JPH0338830B2 - - Google Patents

Abstract

PURPOSE:To cultivate cells efficiently, by making constitution wherein plural culture units are piled, cells are subjected to suspension culture, a settling zone is set in each culture unit and the cells are separated from an oil culture solution by gravity. CONSTITUTION:Plural culture units are arranged in piles in the vertical direction in a main body 5 of a culture device in such a way that a culture solution can be circulated through each culture unit. Further each culture unit is divided into a suspension culture zone for cells and a settling zone 7 by a partition wall 6 so that the culture units are communicated through the lower parts. The culture device 5 is charged with the culture solution from a conduit 1 and oxygen from a conduit 3, cells are kept in a suspension state by a stirrer 8 and the cells are multiplied by culture. Then, the cells are separated from the culture solution at the settling zone 7 by gravity, the culture solution containing no cells is discharged from conduits 2-1-2-4 and the cells are settled on the bottom of the device 5.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial Application Field The present invention relates to an apparatus for culturing and proliferating cells. More specifically, the invention relates to an apparatus for culturing cells in suspension.

Cell culture technology, for example, viruses, vaccines,
It is important for the production of antiviral agents such as interferon or biological drugs such as hormones.
Furthermore, in recent years, monoclonal antibodies targeting specific proteins have been produced by culturing hybridomas consisting of antibody-producing cells and myeloma.
The solution of this technology is an industrially important theme.

(b) Prior Art 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 cell culture methods and devices for the same. These proposals can be broadly divided into anchorage culture (anchorage dependent culture).
It is classified into two methods: -dent culture) and suspension culture, and one of these methods is determined depending on the characteristics of the cells to be cultured.

Among these, the following proposals have been made regarding suspension culture. Cultivation methods have been proposed in which a spinner flask is provided with a regulated stirrer, for example by a magnetic stirrer or an impeller on a mechanically driven shaft (U.S. Pat. No. 2,958,517; 3649465).

JP-A-57-65180 discloses that at least one flexible sheet with a relatively large surface area supported on a rotatable shaft is used as an agitator, and the agitator is rotated to ripple the sheet. Suspension culture devices have been proposed, thereby creating the desired gentle agitation for certain fragile cells, such as human diploid cells.

However, in the culture method using the above device,
Since the cells are cultured in a constant amount of nutrients, cell growth and proliferation cease at a relatively low density.

In cell suspension culture, in order to prevent cell growth and proliferation from stopping at a relatively low density and to culture cells in large quantities and at high density, growth is generally inhibited while supplying new culture medium to the culture tank. A method has been proposed in which the old culture solution containing substances is cultured while being drained out of the culture tank, which is usually called the perfusion method.
Report of Fermentation Processes, vol.6
reference). One of the important things when culturing using this method is to efficiently separate the living cells in the suspension solution from the old culture solution, take out the old culture solution out of the culture tank, and put it inside the culture tank. The goal is to maintain the cell growth environment under optimal conditions. Various filters and various formats have been proposed to separate living cells and old culture fluid from the suspension solution, but industrial culture is difficult due to problems such as clogging of the filter and complicated structure of the device. As a device, it is difficult to say that either of them is satisfactory.

JP-A-59-82083 and JP-A-60-9482
In the publication, a culture supernatant discharge pipe that also serves as a cell sedimentation tube and a conduit for adding fresh medium are provided in the culture device, and the medium is added from the conduit and at the same time, the culture supernatant is discharged from the discharge pipe. A high-density culture device for floating cells has been proposed.

In general, floating animal cells are small, ranging from several microns to several tens of microns, and their specific gravity is not significantly different from the specific gravity of the culture medium. In order to advantageously achieve this, it is necessary to increase the sedimentation area, but with the devices disclosed in the above-mentioned two JP-A-Sho, it is not possible to increase the sedimentation area very much.

(c) Object of the Invention Therefore, an object of the present invention is to provide an apparatus that has a simple structure and is capable of separating living cells and a culture medium from a suspension culture medium.

Another object of the present invention is to provide an apparatus and method that can separate living cells and old culture fluid without using a filter, and thus eliminate problems caused by clogging of the filter. It is about providing.

Still another object of the present invention is to provide an industrially advantageous apparatus for suspension culture that can provide a very large sedimentation area for advantageously achieving cell sedimentation.

Still another object of the present invention is to provide an apparatus that can easily remove dead cells and their debris, which are often a problem in suspension culture, out of the culture tank.

Still another object of the present invention is to provide an industrial device using a perfusion method suitable for culturing cells in large quantities and at the same density.

Further objects and advantages will become apparent from the description below.

(d) Structure and Effects of the Invention According to the research of the present inventor, the above-mentioned objects and advantages of the present invention are provided for suspension culture including at least two culture units and having a culture solution supply port. A cell culture device,
The culture unit consists of a cell suspension culture zone, a cell settling zone, and a culture solution outlet from the top of the settling zone. The culture units are separated by partitions communicating through the lower part, the settling zone having a structure formed between the outer wall of the culture unit and the partition, and each culture unit having a structure in which the culture medium can flow through the culture unit. This is achieved by means of a cell culture device characterized in that it is permissibly stacked vertically.

The cell culture device of the present invention has a structure in which two or more culture units, preferably 2 to 10, particularly preferably 2 to 5, are stacked so that the culture solution can flow in the vertical direction. . The culture unit is composed of a cell suspension culture zone, a cell settling zone, and a culture solution outlet from the upper part of the settling zone. a partition in communication through a lower portion of the culture unit, the settling zone having a structure formed between an outer wall of the culture unit and the partition.

In the culture apparatus of the present invention, living cells settle to the bottom in the direction of gravity in the settling zone installed in the culture unit, and old culture solution and cell debris are separated to the top of the settling zone. The culture solution substantially free of living cells can be discharged from the culture solution outlet provided at the upper part of the settling zone. Furthermore, since the apparatus of the present invention is constructed by arbitrarily stacking two or more culture units, it is easy to install a relatively large settling zone volume relative to the actual volume of the culture, and if necessary, the culture unit can be stacked as desired. The culture scale can be changed by increasing or decreasing.

Furthermore, the present invention makes it possible to efficiently separate living cells and culture medium using a simple method without using filters that cause clogging, so it is possible to culture a large amount of cells at the same density. It can be applied advantageously.

The culture unit in the culture apparatus of the present invention includes a cell suspension culture zone that can be defined as an area where cells are suspended to carry out substantial cell culture, and a cell suspension culture zone that can be defined as an area where cells are separated from the culture medium by gravity. It has a settling zone of The cell suspension culture zone and the cell settling zone are separated by a partition so as to communicate through a lower portion of the settling zone in the cell culture tank. With such a structure, in the cell settling zone, the cells that have settled and separated from the culture medium can be reintroduced into the cell suspension culture zone through the lower part of the settling zone,
The area is then recultured.

In the device of the invention, the settling zone within the culture tank is formed between the outer wall of the culture device and the partition. With this structure,
There is an advantage that the effective settling volume of the settling zone within the culture apparatus can be relatively large.

The apparatus of the present invention has a culture solution outlet for discharging old culture solution separated from cells in the settling zone of each culture unit and a culture solution supply port for supplying new culture solution to the culture device. There is one or more locations in any location. It is desirable that such a supply port be provided at the top of the device.

In the culture unit of the present invention, the settling zone is preferably formed between the device side wall and the partition so as to surround the suspension culture zone. In this preferred embodiment, for example, the suspension culture zone and the settling zone are separated by a cylindrical partition. in this case,
More preferably, a substantial portion of the side wall facing the cylindrical partition is cylindrical, and the cylindrical partition and the cylindrical tank side wall are located substantially concentrically.

Furthermore, since the culture unit of the present invention promotes cell settling in the settling zone,
Means may be included within the settling zone to facilitate settling of the cells. Such means are
It is a means for shortening the distance in the direction of gravity over which cells settle, and can be, for example, a settling plate provided within the settling zone in a direction that blocks the direction of gravity.

Furthermore, in the settling zone, a wall board extending in the direction of gravity and partitioning the settling zone is provided in order to minimize the influence of the flow of the culture medium formed in the cell suspension culture zone. You can also do it. It will be readily understood that a wall plate placed in the settling zone at an angle to the direction of gravity functions as a settling plate.

The culture apparatus of the present invention may include means for forcibly flowing the culture medium in the cell suspension culture zone. Such means are
For example, it can be a mechanical stirring means for forcibly suspending the cells in the culture solution. In addition, the culture device is provided with an oxygen-containing gas inlet, or an oxygen-containing gas inlet and an oxygen-containing gas guide tube,
The cells can also be forcibly suspended in the culture zone due to the stirring effect when the oxygen-containing gas introduced through the inlet rises.

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, such as hybridomas. In particular, the culture apparatus of the present invention is suitable for culturing animal cells.

In the suspension type cell culture device 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.

Perfusion culture of cells using the culture device of the present invention is advantageously carried out by withdrawing a culture medium substantially free of cells from the settling zone and introducing a new culture medium into the culture device. .

^At that time ^, the ratio V/ S is preferably in the range of 0.1 to 500 cm, particularly preferably 1 to 100 cm.

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

First, the present invention will be explained with reference to the attached FIGS. 1 to 3. Each of FIGS. 1 to 3 shows an example of a schematic diagram of the apparatus of the present invention.

In FIG. 1, the culture device main body 5 is provided with a settling zone 7 partitioned off by a partition wall 6 inside its inner wall. The culture device 5 is equipped with a culture solution supply port through a new culture solution supply conduit 1, oxygen or oxygen-containing gas is supplied through a conduit 3, and is also provided with an exhaust gas discharge conduit 4. There is. Furthermore, an outlet for discharging the old culture solution is provided in the upper part of the settling zone 7, and the culture solution is discharged to the outside of the culture apparatus through the conduits 2-1 to 2-4.

What is the culture unit referred to in the apparatus of the present invention? In Fig. 1, when the culture apparatus is cut in a cross section at right angles to the longitudinal direction, the area occupied by the culture solution outlet 2-1 to 2-2 is called one culture unit. be able to. FIG. 1 includes four culture units.

In FIG. 1, a stirring device 8 is installed in a culture device 5,
By rotating it, the cells are maintained in suspension.

On the other hand, in FIG. 2, numerals 1 to 8 refer to means and devices having the same functions as those in FIG. The difference from FIG. 1 is that a guide tube 9 is provided in the culture tank 1, and oxygen or oxygen-containing gas is supplied to the lower part of the guide tube 9 through a conduit 3.
By supplying oxygen or an oxygen-containing gas to the lower part of the powder guide tube 9, the suspension liquid rises inside the guide tube 9 from the bottom to the top, and conversely from the top to the bottom outside the guide tube 9. Movement of the suspension liquid occurs, and the suspension liquid as a whole is stirred.

FIG. 3 shows a schematic longitudinal section of an apparatus for introducing oxygen dissolved in fluorocarbon into a culture tank. In the apparatus of Fig. 3, compared to the apparatus of Fig. 1, etc., fluorocarbon with dissolved oxygen is introduced from the fluorocarbon inlet 3-1, and while falling downward, it contacts the culture medium and supplies oxygen to the culture medium. Thereafter, the fluorocarbon is accumulated in the lower part of the culture tank and is discharged from the fluorocarbon discharge port 4-1. In Figure 3, except for 3-1 and 4-1, 1 to 8 correspond to Figure 1.
means the same means and devices as .

1 to 3, a settling zone 7 partitioned by a partition wall 6 is provided inside the inner wall of the culture apparatus, and the stirring effect of the suspension liquid does not substantially reach the settling zone. It seems like there is no such thing. Settling zone 7
Inside, the cells are designed to sink in the direction of gravity, and the old culture medium containing growth inhibitors, which is essentially free of cells, is present at the top.

The settling zone only needs to be an area in which the linear velocity of the culture solution is slower than the sedimentation velocity of the cells and where the suspension solution in the culture tank cannot be agitated, and there are no restrictions on its shape and structure. I don't accept it.

In the apparatus shown in FIGS. 1 to 3, a settling zone 7 and a suspension culture zone are separated by a cylindrical partition 6, and a substantial portion of the tank side wall facing the cylindrical partition 6 is cylindrical. , and the cylindrical partition wall and the cylindrical tank side wall are located substantially concentrically.

In each culture unit in the powder apparatus,
S, which is the basis for calculating V/S (where V is the volume of culture medium in the suspension culture zone and S is the effective settling area of cells), is expressed by the following formula.

S=π/4(D ² - d ² ) Here, D is the diameter of the cylindrical tank side wall, and d
is the diameter of the cylindrical partition wall 6. Further, V should be understood as the volume obtained by subtracting the volume of the space where no culture solution exists and the volume of the settling zone from the volume of the culture tank.

Furthermore, a culture device is generally equipped with a device that measures oxygen, carbon dioxide, nutrient concentrations, and PH values 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 the accompanying drawings.

When culturing is carried out using the apparatus of the present invention, a new culture medium contains nutrients such as glucose and protein, various amino acids, inorganic salts, antibiotics, and other necessary ingredients for cell culture in an aqueous solution. However, it may or may not further contain serum.

This new culture solution is supplied from the culture solution supply port 1 to the suspension culture device, for example, from the culture solution supply port 1 provided at the top of the culture device as shown in FIGS. 1 to 3. Alternatively, it may be fed directly into the suspension liquid through a conduit.

Furthermore, when performing culture using the apparatus of the present invention, it is necessary to supply new culture medium and drain old culture medium so that the liquid level in the culture apparatus is maintained at a substantially constant level. Although desirable, it is not necessary. The supply of new culture solution and the discharge of old culture solution can be performed independently, continuously, or intermittently.

In order to maintain the oxygen concentration in the suspension liquid constant, as a method of supplying oxygen, as shown in FIGS. 1 and 2, oxygen or an oxygen-containing gas in the suspension liquid may be directly supplied. Alternatively, other supply means may be used. Another supply means is, for example, a method using an oxygen carrier as shown in FIG. As the oxygen carrier, a liquid compound capable of dissolving oxygen without substantially mixing with water is used, examples of which include various fluorocarbons used as materials for artificial blood. When such a fluorocarbon is used as an oxygen supply means, the fluorocarbon in which oxygen is dissolved may be added from the top of the suspension liquid in the form of droplets or a thin film.

Stirring methods include a mechanical stirring method such as the rotation of a stirring blade shown in Figure 1, a draft effect method using a guide cylinder shown in Figure 2, and a method using dissolved oxygen as shown in Figure 3. Examples include a method of simultaneously supplying oxygen and stirring by using a carrier. Of course, two or more of these stirring means can be used in combination.

In addition, when carrying out culture, the ratio of supplying new culture solution to the effective culture volume (V) of the culture tank (supply amount of new culture solution/V) should be determined per day.
A suitable range is from 0.2 to 10, preferably from 0.5 to 5.

Thus, the apparatus of the present invention can easily separate old culture fluid that does not contain living cells in suspension culture of cells, and can also remove cell debris from the suspension fluid together with the old culture fluid. Furthermore, the device of the present invention has a simple structure and is not complicated to operate, making it suitable as an industrial device.
It is particularly advantageous for mass culture and high density culture of cells.

[Brief explanation of the drawing]

1 to 3 each show an example of a schematic diagram of the culture apparatus of the present invention.

Claims (1)

[Claims] 1. A cell culture device for suspension culture comprising at least two culture units and having a culture medium supply port, the units comprising a cell suspension culture zone, a cell settling zone, and an upper part of the settling zone. In the culture unit, the suspension culture zone and the settling zone are separated by a partition so as to communicate through a lower part of the settling zone, and the settling zone is connected to the settling zone. A cell culture device having a structure formed between an outer wall of a culture unit and the partition, wherein each culture unit is vertically stacked to allow circulation of culture solution. .