JPH078230B2 - Cell culture device - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a cell culture device, and more particularly to a cell culture device capable of efficiently culturing cells without damaging them by shearing.

[Conventional technology]

To cultivate animal cells, plant cells, etc. at high density and to produce useful substances with high efficiency using this cell culture, supply of oxygen, supply of nutrients, removal of waste products, pH of medium, dissolved The adjustment of the carbon dioxide concentration and the like is an important factor, and stirring in the culture tank is important for controlling each of the above factors to bring them into an appropriate state.

Here, as a conventional cell culture device, a medium in which cells or microcarriers to which cells are attached are suspended in a culture tank is stored, and oxygen is supplied to the culture tank to dissolve oxygen in the culture tank. There has been a cell culture device of a type in which a culture medium is stirred by a stirring member having a screw stirring blade. In addition, in order to supply oxygen more efficiently, a bubble cylinder type cell culture in which oxygen is supplied in a bubble state from the oxygen supply part provided at the bottom of the culture tank to increase the dissolved oxygen concentration (DO). There was a device. However, since animal cells do not have cell membranes like microorganisms, they are generally weak in shearing force, and are also sheared by turbulent flow generated when oxygen bubbles form in the medium or oxygen bubbles present in the medium disappear. There is a risk.

For this reason, it is preferable to supply oxygen into the medium in a state where it does not foam, and a tube using silicon, which is a polymer porous material, is arranged on the inner wall of the culture tank to realize this, and this tube is used to A cell culture device for supplying oxygen to a medium has been developed.

[Problems to be Solved by the Invention]

However, compared with the conventional bubble-tube type cell culture device, the oxygen supply using the above-mentioned silicon tube has a small supply ability, and the area of the silicon tube is increased and the medium is supplied in order to sufficiently supply oxygen into the medium. There is a problem in that it is necessary to sufficiently stir, and thus the stirring speed becomes high, and a turbulent flow of the medium occurs in the culture tank and the cultured cells are likely to be sheared.

The present invention has been made in order to solve the above-mentioned problems, and has a high oxygen supply ability, and a turbulent flow of a medium in a culture tank is extremely small, which enables efficient cell culture. The purpose is to supply.

[Means for Solving the Problems]

In order to achieve this object, the present invention provides a sealable tank for storing a medium in which cells or microcarriers to which cells are attached are suspended, and a tube made of a polymeric porous material disposed in the tank. A cylindrical oxygen supply member formed by being wound around a support member so as to form a cylindrical shape, and an agitating member arranged so as to be located in a cylindrical inner region of the cylindrical oxygen supply member, The medium was configured to circulate in the region between the inner wall of the tank and the outer wall of the cylindrical oxygen supply member and the inner region of the cylinder.

[Action]

Oxygen is supplied to the medium in which cells or microcarriers to which cells are attached are suspended in a tank that can be sealed from a tube made of a polymeric porous material that constitutes the cylindrical oxygen supply member without forming bubbles. Since the cylindrical oxygen supply member is formed by winding the tube around the support member so as to form a cylindrical shape, the area in contact with the culture medium is large and the cylindrical inner region of the cylindrical oxygen supply member is By the stirring member arranged so as to be located at, the culture medium flows so as to circulate in the region between the inner wall of the tank and the outer wall of the cylindrical oxygen supply member and the inner region of the cylinder. As a result, sufficient oxygen is supplied to the medium, turbulent flow of the medium is suppressed, the shearing force on the cells is minimized, and highly efficient cell culture becomes possible.

〔Example〕

An embodiment embodying the present invention will be described below with reference to the drawings.

FIG. 1 is a partial sectional view showing a cell culture device according to the present invention. In FIG. 1, a cell culture device 1 includes a tank 2 for storing a medium 10, a cylindrical oxygen supply member 3 arranged in the tank 2 for supplying oxygen to the medium 10, and a cylindrical oxygen supply. A stirring member 4 for stirring the culture medium 10 is provided in the cylindrical inner region of the member 3.

The tank 2 has a tank main body 21 and a sealing member 22 for sealing the tank main body 21. The tank main body 21 is made of a material such as glass or metal, and a jacket portion 23 having openings 24 and 25 is provided on the outer wall portion thereof. Then, the temperature-controlled water is circulated through the jackets 23 through the openings 24 and 25, so that the culture medium stored in the tank 2 is maintained at a predetermined temperature. In addition, the tank body 21
A sensor mounting opening 26 is provided at a predetermined position on the outer wall of the sensor, and the sensor is inserted into the tank body 21 through the sensor mounting opening 26 to measure pH, DO, temperature, etc. of the medium. It Further, in the present invention, the medium filters 5a and 5b may be provided inside the tank body 21 to perfuse the medium in the tank body 21. That is, the medium is supplied into the tank main body 21 via the medium filter 5a, and the medium is recovered via the medium filter 5b. In this case, medium filter 5
Filters a and 5b are set in a range that allows the medium to pass but not the cells or microcarriers to which the cells adhere. When the collecting medium filter 5b is clogged with microcarriers or the like, the filter can be backwashed by using the medium filter 5b for supplying and the medium filter 5a for collecting. A sampling opening 28 is provided at the bottom of the tank body 21. Furthermore, in the present invention, an inlet for an acidic or alkaline solution for adjusting the pH of the medium, a window for observing the inside of the tank 2, oxygen for dissolving a predetermined amount of oxygen in the medium prior to cell culture. Oxygen supply part (sparger) etc. which supplies small bubbles from the bottom of the tank body
21 may be provided.

The sealing member 22 is made of a material such as metal, and the tank body
It is for sealing the upper openings of 21. The sealing member 22 is provided with pressurizing air nozzles 7a and 7b.
Then, the culture medium 10 formed by the sealing by the sealing member 22.
Oxygen is supplied to the gas phase portion between the sealing member 22 and the sealing member 22 from the pressurizing air nozzle 7a to bring the gas phase portion into a pressurized state to increase the supply of oxygen to the medium and to the inside of the tank 2. It is possible to prevent the contamination of various bacteria. In addition, excess oxygen can be extracted from the gas phase portion by the pressurizing air nozzle 7b so as to prevent excessive pressurization.

The cylindrical oxygen supply member 3 is formed by winding a tube 32 around a support member 31 so as to form a cylindrical shape. The support member 31 includes a plurality of rod members 31a, 31 suspended from the sealing member 22.
The rods 31a are arranged at predetermined intervals so as to form a cylindrical shape. tube
As 32, a tube made of a polymeric porous material such as a silicon tube or a Teflon tube can be used. Such a tube 32 has an outer diameter of 2.0 to 10 mm and an inner diameter of 1.0.
~ 9.0 mm, wall thickness 0.2 ~ 1.0 mm is preferable. Then, the tube 32 is wound in a single transverse direction so that the tubes 32 adjacent to the support member 31 arranged so that each rod 31a forms a cylindrical shape as described above are in contact with each other,
A cylindrical winding portion 33 having an opening in the vertical direction of the drawing is formed. Further, the ends of the tubes 32 are respectively sealed members 22.
Is connected to the oxygen supply air nozzles 35a and 35b provided in the. The outer diameter of the cylindrical winding portion 33 can be determined from the relationship between the inner diameter of the tank body 21 described above and the diameter of the stirring blade of the stirring member described later, which will be described later. Further, the cylindrical winding portion 33 is preferably submerged in the stored medium 10, and the ratio of the height of the cylindrical winding portion 33 to the depth of the medium 10 is preferably about 2: 1. Then, for example, oxygen supplied from the oxygen supply air nozzle 35a to the tube 32 is supplied to the medium without passing through the wall surface of the tube 32 and generating bubbles while passing through the cylindrical winding portion 33, and excess oxygen is oxygen. It is discharged from the supply air nozzle 35b.
The tube 32 may supply oxygen to the medium by using air.

The stirring member 4 includes a shaft 41 and a spiral type screw stirring blade 42 spirally formed on the shaft 41. The shaft 41 has a bearing portion 27 provided on the sealing member 22 and a bearing 45,
It is attached to the lower end of a drive shaft 43 that is pivotally supported via 45. The diameter R ₃ of the spiral screw stirring blade 42 is, as described above, the inner diameter R ₁ of the tank body 21 and the outer diameter of the cylindrical winding portion 33.
It is set based on the relationship with R _2, and usually R ₁ : R ₂ : R ₃ = 10: 6: 5-1
A range of 0: 8: 7 is preferred. Further, the spiral pitch P of the spiral screw stirring blade 42 is
P from the relationship between the diameter _{R 3: R 3 = 1:} 1~1: it is preferable to set at 2 range. Further, the height H of the cylindrically wound portion 33 of the cylindrical oxygen supply member 3 and the axial length L of the spiral screw stirring blade 42 are in the range of H: L = 1: 1 to 1 to 1.5. Is preferred. Then, such a stirring member 4 is inserted and held in the cylindrical winding portion 33 of the cylindrical oxygen supply member 3 so that the spiral screw stirring blade 42 is submerged in the medium 10. A power transmission pulley 44 is provided on the upper end of the drive shaft 43, and the inside of the tank 2 is kept airtight by a seal portion 46 provided near the lower end of the drive shaft 43. Then, when the drive shaft 43 is rotated in the direction of arrow A by the drive source via the power transmission pulley 44, the spiral screw stirring blade is formed in the medium 10 inside the cylindrical winding portion 33 of the cylindrical oxygen supply member 3.
42 causes an upflow (arrow B). Then, the flow of the culture medium flows between the tank body 21 and the cylindrical oxygen supply member 3 as shown by an arrow C, reaches the bottom of the tank body 21, and becomes an upward flow again by the spiral screw stirring blade 42. The medium 10 is circulated.

FIG. 2 is a schematic perspective view showing another cylindrical oxygen supply member used in the cell culture device of the present invention. In FIG. 2, the cylindrical oxygen supply member 3 ′ includes a support member 36 and a tube 32.
It has and. The support member 36 includes a plurality of bar members 36a, 36a, ... Suspended from a sealing member (not shown), and the bar members 36.
It is composed of a pair of ring members 36b, 36b provided at the lower end portion and the intermediate portion of a, 36a, ... so as to be orthogonal to the axial direction of the rod member 36a. The tube 32 is a set of ring members arranged in parallel at a predetermined interval above and below.
A cylindrical winding portion 37 is formed in such a manner that the cylindrical winding portion 37 is wound in the vertical direction so as to bridge over between 36b and 36b and has an opening in the vertical direction of the drawing. Here, the tube 32 is the same as the tube 32 in the embodiment shown in FIG. 1, and the ends of the tube 32 are connected to oxygen supply air nozzles (not shown) provided in the sealing member. . Tube like this
The cylindrical oxygen supply member 3 ′ formed by winding 32 in the vertical direction is different from the cylindrical oxygen supply member 3 shown in FIG. 1 formed by winding the tube 32 in the horizontal direction in the tank 2 The unevenness in the flow direction of the culture medium generated inside is reduced, and the turbulent flow of the culture medium can be reduced to further reduce the shear fracture of cells due to stirring.

The cylindrical oxygen supply member used in the cell culture device of the present invention is not limited to the cylindrical oxygen supply member in which the cylindrical winding portions 33 and 37 have a single-layer structure as shown in FIGS. 1 and 2. Instead, the cylindrical winding parts 33, 37 may be a cylindrical oxygen supply member having a multiple structure. Here, the multiple structure will be described by taking the double structure of the cylindrical winding portion 33 as an example. Fig. 3 is a schematic sectional view of a double-layered cylindrical oxygen supply member 3 ". In Fig. 3, a plurality of rod members 31a, 31a of a supporting member 31 suspended from a sealing member (not shown). , Are arranged at a predetermined interval so as to form a double-cylindrical shape, and the tube 32 is first wound laterally around each of the outer rod members 31a in a single lateral direction to form an outer cylindrical winding portion 33a. Then, the inner cylindrical member 33a is similarly wound around the inner bar member 31a in the lateral direction in the same manner to form the inner cylindrical winding portion 33b. With the double structure of the turning portion 33a and the inner cylindrical winding portion 33b, the oxygen supplying ability of the cylindrical oxygen supplying member is remarkably improved.

FIG. 4 is a schematic perspective view showing another stirring member used in the cell culture device of the present invention. In FIG. 4, a stirring member 4'is provided with a shaft 47 and two sets of propellers which are provided so as to face the shaft 47 via a support member 49 so as to form a predetermined angle with respect to the axial direction of the shaft 47. A mold screw stirring blade 48 is provided. And the shaft 47 is the first
Like the shaft 41 shown in the figure, it is attached to the lower end of a drive shaft (not shown). Propeller type screw stirring blade 48
The width W is set from the relationship between the inner diameter R ₁ of the tank body 21 and the outer diameter R ₂ of the cylindrical winding portion 33, similar to the spiral screw stirring blade 42 described above, and usually R ₁ : R ₂ : W The range of 10: 6: 5 to 10: 7: 6 is preferable. Further, the mounting angle of the propeller type screw stirring blade 48 with respect to the axial direction of the shaft 47 is 15 to 45.
A degree of about is preferable. And such a stirring member 4 '
Is inserted and held in the cylindrical winding portion 33 of the cylindrical oxygen supply member 3 so that the propeller type screw stirring blade 48 is submerged in the medium 10. In the illustrated example, the two propeller type screw stirring blades 48 are mounted on the shaft 47 so as to face each other, but three or more propeller type screw stirring blades 48 may be mounted on the shaft 47. In addition, in the illustrated example, two sets of propeller type screw stirring blades 48 are provided on the shaft.
Although mounted on 47, three or more sets of propeller type screw stirring blades 48 may be provided.

Next, an example of a cell culture system using the cell culture device of the present invention will be described.

FIG. 5 shows a cell culture system using the cell culture device as shown in FIG. In FIG. 5, the tank 51 of the cell culture device 50 has a tank body 52 in which the medium 10 is stored, which is sealed by a sealing member 53. Then, in the jacket portion 54 provided on the outer wall portion of the tank body 52, water whose temperature is adjusted by the constant temperature bath 60 is circulated by the pump 61,
The medium stored in 51 is maintained at a predetermined temperature. Also,
A sensor 65 is inserted into the tank body 52 through the sensor mounting opening 55 of the tank body 52.

Oxygen is supplied to the medium in the tank 51 through a tube 57 made of a polymeric porous material. That is, the oxygen supplied from the oxygen cylinder 70 to the tube 57 made of the polymeric porous material through the oxygen supply air nozzle 56a passes through the wall surface of the tube 57 while passing through the cylindrical winding portion 58 to generate bubbles. Without being supplied to the medium, excess oxygen is discharged from the oxygen supply air nozzle 56b. At the same time, an oxygen cylinder is placed in the gas phase between the culture medium 10 and the sealing member 53.
Oxygen is supplied from 70 through the pressurizing air nozzle 59a,
It is possible to increase the supply of oxygen to the medium and prevent contamination of miscellaneous bacteria in the tank 51 by setting the gas phase portion in a pressurized state. Also, excess oxygen is compressed by an air nozzle.
Pulled out from 59b.

Further, the perfusion of the medium in the tank 51 is performed through the medium filters 75a and 75b arranged inside the tank main body 52.
That is, a medium tank 76 that aseptically stores fresh medium.
The medium is supplied from the pump 77 into the tank main body 52 through the medium filter 75a. At the same time, the excess medium is collected by the pump 78 via the medium filter 75b in the collection tank 79 together with the metabolites. Since the perfusion of the medium is performed via the medium filters 75a and 75b, cells or microcarriers to which cells are attached remain in the tank 51, and wasteful discharge of microcarriers and the like is prevented. Further, when the collecting medium filter 75b is clogged by microcarriers or the like, the medium can be backwashed by using the medium filter 75b for supplying and the medium filter 75a for collecting.

In the cell culture system as described above, a predetermined DO
When the cells are added to the medium described above and the cells are adherent cells, the cells are seeded so that the microcarriers are evenly distributed. After that, main culture is performed, but usually, the medium is not perfused in the initial culture process, but the cells are grown by the batch treatment to increase the cell density to some extent, and then the medium is perfused. In the cell culture device of the present invention, the DO of the medium is maintained at a high level due to the sufficient supply of oxygen, and the turbulent flow of the medium is suppressed to minimize the shearing force on the cells. It enables efficient cell culture.

Next, the present invention will be described in more detail with reference to experimental examples.

Experimental Example-1 Two kinds of carriers C-1 and C-2 shown in Table 1 were prepared, and four kinds of cell culture devices shown in Table 2 (D-1 (Example), D-2 In Examples (Example), D-3 (Comparative Example), and D-4 (Comparative Example)), the minimum rotation speed of the stirring member required for carrier flow was examined for each carrier. Where D-1
(Example) is equipped with a cylindrical oxygen supply member and a stirring member having a spiral screw stirring blade as shown in FIG. D-2 (Example) is the stirring member of D-1 (Example), which has a propeller type screw stirring blade shown in FIG. Also, D-3
(Comparative Example) uses a conventional oxygen supply member in which a silicon tube, which is a polymer porous material, is arranged on the inner wall of the culture tank, and uses the same stirring member as D-1 (Example) only for the stirring member. , D-4 (comparative example) is D-3 (comparative example)
4 is a stirring member having a propeller type screw stirring blade shown in FIG. The tank of each cell culture device had the same shape and a volume of 10 and an inner diameter of 220 mm. The outer diameter of the cylindrical oxygen supply member is 110 mm, the diameter of the spiral screw stirring blade is 80 mm,
The width of the propeller type screw stirring blade was 80 mm. Then, 7 mediums were stored in the tank, and 35g of the carrier was put into this medium.

The results of the tests conducted under the above conditions are shown in Table 2. As shown in Table 2, in the cell culture device (D-1 (Example), D-2 (Example)) according to the present invention, the flow of the carrier C-1 was possible at low rotation, and the particles were Even carrier C-2 having a large diameter and a large specific gravity was able to flow at a rotation speed of about 30 rpm. From this, the cell culture device (D-1 (Example), D-2 (Example)) according to the present invention is suitable for culturing cells that are weak in shearing force, and the stirring member can be used even for carriers that are difficult to flow. It can be said that the culture device has high versatility because it can generate stable flow at low speed rotation.

Experimental Example-2 Four types of cell culture devices used in Experimental Example-1 (D-1 (Example), D-2 (Example), D-3 (Comparative Example), D-4 (Comparative Example)) For each of the above, the cell culture system shown in FIG. 5 was constructed, and cell culture was performed using the following cells, medium and microcarriers.

・ Cells… CHOK1 cells (adherent cells) ・ Media… DMEM / F-12 mixed medium with non-essential amino acids added ・ Medium pH… 7.2 ・ Medium temperature… 37 ℃ ・ Microcarriers… Pharmacia Cytodex 3 / Collagen Coated cross-linked dextran Cells were seeded prior to culture. For cell seeding, 20 cells are aseptically put into the microcarriers and medium 3 which are stored in the tank of the cell culture device and are in a previously sterilized state.
The mixture was intermittently stirred at a minute interval for about 3 hours (rotation speed 30 rpm).
The medium used was one in which oxygen was previously supplied (supplied by 5% CO ₂ + 95% air) to equilibrate the dissolved oxygen concentration (DO). After seeding the cells, the main culture was carried out with the amount of medium being 7. The microcarrier concentration at this time is 5 g /
The cell seeding density was 4 × 10 ⁵ cells / ml. In the main culture, fetal bovine serum (FBS) was added to the medium at 5 vol% until the 6th day as the initial culture process (cell growth phase),
The culture was performed without perfusion of the medium. From the 7th day onward, the medium was perfused according to the dilution rate shown in FIG. During this period, oxygen was supplied by 5% CO ₂ + 95% air at a flow rate of 500 ml / min and a pressure of 0.8 kg / cm ² via an oxygen supply member. In addition, 5% in the gas phase part on the top of the medium
Aeration was performed with CO ₂ + 95% air at a flow rate of 100 ml / min.

Changes in cell density and oxygen dissolved concentration (DO) in the above culture are shown in FIGS. 7 and 8.

As shown in FIGS. 7 and 8, in the culture using the cell culture devices D-1 (Example) and D-2 (Example) according to the present invention, the cell density was 15 days after the start of the culture. 10 ⁷ cells / ml
As shown above, the dissolved oxygen concentration (DO) was maintained at a high level of 3 ppm or more. On the other hand, in the cell culture using the cell culture devices D-3 (Comparative Example) and D-4 (Comparative Example), the cell density was 2 × 10 ⁶ to 4 × 10 ⁶ cells even after 15 days from the start of the culture. / ml or less, and the dissolved oxygen concentration (DO) gradually decreased to 2 ppm or less after the 9th day, and the cell density gradually decreased accordingly.

Experimental Example-3 Four types of cell culture devices used in Experimental Example-1 (D-1 (Example), D-2 (Example), D-3 (Comparative Example), D-4 (Comparative Example)) For each of the above, the cell culture system shown in FIG. 5 was constructed, and cell culture was performed using the following cells and medium.

・ Cells: Namalwa cells (non-adherent cells) ・ Media: RPMI-1640 medium containing FBS at 5 vol% ・ Medium pH: 7.2 ・ Medium temperature: 37 ° C Medium (supplied with 5% CO ₂ + 95% air) to equilibrate the dissolved oxygen concentration (DO) 7
Cells were added to the cells at a cell density of 5 × 10 ⁵ cells / ml. From the 6th day after the start of culture, the gas phase above the medium was aerated with 5% CO ₂ + 95% air at a flow rate of 500 ml / min, and during this period, culture was performed without perfusion of the medium. . From the 7th day onward, the medium was perfused according to the dilution rate shown in FIG. From the 7th day, oxygen was supplied through the oxygen supply member under the conditions of an oxygen flow rate of 200 ml / min and a pressure of 0.5 kg / cm ² .

Changes in cell density and oxygen dissolved concentration (DO) in the above culture are shown in FIGS. 10 and 11.

As shown in FIGS. 10 and 11, in the culture using the cell culture devices D-1 (Example) and D-2 (Example) according to the present invention, the cell density was 10 days after the start of culture. 5 x 10 ⁶ ~ 7
X10 ⁶ cells / ml or more, dissolved oxygen concentration (DO) of 3 ppm
The above high condition was maintained. On the other hand, in the culture using the cell culture devices D-3 (Comparative Example) and D-4 (Comparative Example), the cell density was 1.5 × 10 ⁶ to 2 × 10 ⁶ days after the start of the culture.
^{It was 6} cells / ml or less, and the dissolved oxygen concentration (DO) gradually decreased to 2 ppm or less after the 10th day, and the cell density gradually decreased accordingly.

〔The invention's effect〕

As is apparent from what has been described in detail above, according to the present invention, the culture medium is divided between the tank inner wall and the cylindrical oxygen supply member outer wall by the stirring member arranged so as to be located in the cylindrical inner region of the cylindrical oxygen supply member. By circulating the flow between the region between and the inner region of the cylinder, the turbulent flow of the medium is suppressed and the shearing force to the cells is minimized, and the cylindrical oxygen supply member is made of a polymeric porous material. Since the tube is wound around the support member to form a cylindrical shape,
Since the area in contact with the medium is large and sufficient oxygen is supplied to the medium, the effect of enabling highly efficient cell culture is achieved.

[Brief description of drawings]

FIG. 1 is a partial sectional view showing a cell culture device according to the present invention,
FIG. 2 is a schematic perspective view showing another cylindrical oxygen supply member used in the cell culture device of the present invention, FIG. 3 is a schematic sectional view of a double-structured cylindrical oxygen supply member, and FIG. 4 is the present invention. 5 is a schematic perspective view showing another stirring member used in the cell culture of FIG.
The figure shows a cell culture system using the cell culture device as shown in FIG. 1, FIGS. 6 and 9 show dilution rates in medium perfusion, and FIGS. 7 and 10 show cell culture. Showing changes in cell density in Fig. 8, Fig. 11 and Fig. 11
The figure shows changes in dissolved oxygen concentration (DO) in cell culture. 1 ... Cell culture device, 2 ... Tank, 3 ... Cylindrical oxygen supply member, 4 ... Stirring member, 5a, 5b ... Medium filter, 10 ... Medium, 21 ... Tank body, 22 ... Sealed Element,
23 …… Jacket part, 31 …… Supporting member, 31a, 31a …… Bar, 32 …… Tube, 42 …… Spiral type screw stirring blade.

Claims (3)

[Claims] 1. A closable tank for storing a medium in which cells or microcarriers to which cells are adhered are suspended, and a tube made of a polymeric porous material disposed in the tank has a cylindrical shape. The cylindrical oxygen supply member formed by being wound around the support member, and the stirring member arranged so as to be located in the cylindrical inner region of the cylindrical oxygen supply member, wherein the culture medium is the inner wall of the tank. A cell culture device, which circulates in a region between the outer wall of the cylindrical oxygen supply member and the inner region of the cylinder. 2. The cell culture device according to claim 1, wherein the stirring member has a spiral screw stirring blade. 3. The cell culture device according to claim 1, wherein the cylindrical oxygen supply member has a multiple structure.