JPS6212990B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial Application Field The present invention relates to a culture device for culturing and proliferating cells. More specifically, it relates to equipment suitable for large-scale cell culture, including cell culture in a suspension-type cell culture system designed to efficiently supply nutrients to cells while removing by-product substances. It is related to the device.

(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 a suspension-type cell culture device, and there have recently been several proposals regarding methods for culturing cells by suspension culture, such as using a magnetic stirrer or a mechanically driven shaft. A culture method has been proposed in which a spinner flask is provided with a controlled agitation function using an impeller (see, for example, Japanese Patent Application Laid-Open No. 57-65180).

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 device that is capable of culturing in large quantities and at high density using the suspension culture method. We have arrived at the present invention.

That is, the present invention is a suspension-type cell culture device, and the culture device includes a wall membrane that substantially permeates nutrients and growth-inhibiting substances necessary for cell culture, but does not substantially permeate cells. The capillary bundle consists of a large number of hollow fibers having a capillary bundle, and both ends of the capillary bundle house at least one unit fixed by a fixing layer, and both ends or one end of the unit have a common inlet and outlet for the cell culture medium. This is a cell culture device characterized in that a conduit for supplying or discharging a cell culture medium is connected so as to have a cell culture medium.

Furthermore, in the cell culture device of the present invention, at least one pair of the units is housed in the culture device, and the pair of units mutually prevents cell culture fluid from flowing into and out of the culture device. It is also possible to use a cell culture device that repeats alternating cycles.

Thus, in the culture apparatus of the present invention, the unit is submerged in the liquid of the suspension type culture apparatus, and a common inlet and outlet for the cell culture medium provided at one end or both ends of the capillary bundle in the unit. Fresh culture medium is supplied through the conduit and into the suspension liquid through the wall membrane of the hollow fibers. Then, after a certain period of time, the supply is stopped, and the old culture fluid containing waste products, metabolites, and other growth-inhibiting substances produced by the cells flows from the suspension fluid through the wall membrane to the hollow fibers through the same inlet and outflow ports, and then flows through the conduit. It is operated so that it flows out of the culture solution. That is, the unit in the culture apparatus of the present invention is capable of supplying new culture solution into the suspension solution and discharging the old culture solution from the suspension solution through the wall membrane of a large number of hollow fibers housed therein. Since this is repeated alternately, the pores of the hollow fiber wall membrane will not be clogged with cells or other solid matter, resulting in a substantial loss of wall area efficiency, and it is advantageous to supply new culture medium and dispose of old culture medium. This allows extremely high-density cell culture.

In particular, two or more units of the present invention can be immersed into a suspension liquid separately or in a suitable combination, and two or more units can be easily connected in series or parallel for use. It is possible to easily adapt the wall area of the hollow fiber according to changes in type and density, culture conditions, and scale of culture.

In addition, in the present invention, at least one pair of units is used in combination, and one pair of units is used to supply new culture solution and the other to drain old culture solution in the suspension solution of the culture device. It can be operated such that the flow of the culture medium is alternately reversed.

The cell culture device 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 device 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 apparatus of the present invention is suitable for culturing animal cells.

Additives used in cell culture, such as inorganic salts, various amino acids, antibiotics, etc., can be added to the new culture solution. Further, serum can be added, or a so-called serum-free medium without serum can be used as the culture medium. Moreover, a part of the old culture solution can be recycled and used in the new culture solution.

The hollow fiber capillary constituting the unit of the present invention may be made of various polymers, such as cellulose acetate,
Examples include polysulfone, polyacrylonitrile, and fluorine-based polymers.

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
10000 or less is desirable.

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.

Attached FIG. 1 shows an example of a schematic diagram showing the entire cell culture device of the present invention, and FIG.
2-B and 2-C each show an example of a schematic diagram of a unit in the present invention.

FIG. 1 shows an apparatus for culturing by operating two units 3-a and 3-b as a pair. In FIG. 1, 4 is a culture tank main body in a suspension type culture apparatus; Fresh culture solution containing nutrients etc. is sent from the supply tank 6 by a pump 7 through a supply conduit 8 and via a switching valve 9 to one unit. On the other hand, the old culture solution flows out from the other unit and is sent via the switching valve 9 through the discharge conduit 10 to the storage tank 12 for the old culture solution by means of a pump 11. The pair of units in FIG. 1 are submerged in suspension fluid,
One unit is operated so that new culture medium is fed into the suspension and the other unit is operated so that old culture is discharged, and the switching valve 9 is switched so that their flows are alternately reversed. ing. By operating the pair of units alternately in this way, it is possible to continuously supply new culture solution and drain old culture solution at all times, and also to maintain the liquid level of the suspension in the culture tank at a constant level. can be easily maintained.

In the two units 3-a and 3-b shown in FIG. 1, a capillary bundle consisting of a large number of hollow fibers is housed in a cylindrical support 1, and both ends of the capillary bundle are fixed by a fixing layer. . The upper part serves as an inlet and outlet for the culture solution, and the lower part has a sealed structure. A large number of holes 2 are provided on the side surface of the support 1 of the unit, through which the suspension liquid flows and comes into contact with the wall membrane of the hollow fiber capillary bundle. Although the hole 2 in the support 1 of the unit shown in FIG. 1 is circular, the hole 2 may have any other shape as long as the suspension fluid can flow therethrough. For example, it may be triangular, square, rectangular, elongated, oval, or a combination thereof. Support 1
It is sufficient that the hollow fiber capillary bundle can be supported, and does not need to be cylindrical as shown in Fig. 1, and can effectively bring the hollow fiber capillary wall membrane and the suspension liquid into contact with each other. It suffices if it has a function to do so. Furthermore, the support may have a connection function at its end for connecting two or more units in series or in parallel.

The culture tank 4 may be equipped with a stirring device 5 as shown in FIG. 1 so that the cells are effectively suspended in the suspension liquid. Stirring is not limited to a rotary stirring blade as in 5, but may also be a magnetic stirrer. Furthermore, other stirring effects that can effectively suspend the cells in the suspension liquid may be used.

Although the culture apparatus 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. Further, the units may not be used as a pair, but an independent unit may be provided and the culture solution may be repeatedly supplied and discharged from this unit. It goes without saying that a large number of these independent units can be used.

Furthermore, a culture tank is generally equipped with a device that measures the concentration of oxygen, carbon dioxide, nutrients, and PH value and maintains them within a certain range, and the cell culture device of the present invention also includes 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 aqueous solutions containing nutrients such as proteins, various amino acids, inorganic salts, antibiotics, and other components necessary for cell culture are used.
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 can be supplied dissolved in new culture medium.
Furthermore, it may be supplied directly to the suspension liquid.

2-A, 2-B and 2-C all show examples of various forms of the unit of the present invention. In these figures, the arrow → indicates a conduit connected to a common outflow inlet of the culture medium which is connected to the switching valve 9 in the incubator of FIG.

The unit shown in Fig. 2-A is basically of the same form as the unit shown in Fig. 1, in which both ends of a capillary bundle consisting of a large number of hollow fibers are fixed by a fixed layer.
One end is provided with a common inlet and outlet for the culture solution, and the other end has a sealed structure. In Figure 2-B, both ends of the capillary bundle are fixed to the fixed layer, and inlets and outlets for the culture solution are provided at both ends, and conduits are connected to each of these inlets and outlets, and these conduits are connected to allow a common culture solution to flow through the inlet and outlet. It has a structure that forms an inlet and an outlet. In addition, Fig. 2-C shows a structure in which both ends of the capillary bundle are fixed by one common fixed layer, and both ends have a common inlet and outlet for the culture solution. are doing. Each of the units described above is an example of a basic type, and other slight changes are of course allowed. These units can be used in any combination.

The units of the present invention are operated by alternately supplying new culture medium and discharging old culture medium, and the intervals are determined depending on the cell type, density,
Although it depends on culture conditions etc., generally 1
It can be switched in the range of minutes to 1 day, preferably 5 minutes to 10 hours.

According to the culture apparatus of the present invention described above, in suspension-type culture, it is possible to continuously or semi-continuously supply new culture solution and drain old culture solution, so that it is possible to perform high-density and large-scale cultivation. Cells can be cultured. 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

[Brief explanation of the drawing]

FIG. 1 shows an example of the schematic diagram showing the entire cell culture device of the present invention, and FIG. 2-A, FIG. 2-B, and FIG. This figure shows an example of the form of a unit used in the device.

Claims (1)

[Scope of Claims] 1. A suspension-type cell culture device, which includes a wall that substantially permeates nutrients and growth-inhibiting substances necessary for cell culture, but substantially impermeable to cells. It consists of a capillary bundle made up of a large number of hollow fibers having membranes, and at least one unit fixed by a fixing layer is housed at both ends of the capillary bundle,
A cell culture device characterized in that a conduit for supplying or discharging a cell culture solution is connected to both ends or one end of the unit so as to have a common inlet and outlet for the cell culture solution. 2. At least one pair of the units is housed in the culture device, and each of the pair of units alternately causes the cell culture solution to flow into and out of the culture device. The cell culture device described in Section 1.