JPS60156378A - Culture medium of cell - Google Patents

Abstract

PURPOSE:In cell culture of suspension type, to make mass production of cell with high density possible, by making it possible to feed a culture solution successively and to remove an effect matter, a metabolic product, and a growth inhibitory substance continuously. CONSTITUTION:A culture solution in the feed tank 6 is continuously fed from the culture solution feed conduit 8 to one end of the unit 1 immersed in a culture solution in the culture medium 4 of cell of suspension type by the pump 7. In the unit 1, a capillary bundle consisting of a great number of hollow systems having wall membranes to permeate a nutritive component required for cell culture, an effect matter, a metabolic product, and a growth inhibitory substance, but not to permeate cells is supported by the supporting member 3. The culture solution is passed through the capillary bundle, fed to the culture medium 4. Meanwhile, the effect matter, the growth inhibitory substance, etc. are taken out from the other end of the unit 1 by the conduit 8'.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Use The present invention relates to a culture vessel for culturing and proliferating cells. More specifically, it relates to a container suitable for carrying out cell culture on a large scale, and relates to a cell culture vessel in a suspension type cell culture system, which is made for the purpose of efficiently supplying nutrients to cells and removing by-product substances. It is something.

City) Conventional technology For example, viral cell culture on a large scale.

Vaccines, antiviral drugs such as interferon.

It is also essential for the production of biological drugs such as hormones.

Particularly in recent years, the production of mononuclear 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.

Conventional cell culture is generally carried out on a laboratory scale using Petri dish test tubes, culture bottles, and the like.

On the other hand, in recent years, as a method for mass culturing cells and a device for that purpose,
Several proposals have been made. These proposals can be broadly divided into two types:
(ent culture) and suspension culture (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, regarding the culture of animal cells, -+g was unclear.

On the other hand, there have been several recent proposals regarding the method of culturing cells by suspension culture, for example using a magnetic stirrer or an impeller on a mechanically driven shaft, using a controlled stirring geometry in a spinner flask. For example, a culture method using a method for cultivating a sterilized cell has been proposed (see, for example, Japanese Unexamined Patent Publication No. 57-65180).

However, in the above method, since the cells are cultured in a constant amount of nutrients, the growth and proliferation of the 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 an incubator that is capable of large-scale, high-density culture using suspension culture. result,
We have arrived at the present invention.

vinegar! [In other words, the present invention is a suspension type cell culture vessel, and the culture vessel has a wall membrane that substantially permeates nutrients necessary for cell culture and growth-inhibiting substances, but does not substantially permeate cells. a capillary bundle consisting of a large number of hollow fibers having a capillary bundle supported by a support for protecting the capillary bundle;
The support and the end of the capillary bundle house at least one unit fixed by a fixing layer, a cell culture medium inlet connected to a conduit at one end and a cell culture medium inlet connected to the conduit at the other end. This is a cell culture vessel equipped with a cell culture solution outlet.

Furthermore, the cell culture device of the present invention includes at least two of the units.
are connected in series or in parallel, thereby making it possible to arbitrarily increase or decrease the membrane permeation area, one end of at least one of the units having a cell culture medium inlet connected to a conduit and another A cell culture solution outlet may be provided at one end of the unit.

Thus, in the incubator of the present invention, the unit is submerged in the solution of the η″ suspension type incubator, and new culture solution 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 mold through the wall membrane, and old culture fluid containing waste products, 2 metabolic products produced by the cells, and other substances that inhibit life passes through the wall membrane to the other end of the unit. Since the cells flow out of the incubator through a certain capillary bundle, it is easy to culture a large amount of cells at high density in the suspension solution.In particular, the unit of the present invention allows two or more cells to be cultured separately in the suspension solution. However, two or more cells can be easily connected in series or in parallel, allowing easy infiltration of capillary tubes according to changes in cell type and density, culture conditions, and culture scale. It is possible to adapt the area of the wall membrane.

The cell culture device of the present invention is applied to suspension type cell culture, and suspension type means that cells are grown in a suspension state by themselves or by supporting them on microcarriers and floating in an aqueous medium. It refers to the culture method, and the cells are plant cells and animal cells.

It may be any microbial cell, and may also be a cell modified artificially or by genetic manipulation.

In particular, the culture vessel of the present invention is suitable for culturing animal cells. The culture solution contains inorganic salts and glucose.

Additives used in cell culture, such as various amino acids and antibiotics, can be added. Further, serum can be added, or a so-called serum-free medium without serum can be used as the culture medium.

Furthermore, the cells may be suspended per se, or they may be attached onto the surface of microcarriers,
A method of growing by suspending the deposits may be used, or a method using microcapsules may be used.

The hollow fiber capillary constituting the unit of the present invention may be made of various polymers, such as cellulose acetate-1, polysulfone, polyacrylonitrile, fluorine-based polymers, etc. .

The wall of the hollow fiber capillary has many micropores, and the wall of the capillary contains nutrients and cellular waste.

There are many theories that there are pores of a size that allow metabolic products to pass through, but not cells or microcarriers or microcapsules to which they are attached. When cells are cultured in suspension, the size of the pores depends on the size of the cells, but generally the average pore size is 10 μm or less, preferably 8 μm or less. 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 large enough to prevent them from passing through.

It is also desirable that the capillary wall membrane has the ability to permeate water to some extent. That is, the wall membrane has a water permeability coefficient (d/end・hr・mmHg) of 10 or more, preferably 1
Advantageously, it is greater than or equal to 00. On the other hand, there is no particular upper limit, but it is preferably 20,000 or less, preferably 10,000 or less.

Furthermore, the wall membrane contains nutrients and cellular waste products.

Compounds with small molecular weight such as metabolic products pass through, but
It is also possible to use a lI9, for example an ultrafiltration membrane, which does not permeate compounds with large molecular weights (for example, 1000 or more, preferably 5000 or more).

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, FIG. 1 is a side external view of an example of a unit used in the cell culture device of the present invention, and FIG. The figure 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.

In FIG. 2 showing the unit of FIG. 1 and its sectional view, 2 is a hollow fiber, 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 shown in FIG. It is supposed to be done. 1 in this figure 1
Although the hole 1' in the microtube is circular, it is sufficient that the culture solution can flow through the hole, and the hole may have a square shape.

For example, as shown in Figure 4, square, rectangle, i
It can be a long rectangle, and its square triangle.

It may be elliptical or a combination thereof. Support 1
It is sufficient that it is capable of supporting the hollow fiber capillary bundle, and it does not need to be cylindrical as shown in Figure 1, and has the function of bringing the culture solution into effective contact with the capillary wall membrane of the hollow fibers. That's fine.

Furthermore, the support 1 can also have a connection function at its end for connecting the supports in series or in parallel.

In Fig. 3, 4 is the main body of the floating culture tank, and a new culture solution containing nutrients is fed from the supply tank 6 via a culture solution supply conduit 8 by a pump 7 to a unit installed in a hanging WJ'a in the culture tank. is supplied to one end of the 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 5 J: sea urchin rotary type stirrer, but may also be a magnetic stirrer. Furthermore, a means having a stirring effect capable of effectively suspending cells in the suspension liquid may be used.

On the other hand, the culture solution introduced from one end of Unit 1 passes through the hollow fiber capillary bundle, and new culture solution containing nutrients etc. is supplied to the suspension solution through the wall membrane. The old culture solution containing products passes through the capillary tube and exits the floating culture tank through the other end of the unit via conduit 8'.
It is extracted by

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

Furthermore, the concentration of oxygen, carbon dioxide and nutrients in the culture tank,
Although devices for measuring p+-+ values and maintaining them within certain ranges are generally installed, it goes without saying that the cell culture vessel of the present invention may also be equipped with these devices. However, these accessories are omitted from FIG.

The new culture solution to be supplied to the culture tank of the present invention is an aqueous solution containing nutrients such as glucose and protein, 1 various amino acids, inorganic salts, antibiotics, and other components necessary for cell culture. However, it may or may not contain serum. All of the necessary components may be supplied as a culture solution through Units 1 to 1, but some of the components may also be introduced into the suspension solution by the supply means described above. Generally, cell culture requires oxygen, carbon dioxide, etc., and these may be supplied dissolved in a new culture solution, or may be supplied directly to the suspension solution.

Figure 4 shows an example in which four units of the present invention are connected in series, and the support of each unit has holes of various shapes as shown in Figure 4 on its side. and each unit (31, unit connection conduit 9
are connected by.

3 is a short tube.

According to the cell culture device of the present invention described above, in suspension-type culture, the culture solution can be continuously supplied, and waste products 9 and metabolic products can be continuously supplied.

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 used by simply combining two or more units in series or in parallel, or by combining two or more units independently depending on the scale of the device, type of cells, culture conditions, etc. However, it has the advantage of being easy to use.

[Brief explanation of drawings]

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)

[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 impermeates 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 in which the membrane permeation area can be increased or decreased, and the cell culture solution is connected to the end of the tube in at least one of the eyes. 2. The cell culture device according to item 1, wherein the inlet and the cell culture solution outlet are provided at the end of the cell culture medium.