JPH074226B2 - Cell culture device and cell culture method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention relates to an apparatus and a method for growing cells in culture. More particularly, it relates to an apparatus and method for culturing cells in a suspended state.

Cell culture techniques are important for the production of antiviral agents such as viruses, vaccines, interferons or biopharmaceuticals such as hormones. Furthermore, in recent years, the production of monoclonal antibodies targeting specific proteins and the like is based on the culture of hybridomas with antibody-producing cells and myeloma, and the solution of the technique is an industrially important theme.

(B) Conventional Technology Conventionally, cell culture is generally carried out on a laboratory scale using a petri dish, a test tube, a culture bottle and the like.

On the other hand, in recent years, as a cell culture method and a device therefor,
Several suggestions have been made. These proposals can be broadly divided into an anchorage dependent culture and a suspension culture, that is, a suspension culture.
ture), and these methods are determined depending on the characteristics of the cells to be cultured.

Of these, the following proposals have been made regarding suspension culture. For example, a culture method has been proposed in which a stirring function is provided in a spinner flask by a magnetic stirrer or an impeller on a mechanically driven shaft (US Pat. Nos. 2,958,517 and 3,6,6).
49,465 specification).

In JP-A-57-65180, at least one relatively large surface area flexible sheet supported on a rotatable shaft is used as an agitator, and the agitator is rotated to wave the sheet. Suspension culture devices have been proposed which are allowed to stand and thereby create the desired gentle agitation for certain cells, such as human diploid cells.

However, in the culturing method using the above-mentioned apparatus, since the cells are cultivated in a certain amount of nutrients, the growth and growth of the cells is stopped at a relatively low density.

In suspension culture of cells, in order to prevent the growth growth of cells from stopping at a relatively low density and to culture the cells in a large amount and at a high density, generally a growth inhibitor while supplying a new culture solution to the culture tank. A method of culturing while discharging an old culture solution containing spores from the culture tank, that is, a method commonly called a perfusion method has been proposed (Annu
al Reports on Fermentation Processes. Vol.6). One of the important things in culturing using this method is that the living cells in the suspension liquid and the old culture liquid are efficiently separated, and the old culture liquid is taken out of the culture tank, To maintain the cell growth environment under optimum conditions.
Various filters and various formats have been proposed to separate living cells and old culture fluid from the suspension fluid, but industrial culture has been proposed in terms of filter clogging and device structure complexity. It is hard to say that all the devices are satisfactory.

In JP-A-59-82083 and JP-A-60-9482, a culture supernatant discharge tube also serving as a cell precipitation tube and a conduit for adding fresh medium are provided in the culture device, and the medium is fed from the conduit. A high-concentration culture device for floating cells has been proposed in which culture is performed while adding and simultaneously discharging the culture supernatant from the discharge tube.

In general, buoyant animal cells are as small as a few microns to a few tens of microns and their specific gravity is not so different from the specific gravity of the medium. Therefore, in the above-mentioned device for separating floating cells and medium by gravity, cell sedimentation is advantageous. In order to achieve this, it is necessary to increase the settling area, but the apparatus disclosed in the above two Japanese Patent Laid-Open Publications cannot make the settling area too large.

(C) Object of the Invention Accordingly, an object of the present invention is to provide an apparatus and method capable of separating living cells and a culture solution from a suspension culture solution with a simple structure.

Another object of the present invention is an apparatus and method capable of separating live cells and old culture solution without using a filter, and thus an apparatus and method which eliminates the problem caused by clogging of the filter and the like. To provide.

Still another object of the present invention is to provide an industrially advantageous apparatus and method for suspension culturing which can have a very large sedimentation area for achieving cell sedimentation.

Still another object of the present invention is to provide an apparatus and method capable of easily removing 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 apparatus and method by a perfusion method suitable for large-scale and high-density culture of cells.

Still another object is a cell culture apparatus for perfusion culture comprising a cell culture tank for suspension culture, the cell culture tank comprising a molecular oxygen supply zone, a cell settling zone, and a settling zone. Of the culture solution discharge port and the culture solution supply port, the molecular oxygen supply zone and the settling zone are partitioned by a partition so as to communicate through the lower portion of the settling zone in the cell culture tank, An object of the present invention is to provide a cell culture device characterized in that the settling zone is formed between a side wall of the cell culture tank and the partition, and main culturing and settling of cells are performed in the settling zone of the cells. .

(D) Structure and Effect of the Invention That is, the present invention is a cell culture device for suspension culture, which comprises at least two culture units and has a supply port for a culture solution, the unit comprising a cell circulation zone, The settling zone of cells and the outlet of the culture solution from the upper part of the settling zone, the distribution zone and the settling zone in the culture unit are partitioned by a partition so as to communicate through the lower part of the settling zone, The settling zone is formed between outer partitions of the outer wall of the culturing unit, and in the lower part of the culturing unit, the following properties (a) are substantially immiscible with water (b) have a density higher than that of water and (C) A tray for holding a liquid medium having substantially no inhibition of the growth of animal cells and stirring the liquid medium. A stirring means is provided, each culture unit is vertically stacked to allow the flow of the culture solution through the flow zone, and the main culturing and settling of cells in each unit is performed in the settling zone of the cells. In a cell culture device characterized by being performed and a method of perfusing cells with the cell culture device, each tray in the culture unit is filled with the liquid medium in layers, and the liquid medium is Culturing cells using the cell culture device characterized by extracting a culture liquid substantially free of cells from the upper part of the settling zone while stirring the layer and introducing a new culture liquid into the culture device Is the way to do it.

According to the present invention, in the settling zone arranged in the culture tank, living cells settle in the lower part in the direction of gravity,
Since old culture medium and cell debris will be separated into the upper part of the settling zone, discharge the culture medium containing substantially no viable cells from the culture medium outlet provided in the upper part of the settling zone. You can Therefore, the present invention is
Since it is possible to efficiently separate living cells from the culture solution by a simple method without using a filter that causes clogging, it can be applied to large-scale and high-density culture of cells.

The culture device of the present invention has a cell suspension culture zone that can be defined as an area in which cells are suspended and substantial cell culture is performed, and a cell settling zone that can be defined as an area in which cells are separated from a culture solution by gravity. ing. The cell suspension culture zone and the cell settling zone are partitioned by a partition so as to communicate with each other through the lower portion of the settling zone in the cell culture tank. Due to such a structure, the cells settle in the settling zone and the cells separated from the culture medium are reintroduced into the suspension culture zone of the cells through the lower part of the settling zone, and recultured there.

In the apparatus of the present invention, the settling zone in the culture tank is formed between the side wall of the culture tank and the partition. Such a structure has an advantage that the effective settling area of the settling zone in the culture tank can be made very large.

The apparatus of the present invention has a culture solution outlet for discharging an old culture solution in which cells have been separated in a settling zone and a culture solution supply port for supplying a new culture solution to the culture zone, in the above-mentioned culture tank. There is.

In the culture tank of the apparatus of the present invention, the settling zone is preferably formed around the suspension culture zone between the tank 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 partitioned by a cylindrical partition. In this case, more preferably, a substantial portion of the tank side wall facing the cylindrical partition is cylindrical, and the cylindrical partition and the cylindrical tank side wall are located substantially concentrically.

Further, the device of the present invention promotes the settling of cells in the settling zone, and thus can have a means for promoting settling of cells in the settling zone.
Such means is a means for shortening the distance in the gravity direction in which the cells settle, and can be, for example, a settling plate provided in the settling zone in a direction blocking the gravity direction.

Further, in the settling zone, for the purpose of minimizing the influence of the flow of the culture solution formed in the suspension culture zone of cells, a baffle that extends in the direction of gravity to partition the settling zone may be provided. . It will be easily understood that the baffle provided in the settling zone at an angle to the direction of gravity acts as a sinking plate.

The culture tank of the device of the present invention may be provided with a means for forcibly flowing the culture medium in the suspension culture zone of cells. Such means can be, for example, a mechanical agitation means for forcibly suspending the cells in culture. In addition, an oxygen-containing gas introduction port, or an oxygen-containing gas introduction port and an oxygen-containing gas guide tube are provided in the culture tank, and cells are added to the culture solution by the stirring effect when the oxygen-containing gas introduced from the introduction port rises. It can be forced to float.

The oxygen supply in the culture apparatus of the present invention may be performed by a method that provides a dissolved oxygen concentration according to the cell type, and any method can be used as long as the settling of the cells is not disturbed. For example, as described above, an oxygen-containing gas introduction port in the culture tank, or a method of providing an oxygen-containing gas introduction port and an oxygen-containing gas guide tube, a method of introducing an oxygen-containing gas from the introduction port to keep the dissolved oxygen concentration constant, Alternatively, a liquid medium that has the following properties of dissolving oxygen to some extent (a) is substantially immiscible with water, (b) has a density higher than that of water, and (c) does not substantially inhibit the growth of animal cells, for example, various types A method for keeping the dissolved oxygen concentration constant by saturating oxygen in a fluorocarbon used as a heat medium, an electrically insulating material, and various fluorocarbons used as an artificial blood and dropping it from the upper part of the culture tank. 61-1383)) etc.

The cell culture device of the present invention is applied to suspension-type cell culture, in which the cells themselves are suspended in an aqueous medium or suspended while being supported on a microcarrier (microcarrier). It also refers to various suspension cultures in which cells are grown in microcapsules. In particular, the present invention is advantageously used in a method of culturing cells themselves while suspending them.

The cells cultured in the cell culture device of the present invention may be plant cells, animal cells, microbial cells, etc., or may be cells artificially or genetically modified, such as hybridomas. In particular, the culture device of the present invention is suitable for culturing animal cells.

In the suspension type cell culture tank of the present invention, cells to be cultured are cultured in a state of being suspended in the culture medium. The culture medium is an aqueous medium consisting essentially of water, to which various inorganic salts, vitamins, scavengers, glucose, amino acids, antibiotics, and other additive components normally used in cell culture are added. Serum can be added to the culture medium, or so-called serum-free medium without serum can be used as the culture medium.

In addition, the culture solution used in the present invention may be one that has been subjected to pyrogen removal treatment.

The method of the present invention in which cells are perfused using the culture apparatus 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 suspension culture zone. .

At that time, the ratio of the effective settling area (S (cm ² )) of cells defined by the settling zone to the volume (V (cm ³ )) of the culture solution in the suspension culture zone, V / S is preferably 0.1. ˜500 cm, particularly preferably 1 to 100 cm.

In addition, the supply of a new culture solution and the discharge of an old culture solution can be performed independently and continuously or intermittently.

The cell culture device of the present invention has at least two culture units, preferably 2 to 10 and more preferably 2 to 5 culture units.

Further, the culturing device has a structure in which, during culturing, a main culturing and settling of cells is performed in a settling zone, and therefore, the main function is a molecule rather than a culturing zone for performing the main culturing as in the device of the present invention. It has a molecular oxygen supply zone for receiving supply of gaseous oxygen.

In such a culture apparatus, the area occupied by the settling zone is, for example, at least the same as the area occupied by the molecular oxygen supply zone in the cross section in the direction perpendicular to the gravity direction of the culture apparatus. It is also preferred that the outer wall of the settling zone has a diameter of at least 10 cm. Therefore, in the large area of the settling zone,
In order to carry out the main agitation for cultivation and effective settling of cells, the cultivation of cells using the above apparatus is usually carried out as follows.

That is, a liquid medium having the following properties at the bottom of the culture tank of the above culture device: (a) is substantially immiscible with water (b) is denser than water and (c) does not substantially inhibit the growth of animal cells. The cell is formed, the layer of the liquid medium is stirred, and the stirring effect of the liquid medium is transmitted to the culture solution located on the liquid medium to carry out the culture, and the cells are substantially free from the settling zone. It is carried out by a method characterized in that the culture solution is extracted and a new culture solution is introduced into the culture tank.

For example, perfluorocarbon is preferably used as the liquid medium.

As such a perfluorocarbon, one that is liquid at room temperature is advantageous, and commercially available ones can be widely used. For example, various heat carriers, fluorocarbons used as electrical insulating materials, and various fluorocarbons used as artificial blood can be used. Specific examples thereof include, for example, perfluoroalkanes having 8 or more carbon atoms, perfluorocycloalkanes (for example, perfluorodecalin, perfluoromethyldecalin, and carbon atoms 3).
To perfluoroalkylcyclohexane having 5 to 5 alkyl substituents, perfluoroalkyltetrahydrofuran having 5 to 7 carbon substituents, alkyltetrahydrofuran having 4 to 6 carbon atoms, and 4 to 6 carbon atoms
And perfluoroadamantanes (eg, perfluoroadamantane, perfluoromethyladamantane, perfluorodimethyladamantane, perfluorodimethylethyladamantane, perfluorodiethyladamantane, etc.) having an alkyl substituent. The fluorocarbon may contain various groups such as a tertiary amino group.

When the above-mentioned liquid medium is used as the oxygen supply method, it is preferably the same as the liquid medium forming the layer at the bottom of the culture tank.

The above-mentioned culture tank and culture method used in the present invention will be described below with reference to the accompanying drawings. The drawing shows a schematic view of the culture tank.

In FIG. 1, the culture device body 1 includes five culture units.

Cylindrical partitions 2, 3, 4, 5 and 6 penetrating each culture unit are provided at approximately the center of each culture unit.
The cylindrical partition of each culture unit has a lower portion cut off as shown in FIG. 1 so that the cell flow zone 7 formed in the partition and the settling zone 8 formed outside thereof can communicate with each other. Inside each cylindrical partition, a stirring rod 10 having a stirring blade 9 for stirring the inside of the cell circulation zone of each unit is provided penetrating each unit. Further, in each cylindrical partition, conduits 11,1 for supplying oxygen or oxygen-containing gas into the cell flow zone of each unit.
2, 13, 14 and 15 are provided, and further sensors 16, 17, 18, 19 and 20 for detecting the oxygen concentration in the zone are provided.

The supply of new culture solution to the device is performed from the culture solution supply conduit 30, and the withdrawal of the culture solution is performed from conduits 21 and 2
Through 2,23,24 and 25.

A liquid medium such as perfluorocarbon is introduced at the bottom of each culture unit to form a liquid medium layer. The dotted line 26 in FIG. 1 indicates the interface between the liquid medium layer and the culture solution. Baffle plate 27 for inclusion in a liquid medium
Is provided in each unit, and a stirring blade 29 fixed to a support rod 28 extending from the stirring rod 10 is also provided in each unit so that the liquid medium can be stirred.

Thus, when the stirring rod 10 rotates, the stirring blade 9 stirs the culture solution in the cell circulation zone, and further the stirring blade 29 stirs the liquid medium. The stirred liquid medium is hindered from being rotated by the baffle plate 27, so that the surface thereof becomes wavy. This ripple is transmitted to the culture medium in the settling zone located above the liquid medium, and the cells in the culture medium are maintained in a suspended state.

In FIG. 2, the culture device body 1 includes three culture units.

Cylindrical partitions 2, 3 and 4 penetrating each culture unit are provided at approximately the center of each culture unit. As shown in FIG. 2, the cylindrical partition of each culture unit is cut at its lower part so that the cell flow zone 7 formed inside the partition and the settling zone 8 formed outside thereof can communicate with each other. Inside each cylindrical partition, a stirring rod 10 having a stirring blade 9 for stirring the inside of the cell circulation zone of each unit is provided penetrating each unit. Also,
In each cylindrical partition, conduits 11, 12 and 13 for supplying oxygen, an oxygen-containing gas or a liquid medium saturated with oxygen into the cell flow zone of each unit are provided, and the oxygen concentration in the zone is controlled. Sensors 16, 17 and 18 for detecting
Is provided. Further, the withdrawal of the liquid medium in which the amount of dissolved oxygen is lowered is performed through the conduits 31, 32 and 33 from each unit.

The supply of new culture solution to the device is performed from the culture solution supply conduit 30, and the withdrawal of the culture solution is performed from conduits 21 and 2 from each unit.
Through 2 and 23.

A liquid medium such as perfluorocarbon is introduced at the bottom of each culture unit to form a liquid medium layer. The dotted line 26 in FIG. 2 indicates the interface between the liquid medium layer and the culture solution. Baffle plate 27 for inclusion in a liquid medium
Is provided in each unit, and the stirring blade 29 having the magnets of each stage is rotated by the magnet attached to the stirring blade 9.

Thus, when the stirring rod 10 rotates, the stirring blade 9 stirs the culture solution in the cell circulation zone, and further the stirring blade 29 stirs the liquid medium. The stirred liquid medium is hindered from being rotated by the baffle plate 27, so that the surface thereof becomes wavy. This ripple is transmitted to the culture medium in the settling zone located above the liquid medium, and the cells in the culture medium are maintained in a suspended state.

That is, according to the present invention, it is possible to carry out an industrially advantageous suspension culture in which a sedimentation area for achieving cell sedimentation can be very large, and further culture of a large amount of cells and a high density perfusion method can be performed. It is easily possible and its industrial value is great.

Hereinafter, the present invention will be described in detail with reference to examples.

Example 1 (1) Shape of culture tank A stack of five units having a volume of about 10 as shown in FIG. 1 was used as a culture tank. As shown in FIG. 1, this container is provided with a settling zone where the force of stirring does not act. Approximately 1.2 fluorocarbons are put in the lower part of each unit so that the stirring blade 9 and the baffle plate 27 are completely submerged, and the structure is such that the fluorocarbons are stirred.

The H: D ratio in the fermentor of Figure 1 is 5: 1.

(2) Composition of culture medium As a basal medium, RPMI1640 medium, Ham-F12 medium and Dulbecco's modified Eagle medium were mixed at a ratio of 2: 1: 1 to which amino acids, glucose and the like were further enhanced (hereinafter,
e-RDF) was used, and insulin, transferrin, ethanolamine, and selenious acid (ITES) were added as growth factors. The amount of insulin added is 9 μg / ml, transferrin is 10 μg / ml, ethanolamine is 10 μM,
Selenious acid was 20 nM.

(3) Culture method 50 culture solutions were put into the culture tank shown in FIG.
Culture was performed by rotating at rpm. At this time, the cells are present in the container as a suspension of about 20. The cells are mouse-human hybridoma C23 whose parent strain is mouse myeloma cell P3U1.
The strain was seeded at 1.1 × 10 ⁵ cells / ml. This cell is an immunoglobulin G (IgG) -producing strain. Oxygen gas was blown into the culture solution from the nozzle, and dissolved oxygen was
Adjusted to ~ 4ppm. The exchange of the culture solution was started on the third day from the start of the culture and was performed at a flow rate of 10 / dey to 40 / day. The culture tank was set so that it was kept at 37 ° C.

(4) Culture results The culture results are shown in Table 1 below.

Example 2 (1) Shape of culture tank The same culture tank as in Example 1 was used.

(2) Culture medium As a basal medium, RPMI1640 medium, Ham-F-12 medium and Dulbecco's modified Eagle medium were mixed at a ratio of 2: 1: 1 to which amino acids, glucose and the like were further enhanced (hereinafter
eRDF) was used.

This basal medium was further fractionated with an ultrafiltration membrane having a molecular weight cut off of 10,000 (Periyun cassette system, Millipore, Japan), and the one having a molecular weight of 10,000 or less was used as a basal medium containing no pyrodiene. The ultra-perforated membrane was used after being impregnated with 1N sodium hydroxide solution for 24 hours and thoroughly washed with pure water.

Four growth factors dissolved in pure water containing no pyrodiene were added to this basal medium to prepare the following concentrations.

Insulin 9 μg / ml Human transferrin 10 μg / ml Ethanolamine 10 μM selenite 20 nM Pyrodiene was not contained in the medium thus prepared.

The medium containing no pyrodiene means a medium having an endotoxin concentration of 0.01 nanogram / ml or less in the LIMULUS test.

(3) Culturing method 50 culture solutions containing no pyrodiene were placed in the culture tank shown in FIG. 1 and cultured by rotating the stirring blade at about 20 rpm. At this time, the cells are present in the container as a suspension of about 20.
The cell is a mouse whose parent strain is mouse myeloma cell P3U1-
Human hybridoma H1 strain was seeded at 1.3 × 10 ⁵ cells / ml. This cell is an immunoglibulin G (IgG) -producing strain. Oxygen gas was blown into the culture solution through a nozzle, and the dissolved oxygen was adjusted to 3 to 4 ppm.
The exchange of the culture medium starts from the third day of the culture and starts from 10 / day to 40
The flow rate was / day. The culture tank was set so that it was kept at 37 ° C.

(4) Culture results The culture results are shown in Table 2.

The endotoxin concentration in the culture supernatant was 0.01 nanogram / ml or less.

Example 3 (1) Shape of culture tank A stack of three units having a volume of about 385 ml as shown in FIG. 2 was used as a culture tank. As shown in FIG. 2, this container is provided with a settling zone where the force of stirring does not act. At the bottom of this culture tank, a stirring blade 9 and a baffle plate
Approximately 200 ml of fluorocarbon is placed so that 27 and 27 are completely submerged, and the structure is such that this fluorocarbon is stirred.

(2) Composition of culture medium As a basal medium, RPMI1640 medium, Ham-F12 medium and Dulbecco's modified Eagle medium were mixed at a ratio of 2: 1: 1 to which amino acids, glucose and the like were further enhanced (hereinafter,
e-RDF) was used, and insulin, transferrin, ethanolamine, and selenious acid (ITES) were added as growth factors. The amount of insulin added is 9 μg / ml, transferrin is 10 μg / ml, ethanolamine is 10 μM,
Selenious acid was 20 nM.

(3) Culturing method 1.3 culture solution was put into the culture tank shown in FIG.
Culture was performed by rotating at 0 rpm. At this time, the cells are present in the container as a suspension of about 1.0. The cell is a mouse-human hybridoma C2 whose parent strain is mouse myeloma cell P3U1.
Three strains were seeded at 1.6 × 10 ⁶ cells / ml. This cell is an immunoglobulin G (IgG) -producing strain. Oxygen gas was blown into the culture solution from the nozzle,
It was adjusted to be 0.1 to 3 ppm. The exchange of the culture solution was started from the day when the culture was started and was performed at a flow rate of 0.8 / day to 1.9 / day. The culture tank was set so that it was kept at 37 ° C.

(4) Culture results The culture results are shown in Table 3 below.

Example 4 (1) Shape of culture tank The same culture tank as in Example 3 was basically used. However, a cell tower equipped with a bubble column that circulates fluorocarbon and absorbs oxygen was used.

(2) Culture medium The same culture medium as in Example 3 was used.

(3) Culturing method 1.3 culture solution was put into the culture tank shown in FIG.
Culture was performed by rotating at 0 rpm. At this time, the cells are present in the container as a suspension of about 1.0. The cell is a mouse-human hybridoma C2 whose parent strain is mouse myeloma cell P3U1.
Three strains were seeded at 1.0 × 10 ⁶ cells / ml. This cell is an immunoglibulin G (IgG) -producing strain.

Oxygen was supplied by the method using fluorocarbon described in JP-A-61-1383, "Cell culture method". Fluorocarbon saturated with oxygen in the oxygen absorption tower was dropped from the upper part of the culture tank to maintain the dissolved oxygen concentration in the culture tank at 3 ppm. The dropped fluorocarbon was collected at the bottom of the culture tank containing the fluorocarbon, extracted from there and sent to the oxygen absorption tower for recycling. The exchange of the culture solution was carried out at 0.9 to 1.8 / day starting from the third day of the culture. The temperature of the culture tank was maintained at 37 ° C.

(4) Culture results The culture results are shown in Table 4.

[Brief description of drawings]

1 and 2 show an example of a schematic view of a culture device used in the present invention.

Claims (2)

[Claims] 1. A cell culturing apparatus for suspension culturing, comprising at least two culturing units and having a supply port for a culturing solution, the units comprising a cell flow zone, a cell settling zone and the settling zone. Of the culture solution is discharged from the upper part of the culture unit, and the flow zone and the settling zone in the culture unit are partitioned by a partition so as to communicate with each other through the lower part of the settling zone, and the settling zone of the culture unit. It is formed between the partitions of the outer wall, and further, in the lower part of the culture unit, the following properties (a) are substantially immiscible with water (b) have a higher density than water and (c) grow animal cells. A tray for holding a liquid medium having substantially no inhibition and a stirring means for stirring the liquid medium are provided, and each culture unit is provided. The cells are vertically stacked so that the flow of the culture solution through the flow zone is acceptable, and the main culturing and settling of the cells in each unit is performed in the settling zone of the cells. Incubator. 2. A cell culturing apparatus for suspension culturing, comprising at least two culturing units and having a supply port for a culturing solution, said units comprising a cell flow zone, a cell settling zone and the settling zone. Of the culture solution is discharged from the upper part of the culture unit, and the flow zone and the settling zone in the culture unit are partitioned by a partition so as to communicate with each other through the lower part of the settling zone, and the settling zone of the culture unit. It is formed between the outer wall and the partition, and in the lower part of the culture unit, the following properties (a) are substantially immiscible with water (b) are denser than water and (c) grow animal cells. A tray for holding a liquid medium having substantially no inhibition and a stirring means for stirring the liquid medium are provided, and each culture unit is provided. The cells are vertically stacked to allow the flow of the culture solution through the flow zone, and a cell culture device is provided in which the main culture and settling of the cells in each unit is performed in the settling zone of the cells. In the method of perfusion culture of cells using, each tray in the culture unit is filled with the liquid medium in layers, and while stirring the liquid medium layer, the cells are substantially removed from the upper part of the settling zone. A method for culturing cells using the cell culture device, which comprises extracting a culture liquid not contained in the above and introducing a new culture liquid into the culture device.