membrane, compounds with a small molecular weight such as nutrients, cellular waste products, and metabolic products permeate, but compounds with a large molecular weight (e.g.
1000 or more, preferably 5000 or more), it is also possible to use a membrane that does not pass, such as an ultrafiltration membrane. The cell culture device of the present invention will be explained in more detail below with reference to the accompanying drawings. The attached FIG. 1 shows an example of a schematic diagram for carrying out the cell culture method of the present invention, and FIG.
Figure A, Figure 2-B, and Figure 2-C each show an example of a schematic diagram of a unit in the present invention. Fig. 1 shows the case where two units 3-a and 3-b are operated as a pair for culturing. In Fig. 1, 1 is the culture main body in a suspension type culture tank, The aqueous medium containing substances is sent from its supply tank 6 by means of a pump 7 through a supply conduit 8 and via a switching valve 12 or 14 to one unit. On the other hand, the old culture solution is discharged from the other unit and the switching valve 13 or 1
5 and through a discharge conduit 11 by means of a pump 10 to a storage tank 9 for old culture medium. In the two units 3-a and 3-b shown in FIG. 1, both ends of a capillary bundle consisting of a large number of hollow fibers are fixed by a fixed layer. The upper part serves as a liquid inlet and outlet, and the lower part has a sealed structure. The culture tank 1 may be equipped with a stirring device 2 as shown in FIG. 1 so that the cells are effectively suspended in the suspension liquid. It is not limited to a rotary stirring blade as in stirring 2, but a magnetic stirrer may also be used. Furthermore, other stirring effects that can effectively suspend the cells in the suspension liquid may be used. Although the culture tank shown in FIG. 1 is shown in one set in which two units are paired, the number of pairs is not limited to one, and a plurality of sets may also be used. Alternatively, the units may not be used as a pair, but a single independent unit may be used to repeatedly discharge the culture solution and supply the aqueous liquid medium to this unit. It goes without saying that a large number of these independent units can be used. Furthermore, culture tanks are generally equipped with devices that measure oxygen, carbon dioxide, nutrient concentrations, and PH values and maintain them within certain ranges, and the cell culture tank of the present invention also incorporates these devices. Needless to say, it may be provided. However, these accessories are omitted from FIG. The new culture solution supplied to the culture tank of the present invention is
Among these, grapes and other materials containing nutrients such as proteins, various amino acids, minerals, antibiotics, and other components necessary for cell culture are used as an aqueous solution.
Furthermore, it may or may not contain serum. All of these components may be supplied through the unit as a culture solution, but some components may also be introduced into the suspension solution by other supply means. Generally, cell culture requires oxygen, carbon dioxide, etc., but these may be supplied dissolved in the new culture medium.
Further, as shown in the first example, the suspension liquid may be directly supplied from the gas supply pipe 17 through the valve 16. Figures 2-A, 2-B and 2-C all show examples of various forms of units used in the method of the present invention. In these figures, the arrow → indicates the switching valve 1 in the culture tank in Figure 1.
2, 13, 14, or 15, the conduit is connected to a liquid inlet or outlet. The unit shown in FIG. 2-A is basically of the same form as the unit shown in FIG.
A common liquid inlet and outlet are provided at one end, and the other end has a sealed structure. In Figure 2-B, both ends of the capillary bundle are fixed to the fixed layer, liquid inlets and outlets are provided at both ends, conduits are connected to each of these inlets and outlets, and these conduits are connected to form a common liquid inlet. It has a structure that forms a discharge port. Furthermore, Fig. 2-C shows a structure in which both ends of the capillary bundle are fixed by one common fixing layer, and both ends have a common inlet and outlet for liquid. ing. Each of the units described above is an example of a basic type, and other slight changes are of course allowed. A plurality of these units can be used in any combination. In the method of the present invention, one unit is operated while alternately supplying an aqueous liquid medium (for example, fresh culture medium) and draining old culture medium, and the intervals are determined depending on the cell type, density, and culture. Although it depends on the conditions, it can generally be switched within a range of 1 minute to 1 day, preferably 5 minutes to 10 hours. The method of the present invention can also be operated by submerging one unit in the suspension liquid and alternately repeating the supply and discharge of the liquid as described above, or by submerging two or more units to be operated in this way and submerging each unit. May be operated independently. However, it is generally preferred to use two or more units, in which case it is advantageous to operate the units in conjunction with one another. When operating multiple units in conjunction with each other, make sure that the suspension solution in the culture tank does not stop discharging the culture solution to the outside of the tank (in other words, the old culture solution is constantly drained to the outside of the tank). It is more desirable to mutually control the intervals between the discharge and inflow of liquid in each unit so that the liquid discharge and inflow of each unit are carried out.
In addition, two units are submerged in the suspension liquid as a pair, one of which is operated to supply an aqueous liquid medium into the suspension liquid, and the other unit is operated so that old culture is discharged, and their flow is controlled. Supplying an aqueous liquid medium by operating the pair of units alternately in this way by switching the switching valve so that the values are alternately reversed;
The old culture solution can be continuously drained at all times, and the liquid level of the suspension in the culture tank can be easily maintained at a constant level. In carrying out the method of the invention, the interval between the supply of aqueous liquid medium and the discharge of old culture medium in a given unit can be arbitrarily determined. In general, old culture solution can be drained for a long time as long as cells etc. do not block the pores of the hollow fiber wall membrane and reduce the amount of discharge, but it is preferable to drain the old culture solution for at least 30 minutes and within 10 days. When the cell density is low, the time is relatively long, and when the cell density is high, the time tends to be short. On the other hand, supplying an aqueous liquid medium (eg fresh culture medium) generally takes less time than said draining. Generally, 1 minute or more is sufficient. The supply time also depends on the supply amount (per area), and the longer it is, the shorter it is. On the other hand, if it is too small, it will be necessary to make it longer. In particular, it is often advantageous to supply the aqueous liquid medium over a short period of time under slight pressure. According to the culture method of the present invention described above, in suspension-type culture, supply of new culture medium and draining of old culture medium are performed continuously or semi-continuously, so that a large number of cells can be cultured at high density. You can. In particular, in the present invention, even if the cells in the wall membrane of the hollow fibers in the unit are temporarily clogged with cells or other solid matter, the clogging will not occur for a long time, and therefore the culture medium can be exchanged effectively. It is possible to perform extremely high-density culture. Furthermore, the units of the present invention can be easily used by simply combining two or more units, in series or parallel, or by combining two or more units independently to suit the scale of the device, cell type, culture conditions, etc. It has the advantage of being The method of the present invention will be described in detail below with reference to Examples. Example (1) Properties of the unit; One side is sealed as shown in Figure 2-A of the attached drawings using a large number of hollow fibers formed from a polymer alloy consisting of cellulose acetate, acrylic acid polymer, and cellulose nitrate. I created a unit that was The inner diameter of each hollow fiber was 320μ and the outer diameter was 460μ, and the wall membrane had many micropores with an average pore diameter of 0.2μ. The perforated length of the hollow fibers in the unit was 36 mm, and the total effective area (based on the outer diameter) of the hollow fibers was 80 cm ² . The water permeability of the hollow fiber used was 4/m ² .
It was hr・mmHg. (2) Shape of the culture tank; A glass container with an internal volume of 500 ml and having the structure shown in attached Figure 1 was used as the culture volume. This culture tank houses two units as shown in FIG. 1, and is also equipped with a marine type stirrer. In addition, carbon dioxide and oxygen supply pipes are inserted into the culture tank, but these are 5 in Figure 1.
Applies to. (3) Composition of new culture solution: The composition of the new culture solution used was as follows. Units are 1mM unless otherwise specified.
(millimol). NaCl 110.0mM KCl 5.0 MgSO ₄ 0.55 MgCl ₂・6H ₂ O − Ca(NO ₃ ) ₂・4H ₂ O − CaCl ₂・2H ₂ O 0.74 NaH ₂ PO ₄・2H ₂ O − Na ₂ HPO ₄・12H ₂ O 3.41 CuSO・5H ₂ O 3.0×10 ^-6 FeSO ₄・7H ₂ O 8.0×10 ^-4 Fe(NO ₃ ) ₃・9H ₂ O − ZnSO ₄・7H ₂ 0 8.0×10 ^-4 Alanine 0.075 Arginine・HCl 2.76 Asparagine・H ₂ O 0.63 Asparatic acid 0.30 Cysteine・HCl・H ₂ O 0.60 Cystine − Glutamic acid 0.27 Glutamine 6.84 Glycine 0.57 Histidine・HCl・H ₂ O 0.36 Hydroxyproline 0.24 Isoleucine 1.20 Lecine 1.26 Lysine・HCl 1.08 Me Thionine 0.33 Phenylalanine 0.45 Proline 0.48 Serine 0.81 Threonine 0.93 Tryptophan 0.09 Tyrosine 0.48 Valine 0.93 p-Amino benzoic acid 3.7×10 ^-3 Biotin 4.1×10 ^-4 Pantothenate−Ca 2.6×10 ^-3 Cholin chloride 8.8×10 ^-2 Folic acid 4.1×10 ^-3 Inositol 2.6 ×10 ^-1 Niacinamide 1.2×10 ^-2 Pyridoxal･HCl 4.9×10 ^-3 Pyridoxine･HCl 2.5×10 ^-3 Riboflavine 5.6×10 ^-4 Thiamin･HCl 4.7×10 ^-3 Vitamin B ₁₂ 2.5×10 ^-4 Lipoic acid 2.5×10 ^-4 Glutathicne 1.6×10 ^-3 Glucose 19.00 Hypoxanthine 7.5×10 ^-3 Putrescine・2HCl 2.5×10 ^-4 Pyruvate−Na 1.0 Thymidine 7.5×10 ^-3 Linoleic acid 7.5×10 ^-5 Phenol red 5.0mg/L HEPES 1.19g/L NaHCO ₃ 12.50mM Streptomycin 0.1g/L Penicillin G 10 ^-5 U/L Insuline 7.5μg/ml Transphclin 2μg/ml Ethanolamine 10μM/L Na ₂ SeO ₃ 2.5×10 ^-9 M/L (4) Cultivation method: A new culture solution was put into the culture tank so that the liquid volume was 300 ml, and two units were inserted into it. Each unit is connected to a new culture solution supply tank as shown at 6 in FIG. 1 and to an old culture solution storage tank 9 via a conduit via a switching valve. A pump is installed in the middle of each conduit, and the two units are operated by switching valves so that when one unit is supplying new culture solution to the culture tank, the other unit is supplying old culture solution to the tank. They are designed to flow in opposite directions so that they are discharged to the outside. On the other hand, in the culture tank, CO ₂ gas and O ₂ gas are supplied into the suspension liquid, and these supplies are applied to the system pH 6.5 to 7 and the amount of dissolved oxygen to 3.
Controlled to a constant ~4ppm. Mouse myeloma cells in the culture medium of the culture tank
Mouse hybridoma with p3u1 as the parent strain
4C10B6 cell line was added at 1 x ^10.5 cells/ml. This cell is an immunoglobulin (IgG) producing strain. The suspension solution during culture was maintained at 27°C, and a marine type stirrer was rotated at a speed of 60 rpm. After starting the culture, the cell density is 1×10 ⁶ cells/
ml, we started operating the two units, supplying new culture medium and draining old culture medium. The feeding and draining intervals in one unit were approximately every 12 hours, and approximately 300 ml of fresh culture solution was fed to the culture tank per day. However, after 5 days from the start of culture, the supply of this new culture medium was gradually increased to a maximum of 500 ml per day. (5) Culture results: The results of culturing for 9 days were as follows. The final cell density was 2 x ¹⁰⁶ cells/ml when culturing was carried out under exactly the same conditions except that no unit of the present invention was used. 【table】

[Brief explanation of the drawing]

FIG. 1 shows an example of a schematic diagram for carrying out the cell culture method of the present invention, and FIG.
2-B and 2-C respectively show examples of the form of the unit used in the cell culture method of the present invention.

Claims (1)

[Scope of Claims] 1. The suspension solution of a suspension-type cell culture tank is formed of a wall membrane that substantially permeates nutrients and growth-inhibiting substances necessary for cell culture, but does not substantially permeate cells. A capillary bundle consisting of a large number of hollow fibers, each end of which is housed with at least one unit fixed by a fixed layer, and (i) the culture solution in the suspension solution is transferred to the hollow fibers of the unit. (ii) After that, the water is discharged from both ends or one end of the unit through the wall membrane to the outside of the culture tank through a conduit, and (ii)
An aqueous liquid medium that does not adversely affect the cell culture is supplied from outside the culture tank to both ends or one end of the unit through a conduit, and the liquid medium flows into the suspension liquid through the hollow fiber wall membrane, and the liquid medium is discharged. A cell culture method characterized by repeating alternating inflow and inflow. 2 At least two units are housed in the suspension liquid of a suspension-type cell culture tank, each unit is operated so that the discharge and inflow are alternately repeated, and the suspension in the culture tank is 2. The cell culture method according to claim 1, characterized in that the intervals between discharge and inflow of each unit are mutually controlled so that the discharge of the culture medium in the John's solution does not stop.