JPH0229310B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial Application Field The present invention relates to a culture tank for culturing and propagating cells. More specifically, the present invention relates to a suspension-type cell culture tank manufactured for the purpose of culturing cells on a large scale and at high density.

(b) Prior Art Mass cell culture is an important technology for the industrial production of viruses, vaccines, antiviral agents such as interferon, or biological drugs such as hormones. Particularly in recent years, the production of monoclonal antibodies targeting specific proteins has been achieved by mass culturing hybridoma cells by combining antibody-producing cells and myeloma cells, and solving this technology is an industrially essential problem.

In addition, culturing cells on a large scale and at high density is an essential technology for producing the above-mentioned useful substances at low cost, and a rapid solution to this technology is desired.

Conventionally, suspension-type cell culture has generally been carried out on a laboratory scale using culture bottles or spinner flasks.

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 concentration.

In recent years, several proposals have been made for suspension-type high-density cell culture methods and devices for the same. For example, a high-density culture device has been proposed that rotates a cylindrical rotating body that has a filter on a plane parallel to a rotating shaft driven by a magnetic stirrer or motor, and replaces the culture solution while preventing the filter from clogging using centrifugal force. (See US Pat. No. 3,647,632).

However, in the above method, there are restrictions on increasing the filtration area per unit cell or per culture solution beyond a certain level, making it extremely difficult to scale up.

(c) Structure of the Invention Therefore, the present inventors overcame the drawbacks of the conventional methods as described above and proceeded with research on a culture tank that is capable of large-scale and high-density culture using a suspension type cell culture method. As a result, the present invention was achieved.

That is, the present invention is a suspension-type cell culture tank, in which at least one plate-like body having at least partially a filter region through which the culture solution permeates but not cells is provided. This is a suspension-type cell culture tank in which a rotary filter is installed in the culture tank and is attached to a conduit that takes out the cellulose to the outside of the apparatus so as to rotate coaxially with the conduit.

The cell culture tank of the present invention is applied to suspension type cell culture, which means that the cells themselves are suspended in an aqueous medium or that the cells are supported on microcarriers. Refers to various suspension cultures in which cells are grown in suspension or in microcapsules. In particular, the present invention can be advantageously used in a method of culturing cells in suspension.

The cells cultured using the cell culture tank of the present invention may be plant cells, animal cells, microbial cells, etc., or may be cells that have been modified artificially or by genetic manipulation. In particular, the culture tank of the present invention is suitable for culturing animal cells.

In the suspension-type cell culture tank of the present invention, the cell culture to be cultured is cultured in a suspended state in a culture solution. The culture solution is an aqueous medium consisting essentially of water, to which additive components commonly used in cell culture, such as various inorganic salts, vitamins, coenzymes, glucose, amino acids, and antibiotics, are added. 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.

The filter region of the disc-shaped body in the present invention is provided with a large number of pores of a size that allows the culture solution to pass through, but not the cells, the microcarriers to which they are attached, or the microcapsules in which they are contained. When cells are cultured in suspension, the size of the pores depends on the size of the cells, but in general, an average pore size of 10 μm or less, preferably 8 μm or less is suitable. On the other hand, microcarriers
When cells are cultured by adhering to the surface of the microcapsule or when cells are cultured using microcapsules, the pores need to be large enough to prevent them from passing through.

It is also desirable that the filter has the ability to transmit water to some extent. In other words, the filter has a water permeability coefficient (ml/ ^m2・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.

In addition, the filter region includes a membrane that allows small molecular weight compounds such as nutrients, cellular waste products, and metabolic products to pass through, but does not allow large molecular weight compounds (e.g., molecular weight of 1000 or more, preferably 5000 or more) to pass through. It is also possible to use filter membranes.

In the present invention, the disk-shaped body means a/
A ratio of b of 2 or more and 100 or less is suitable, and a ratio of 2 or more and 30 or less is particularly preferred.

Further, the area ratio of the plate-shaped bodies per cross-sectional area of the culture tank perpendicular to the rotation axis of the culture tank is preferably 10% or more and 95% or less, particularly preferably 20% or more and 80% or less.

Appropriate cross-sectional shapes of the plate-shaped body include thin rectangles, rhombuses, triangles, ellipses, and semi-ellipses.

The filter area is provided at least partially on such a disk-like body, and the filter area may be provided on almost the entire surface of the disk-like body, or may be provided on only a part of the disk-like body. If it is a part, it may be the upper surface, the lower surface, or a part of these surfaces.

Hereinafter, the cell culture tank of the present invention will be explained in more detail with reference to the accompanying drawings.

Attached Figure 1 shows an example of the schematic diagram for carrying out culture using the cell culture tank of the present invention, and Figure 2-A shows a diagram of the disc-shaped body used in the cell culture tank of the present invention. Fig. 2-B is an external view of one example, and Fig. 2-B shows the cross-sectional structure of the plate-like body, and Fig. 3-A is an external view of another example of the plate-like body. It shows its cross-sectional structure. Also, Figure 4 shows the first
The figure shows a schematic diagram of a separate cell culture tank.

Fig. 1 shows the case of culturing by rotating the disk-shaped body 5. In Fig. 1, 1 is the main body of a suspension-type culture tank, and the aqueous medium containing nutrients, etc. From the supply tank 2, it is sent by a pump 3 through a supply conduit 4 into the culture tank. The culture tank 1 is equipped with a disk-like body 5 having a filter portion so that the filter area is submerged in the suspension liquid, and this disk-like body is rotated by a drive unit 6 to effectively transfer the cells into the culture medium. to float. Stirring is not particularly necessary, but stirring can be made more effective by using a stirring blade attached to the top, bottom, or middle of the disc. Alternatively, a separately provided magnetic stirrer may be used. Furthermore, other means having a stirring effect that can effectively suspend cells in the culture solution may be used.

On the other hand, the old culture solution is passed through a filter area provided on the lower surface of the plate-like body 5 by the pump 3'.
It passes through the rotating shaft and is taken out of the culture tank through a conduit 4'.

In FIG. 2-B showing the disk-shaped body in FIG. 2-A and its cross section, 7 is a filter area;
It is attached to a conduit 8 coaxial with the rotation axis. 2
- In Figure B, the filter is of the same planar shape and is attached to the bottom surface, but it may have other shapes.
For example, Figure 3-A and Figure 3-B showing its cross section.
As shown in the figure, it may be donut-shaped with filter areas (hatched areas) on both the top and bottom sides.

By making the direction of the rotation axis perpendicular to the plane of the filter area, centrifugal force can prevent the filter from clogging. It is possible to easily increase or decrease the area of the filter. 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 includes these devices. Needless to say, it may be provided. However, these accessories are omitted from FIGS. 1 and 4.

The new culture solution supplied to the culture tank of the present invention is
This includes nutrients such as glucose and protein,
An aqueous solution containing components necessary for cell culture such as various amino acids, inorganic salts, and antibiotics is used, and may or may not further contain serum. All of the necessary components may be supplied as a culture source, but some of the components may also be introduced into the culture medium by other supply means.

Figure 4 shows a row of four disc-shaped bodies connected in series in multiple stages like 9 in the present invention, and the number of disc-shaped bodies and the area of the filter part can be increased or decreased to accommodate the culture tank. It is possible to easily obtain suitable stirring and filtration areas. In FIG. 4, 1, 2, 3, 4, 3' and 4' have the same meanings as in FIG.

According to the cell culture tank of the present invention as described above, it is possible to continuously supply a culture medium in suspension-type cell culture, and to continuously remove waste products, metabolic products, etc.
Since growth-inhibiting substances can be removed and the filter does not become clogged, it is possible to culture a large number of cells at high density over a long period of time.

[Brief explanation of drawings]

FIG. 1 shows an example of a schematic diagram showing the entire cell culture tank of the present invention. FIG. 2-A is an external view of an example of a disk-shaped body used in the cell culture tank of the present invention, and FIG. 2-B is a cross-sectional view thereof. FIG. 3-A is an external view of another example of the disk-shaped body used in the present invention, and FIG. 3-B is a cross-sectional view thereof. FIG. 4 shows a schematic diagram of an example of a culture tank in which the plate-shaped bodies are connected in series in multiple stages.

Claims (1)

[Claims] 1. A sun pendillion type cell culture tank, in which at least one disk-like body having at least partially a filter region through which the culture medium permeates but not cells is coaxially connected to the conduit for taking out the culture medium out of the apparatus. A suspension-type cell culture tank in which a rotatable filter is installed in the culture tank.