JPS6212989B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial Application Field The present invention relates to an incubator for culturing and proliferating cells. More specifically, it relates to a container suitable for carrying out cell culture on a large scale,
This invention relates to a cell culture vessel in a suspension type cell culture system that is designed to efficiently supply nutrients to cells while removing by-product substances.

(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 achieved by mass culturing hybridomas of antibody-producing cells and myeloma, and solving this technology is an industrially important theme.

Conventionally, cell culture is generally 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).
It is classified into two types: dependent culture and suspension culture. However, most of the methods for mass culturing cells are the former method, which relies on adherent culture. This method of adherent culture has problems in supplying nutrients to cells and removing by-product waste, and there are still many hurdles to overcome before creating environmental conditions suitable for cell culture and adopting it for mass culture. It had the right points. In particular, the culture of animal cells has remained unclear.

On the other hand, there have been several recent proposals regarding 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. Cultivation methods equipped with a stirring function have been proposed (for example, in Japanese Patent Laid-Open No. 1983-1989).
(See Publication No. 65180).

However, in the above method, since the cells are incubated in a constant amount of nutrients, their growth and proliferation stop at a relatively low density.

(c) Structure of the Invention The present inventors have overcome the drawbacks of the conventional methods described above and conducted research on an incubator that can perform suspension culture in large quantities and at high density. invention has been achieved.

That is, the present invention is a suspension type cell culture device, and the culture device includes a hollow fiber having a wall membrane that substantially permeates nutrients and growth inhibitory substances necessary for cell culture, but does not substantially permeate cells. The capillary bundle is supported by a support for protecting the capillary bundle, and the support and the end of the capillary bundle have at least one unit fixed by a fixing layer. The cell culture vessel is housed in a cell culture vessel and is provided with a cell culture medium inlet connected to a conduit at one end of the unit and a cell culture medium outlet connected to a conduit at the other end.

Furthermore, in the cell culture device of the present invention, at least two of the units are connected in series or in parallel, so that the membrane permeation area can be increased or decreased as desired, and at least one of the units is connected in series or in parallel. The unit may have a cell culture medium inlet connected to a conduit at one end and a cell culture medium outlet at one end of the other unit.

Thus, in the incubator of the present invention, the unit is submerged in the liquid of the suspension type incubator, and a new culture medium is supplied from one end of the capillary bundle in the unit through the conduit, and the hollow fibers are New culture fluid is supplied to the suspension fluid through the wall membrane, and old culture fluid containing waste products, metabolic products, and other growth-inhibiting substances produced by the cells passes through the wall membrane and is transferred to the other end of the unit. Since the cells flow out of the incubator through the capillary bundle in the suspension, it becomes possible to easily culture a large amount of cells at high density in the suspension liquid. In particular, the unit of the present invention has two
Two or more cells can be immersed separately into the suspension solution, or two or more cells can be easily connected in series or in parallel. It is possible to easily adapt the area of the capillary wall membrane in response to changes.

The cell culture device of the present invention is applied to suspension-type cell culture, and suspension-type cell culture means that the cells themselves or supported on microcarriers are grown in a suspended state while floating in an aqueous medium. The cells may be plant cells, animal cells, or microbial cells, and may also be cells that have been modified artificially or by genetic manipulation. is suitable for culturing animal cells. Additives used in cell culture, such as inorganic salts, glucose, various amino acids, and antibiotics, can be added to the culture solution. Further, serum can be added, or a so-called serum-free medium without serum can also be used as the culture medium.

Furthermore, the cells may be allowed to float themselves, or may be grown by attaching the cells to the surface of microcarriers and suspending the attached matter.Also, the cells may be grown by a method using microcapsules. It may be.

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, the wall membrane is 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 (for example, 1000 or more, preferably 5000 or more) to pass through. It is also possible to use filter membranes.

The incubator of the present invention will be explained in more detail below with reference to the accompanying drawings.

FIG. 3 shows an example of the schematic diagram showing the entire cell culture device of the present invention, the first is a side external view of an example of a unit used in the cell culture device of the present invention, and FIG. 4 shows a sectional view of the unit, and FIG. 4 shows a side external view of another example of the unit of the present invention and an example in which the units are connected in series.

The unit shown in Fig. 1 and its sectional view are shown in Fig. 2.
In the figure, reference numeral 2 indicates hollow fibers, and the hollow fibers form a large number of bundles in the support 1 and are packed in parallel. A large number of holes 1' are provided on the side surface of the support 1 of the unit shown in FIG. 1, and the culture solution flows through these holes 1' and comes into contact with the wall membrane of the hollow fiber capillary bundle. . In the unit shown in FIG. 1, the hole 1' is circular, but the hole may have any other shape as long as the culture solution can flow therethrough. For example, as shown in FIG. 4, it may be a square, a rectangle, or an elongated rectangle, or it may be a triangle, an ellipse, etc., or a combination thereof. The support 1 only needs to be capable of supporting the hollow fiber capillary bundle, and does not need to be cylindrical as shown in Fig. 1. It is enough if you have it. Furthermore, support 1
can also have a connection function at its end when connecting them in series or in parallel.

In Fig. 3, 4 is the main body of the floating culture tank;
Fresh culture medium containing nutrients is supplied from its supply tank 6 by means of a pump 7 via a medium supply conduit 8 to one end of the unit installed in the suspension in the culture tank. The suspension culture tank 4 may be equipped with a stirring device 5 so that the cells are effectively suspended in the suspension liquid. Stirring is not limited to the rotary stirring blade as in 5, but may also be a magnetic stirrer. Furthermore, other means having a stirring effect that can effectively suspend the cells in the suspension liquid may be used. On the other hand, the culture solution introduced from one end of the unit passes through the hollow fiber capillary bundle, and new culture solution containing nutrients is supplied to the suspension solution through the wall membrane, as well as cell waste products and metabolic production. The old culture solution, including substances, permeates into the capillary tube and is removed via the other end of the unit and out of the suspension culture tank by a conduit 8'.

When actually culturing using the incubator of the present invention, the inlet of the new culture solution and the outlet of the old culture solution in the unit are reversed as necessary, and the inlet of the inlet of the old culture solution is It is also possible to take out the liquid and introduce a new culture medium through the outlet.

Furthermore, the culture tank is generally equipped with devices that measure the concentrations of oxygen, carbon dioxide, nutrients, and PH values and maintain them within a certain range, and the cell culture device 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
An aqueous solution containing nutrients such as glucose and protein, various amino acids, inorganic salts, antibiotics, and other components necessary for cell culture is used, but it may also contain serum. You don't have to be there. 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.
These may be supplied dissolved in a fresh culture solution, or may be supplied directly to the sampendion solution.

Fig. 4 shows an example in which four units of the present invention are connected in series, and holes of various shapes as shown in Fig. 4 are provided on the sides of the support of each unit. and each unit is connected by a unit connecting conduit 9. 3 is a short tube.

According to the cell culture device of the present invention described above, in suspension-type culture, a culture solution can be continuously supplied, and waste products, metabolic products,
Since growth-inhibiting substances can be removed, it becomes possible to culture a large number of cells at high density. Furthermore, the units of the present invention can be easily used by simply combining two or more units, in series or in parallel, or by combining two or more units independently to suit the scale of the device, cell type, culture conditions, etc. There is an advantage.

[Brief explanation of the drawing]

FIG. 1 is an external view of an example of a unit used in the cell culture device of the present invention, and FIG. 2 is a sectional view thereof. FIG. 3 shows an example of a schematic diagram showing the entire cell culture device of the present invention.
FIG. 4 shows an example in which the units are connected in series.

Claims (1)

[Scope of Claims] 1. A suspension-type cell culture vessel, which has a wall membrane that substantially permeates nutrients and growth-inhibiting substances necessary for cell culture, but substantially impermeable to cells. A capillary bundle consisting of a large number of hollow fibers, and the capillary bundle is supported by a support for protecting it, and the support and the end of the capillary bundle are connected to at least one of the units fixed by a fixing layer. A cell culture vessel in which a cell culture medium is housed, and the unit is provided with a cell culture medium inlet connected to a conduit at one end and a cell culture medium outlet connected to the conduit at the other end. 2 At least two of the units are connected in series or in parallel to form a unit connecting conduit that can increase or decrease the membrane permeation area, and at least one end of the tube is provided with a cell culture medium. 2. The cell culture device according to claim 1, wherein the inlet and the other terminal are provided with a cell culture solution outlet.