JPS6336774A - Cell culture apparatus - Google Patents

Abstract

PURPOSE:To enable the separation of living cells and culture liquid from a suspension culture liquid using an apparatus having simple structure, by separating a suspension culture zone of cell from a settling zone of cell in a manner to connect the zones with each other at the lower part of a culture tank. CONSTITUTION:A cell culture tank 5 is provided with a suspension culture zone 11 of cell, a settling zone 7 of cell and the ports 2-1-2-4 for discharging culture liquid from the top of the zone 7. The zone 11 and the zone 7 are separated with each other with a partition plate 6 in a manner that the zones are connected with each other through the lower part of the zone 7 in the tank 5. The zone 7 is formed between a side wall of the tank 5 and the partition plate 6. A baffle plate 8 is placed between the inner wall of the tank 5 and the partition plate 6 along the inner wall in a manner that the baffle plate 8 does not form a flow of the culture liquid in the partitioned zone above the zone 7. The zone 11 is furnished with a culture liquid feeding port 1. Living cells and culture liquid can be separated from suspension culture liquid by the use of the apparatus having the above simple structure.

Description

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

Examples of cell culture technology include viruses and vaccines.

Antiviral agents such as interferons are important for the production of biological drugs such as hormones.

Furthermore, in recent years, the production of monoclonal antibodies targeting specific proteins has been achieved by culturing hybridomas made of antibody-producing cells and myeloma, and solving this technology is an industrially important theme.

(b) Prior Art Conventionally, cell culture has generally been carried out on a laboratory scale using Petri dish 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 attached media M (anchorage d(?pen-d
ent culture) and 1 culture, that is, suspension culture.
It is classified into two methods: (e) and one of these methods is determined depending on the characteristics of the cells to be cultured.

The following proposals have been made regarding this back suspension culture. Cultivation methods have been proposed in which a controlled stirring function is provided in the spinner flask, for example by a magnetic stirrer or an impeller on a mechanically driven shaft (U.S. Pat. No. 2,958,517). @
and 3.649.465).

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 sheet is waved by rotating the at least one agitator. Suspension culture devices have been proposed that allow cells to stand up and thereby create the gentle agitation desired for the fragile cells of certain species, such as human diploid cells.

However, in the culturing method using the above-mentioned apparatus, the cells are cultured in a fixed amount of nutrients, so the growth and proliferation of the cells stops at a relatively low density.

In cell glue suspension culture, in order to prevent the growth and proliferation of cells from stopping at a relatively low density and to culture cells in large quantities and at high density, growth inhibitors are generally added while supplying new culture medium to the culture tank. A method has been proposed in which culture is carried out while draining the old culture solution containing
Natural Report-ts of Fermenta
tion Processes, vol. 6).

What is important when culturing using this method is to efficiently separate the living cells in the 1-nospension solution from the old culture solution, take out the old culture solution out of the 18-culture tank,
The goal is to maintain the cell growth environment in the culture tank under optimal conditions. Various filters and various types of filters have been proposed to separate living cells and old culture fluid from the suspension solution, but industrial culture equipment is difficult to use due to problems such as filter mouth clogging and the complexity of the equipment structure. It is hard to say that either of these results is satisfactory.

JP-A-59-82083 and JP-A-60-94
82 @ Publication states that a culture supernatant discharge tube that also serves as a cell sedimentation tube and a conduit for adding fresh medium are provided in the culture device, and the culture medium is added from the conduit and at the same time the culture supernatant is discharged from the discharge tube. A high-density culture device for floating cells has been proposed.

In general, floating animal cells are small, ranging from a few microns to several tens of microns, and their specific gravity is not much different from the specific gravity of the culture medium. In order to advantageously achieve this, it is necessary to increase the settling area, but with the apparatuses disclosed in the above-mentioned two JP-A-Sho, the settling area cannot be made very large.

(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's about doing.

A further object of the present invention is to provide an industrially advantageous device for four-nospension culture, which allows a very large sedimentation area to advantageously achieve cell sedimentation.

Still another object of the present invention is to provide an apparatus that can easily remove dead cells and their broken pieces, which are often a problem in 4-nospension 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 effects of the present invention are such that the cell culture unit has a cell glue suspension culture zone and a cell settling zone. 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 in the cell culture tank, The settling zone is formed between the tank side wall of the cell culture tank and the partition, and a baffle plate is installed between the tank inner wall and the partition plate along the tank inner wall of the settling zone, and the baffle plate is a cell culture device comprising at least a cell culture unit characterized by being installed so that the culture solution in the area delimited by the cell culture unit does not form a flow above the settling zone, the culture device any V of
This is achieved by using a cell culture tank for perfusion culture in which the suspension culture zone is provided with a culture solution supply port.

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. A culture solution substantially free of living cells can be discharged from a culture solution discharge L1 provided above the settling zone. In addition, the apparatus of the present invention may have only one culture unit, but if two or more are arbitrarily stacked, it is easy to set a relatively large settling zone volume relative to the actual culture volume. If necessary, the culture scale can be changed by increasing or decreasing the number of tJl' feeding units.

Roughly speaking, the present invention makes it possible to efficiently separate living cells and culture medium using a simple method without using filters that can cause clogging, making it ideal for culturing cells in a wide open space and at the same density. It can be applied to

The culture unit in the culture apparatus of the present invention includes a cell glue suspension culture zone which can be defined as an area where cells are subjected to lisbend to carry out substantial cell cultivation, and a cell suspension culture zone which can be defined as an area where cells are separated from the culture medium by vehicle force. It has a settling zone of The cell glue 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. Because of this structure, in the settling zone of the cell,
Cells that have settled and been separated from the culture medium are reintroduced into the cell suspension culture zone through the lower part of the settling zone and cultured again in that area.

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. Such a structure has the advantage that the effective settling volume of the settling zone within the culture apparatus can be relatively large.

The apparatus of the present invention includes a culture solution outlet for discharging old culture solution separated from cells in a settling zone of the culture unit and a culture solution supply port for supplying new culture solution to the culture device. Have 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, it is particularly preferred that a substantial portion of the side wall facing the cylindrical partition is cylindrical, and that the cylindrical partition and the cylindrical tank side wall are substantially concentrically located.

Furthermore, in the settling zone in the culture unit of the present invention, a baffle plate is installed along the inner wall of the inner tank between the tank side wall and the partition plate, and the baffle plate allows the culture solution in the area separated by the baffle plate to settle. It is installed so that no flow forms in the upper part of the zone. Specifically, for example, as can be seen from FIG. The area is separated by a baffle plate (7 in the case of Figure 1).
(areas) are separated with no gaps so that the culture solution does not form any flow.

In this way, the baffle plate in the settling zone only needs to be installed at least above the settling zone in order to fulfill its purpose, and its length should be the same or shorter (as viewed from the top) in relation to the partition plate. It is preferable. Although it is desirable that the baffle plate be a flat plate, it may have communicating holes as long as the purpose of not forming a flow of the culture solution is achieved.

The baffle plate does not need to be rough. Although there may be more, 1 to 3 is generally advantageous.

If baffles in the settling zone are not installed, or even if they are installed but there is a gap above them, a flow of culture medium will often occur at the top of the settling zone, which will cause the original flow to occur. The intended separation of cells and culture medium within the settling zone by gravity is not effectively achieved, making it difficult to smoothly discharge the culture medium that does not contain cells.

On the other hand, according to the present invention, since the flow of the culture medium is not formed by the baffle in the inside of the settling zone, especially in the upper part, the cells are effectively settled by gravity, and the culture medium without cells is discharged. This can be done advantageously.

The cell culture device of the present invention is applied to suspension-type cell culture, and "suspension type" means that the cells themselves are suspended in an aqueous medium, or the cells are cultured in a microcarrier (
It refers to various types of suspension culture in which cells are grown in microcapsules while floating on microchirelia. In particular, the present invention is advantageously used in a system in which the cells themselves are cultured in suspension.

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, containing various Alua salts, vitamins, enzyme traps, glucose,
Additives commonly used in cell culture, such as 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.

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

In this case, the effective retrieval area of cells defined by the settling zone in each culture unit [S (cm
2) ] and the accumulation of culture solution in the suspension culture zone [v(CIl13)] tono ratio ■/S force preferably 01
It is in the range of 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 4. Each of FIGS. 1 to 4 shows an example of a schematic diagram of the apparatus of the present invention.

Figure 1. In the culture unit main body 5, a settling zone 7 partitioned off by a partition wall 6 is provided inside the inner wall of the culture unit body 5. The culture unit 5 is provided 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 an exhaust gas discharge conduit 4 is provided. There is. Roughly, the settling zone 7 is partitioned by a cylindrical baffle plate 8 so that no flow occurs, and a discharge port for the old culture solution is provided at the top of each area, and the old culture solution is discharged through the conduits 2-1 to 2-4. The culture solution is drained outside the culture unit.

A stirring device 9 is installed in the culture unit 5, and the cells are maintained in suspension by rotating the stirring device 9.

In Figure 2, the culture unit in the device of the present invention is the area occupied by the culture solution outlet 2-1-1 to 2-2 when the cross section is cut at right angles to the longitudinal direction of the culture device. It can be called a culture unit. In Figure 2, four culture units are included.

On the other hand, in FIG. 3, 1 to 9 mean means and devices having the same functions as those in FIG. 2. The difference from FIG. 2 is that a guide tube 10 is provided in the culture tank 1, and oxygen or oxygen-containing gas is supplied to the F section of the guide tube 10 via a conduit 3. . By supplying oxygen or oxygen-containing gas to the lower part of the guide tube 10, the suspension liquid rises inside the guide tube 10 from the bottom to the top, and conversely outside the guide tube 10, the suspension liquid moves from the top to the bottom. occurs, and the suspension liquid is stirred as a whole.

FIG. 4 shows a schematic longitudinal cross-sectional view of an apparatus for introducing oxygen dissolved in fluorocarbon into a culture tank. In the device shown in Figure 4, compared to the device shown in Figure 2, etc., fluorocarbon with oxygen dissolved in it is introduced from the fluorocarbon inlet 3, and as it falls downward, it comes into contact with the culture medium and supplies oxygen to the culture medium. After that, it accumulates in the F side of the culture tank and is discharged from the fluorocarbon discharge port 4. In addition, in FIG. 4, except for 3 and 4, 1 to 9 mean the same means and devices as in FIG. 2.

In each of the above figures 1 to 4, a settling zone 7 is partitioned by a partition wall 6 inside the inner wall of the culture device.
/JEF,Q GJ, so that the stirring effect of the 4-nospendion liquid does not substantially reach the settling zone. Inside the settling zone 7, the setting 1 is such that the cells settle in the direction of gravity, and the old culture solution containing the growth-inhibiting substance, which is substantially free of cells, is present at the top.

The settling zone only needs to be an area where 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 4, the settling zone 7 and the suspension culture zone are separated by a cylindrical partition 6, and a practical portion of the tank side wall facing the cylindrical partition 6 is cylindrical, The cylindrical partition wall and the cylindrical tank side wall are located substantially concentrically.

Roughly in the settling zone are 8 cylindrical baffles extending in the direction of gravity and delimiting the settling zone to prevent flow from forming in the cell suspension culture zone.

In each culture unit in this device, ■/S (
Here, S, which is the basis for calculating (2) is the volume of the culture medium in the suspension culture zone and S is the effective settling area of cells, is expressed by the following formula.

π S= −(D2−d2 ) Here, D is the diameter of the cylindrical tank side wall portion, and d is the diameter of the cylindrical partition wall 6. In addition, (2) 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.

In addition, the culture equipment contains oxygen, carbon dioxide, and nutrients.
Generally, a device is installed to measure the ratio of pl+ and maintain it within a range, but the cell J&rJ of the present invention
There are no words for how these devices can be installed in the equipment. 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 1-nospension culture device, for example, from the culture solution supply port 1 provided at the L section of the culture device as shown in Figures 1 to 4. . Alternatively, it may be supplied directly into the suspension liquid through a conduit.

Furthermore, when carrying out culture using the apparatus of the present invention, it is necessary to supply new culture medium and drain old culture medium so that the level of the liquid in the culture apparatus remains approximately constant. 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 a constant oxygen concentration in the nosovention liquid, the method of supplying oxygen is as shown in Figure 11.
As shown in FIG. 3, the oxygen or oxygen-containing gas in the suspension liquid may be supplied directly, or may be supplied by other means. Another supply means is, for example, a method using oxygen ↓/rea as shown in Figure 4. As the oxygen carrier, a liquid compound is used that dissolves oxygen without substantially mixing with water, and examples thereof include various fluorocarbons used as raw materials for artificial blood. When such Fluorocarbon is used as an oxygen supplying means, the fluorocarbon in which oxygen is not dissolved may be added from the top of the suspension liquid in the form of droplets or a thin film.

Stirring methods include mechanical stirring as shown in Figure 12 and Figure 2 by rotating a sliding blade, draft effect using a guide cylinder as shown in Figure 3, and the method shown in Map 4. Examples include a method of simultaneously supplying oxygen and stirring by using an oxygen carrier containing dissolved oxygen. Of course, two or more of these stirring means can also be used in combination.

In addition, when culturing, the ratio of supplying new culture solution to the effective culture volume (V) of the culture tank (supply amount of new culture solution/V) is 0.2 to 10 per day, preferably A range of 0.5 to 5 is appropriate.

Thus, the apparatus of the present invention can easily separate old culture fluid that does not contain living cells in cell glue suspension culture, 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 drawings]

1 to 4 each show an example of a schematic diagram of the culture apparatus of the present invention. ]＼ Figure-1 Figure-2 I￥I-3

Claims (1)

[Claims] The cell culture unit has a suspension culture zone for cells, a settling zone for cells, and a culture solution outlet from an upper part of the settling zone, and the suspension culture zone and the settling zone are arranged in the cell culture tank. The settling zone is separated by a partition so as to communicate through a lower part, and the settling zone is formed between a tank side wall of the cell culture tank and the partition, and the settling zone is separated from the tank inner wall along the inner tank wall of the cell culture tank. A cell culture unit characterized in that a baffle plate is installed between the plate and the baffle plate, and the baffle plate is installed so that the culture solution in the area separated by the baffle plate does not form a flow above the settling zone. 1. A cell culture device for perfusion culture, comprising at least one of the above, wherein any suspension culture zone of the culture device is provided with a culture solution supply port.