JPH0269176A - Cell culture apparatus - Google Patents

Abstract

PURPOSE:To prevent a weak cell from injuring in culturing the weak cell such as animal or plant cell by forming a stirring wing for culture medium out of a specific boss member and stirring wing and providing the stirring wing at the upper part in the culture medium. CONSTITUTION:The aimed cell culture apparatus is composed of a feed part 2 having a liquid system L for feeding a culture medium and gas system G for feeding oxygen, etc., culture part 3 for carrying out culture of cell and filter part 4 for removing waste matter, etc. The culture part 3 is composed of a culture tank 5 and stirring wing 6 arranged at the upper part in the culture tank 5. The stirring wing 6 is formed out of a boss member 12 fixed to a driving shaft 11, four plate stirring blades 13 provided in the circumferential part of the lower face of the boss member 12 and ring 14 coupling lower part of outer edge of the stirring blades 13. Descending current near wall face of the culture tank 5 and ascending current in the center part of culture tank 5 can be generated in the culture medium in the culture tank 5 and circulation current evenly flowing in the culture tank 5 can be formed by arranging the stirring wing 6 at the upper part of the culture tank 5.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a cell culture device that performs culture while stirring a culture solution (medium) in a culture tank to disperse cells in the culture solution, and particularly relates to a cell culture device for culturing cells while dispersing them in the culture solution. The present invention relates to a cell culture device suitable for culturing cells.

[Conventional technology]

2. Description of the Related Art As a cell culture device, one is known that performs cell culture by dispersing cells in a culture solution in a culture tank. In order to improve the growth rate and culture efficiency of m-cells in this type of cell culture device, it is necessary to uniformly disperse the cells in the culture solution and supply nutrients, oxygen, etc. to the cells every 4 minutes. To do this, it is necessary to properly stir the culture solution, and the culture solution stirring means is C, a propeller-shaped or turbine-shaped stirring blade, or a paddle-shaped F! Many were used to rotate the worship tools using a drive shaft or magnetic force.

However, animal cells are larger than other microorganisms, and the cell membranes surrounding the cells are fragile and easily destroyed.
Very slow stirring is required, but in order to uniformly disperse the cells in the culture medium using the above-mentioned stirring blades, the rotation speed must be high, which may damage the cells or Furthermore, when cells are cultured by being supported on a microparticle carrier (microcarrier), there is a risk that the cells may fall off from the carrier. On the other hand, at a rotation speed that does not damage the m cell,
The cells could not be uniformly dispersed in the culture medium due to insufficient stirring power.

Therefore, in order to solve this problem, conventional methods have been to provide a large number of stirring IT devices, to add complex movements to the stirring W, or to create a special stirrer as shown in Japanese Patent Application Laid-open No. 160-160 B82. The culture solution was stirred using a stirrer with a different shape.

(Problems to be Solved by the Invention) However, with these methods, the device becomes complicated and expensive, and the construction and maintenance time is 11+.
It was.

Therefore, the present invention has a cylinder configuration that allows for sufficient stirring of 1 Sl of the culture solution, which is easy to manufacture and maintain, and which also prevents damage to cells even when culturing weak cells such as animal and plant cells. The purpose is to provide a cell culture device that does not require

[Means to solve the problem] Achieve the above objectives! In particular, the present invention provides a cell culture device that performs tissue culture by rotating a stirring blade using a drive shaft to stir a culture medium and dispersing Ill cells in the culture medium. a boss member fixed to the Moeki drive fJJ shaft, a plurality of plate-shaped stirrers provided on the outer periphery of the lower surface of the boss member at a distance from the center of rotation, and a lower outer edge of the stirrer. It is characterized in that it is formed by a ring that connects the nutrient solution, and that the agitator is placed at the top of the nutrient solution.

[Function] Form the stirring blades as described above and place them above the culture medium.
When the stirrer is rotated, the culture solution in the center of the stirring blade flows almost horizontally toward the wall of the culture tank due to the action of centrifugal force caused by the rotation of the stirrer. Flows down towards the bottom.

On the other hand, as the stirring blade rotates, the culture solution in the center of the culture tank rises inside the ring and is sucked up to the center of the stirring blade. As a result, the culture solution in the culture tank flows in the culture tank as a downward flow near the wall and an upward flow in the center without a whirlpool layer, so that cells are uniformly dispersed in the culture solution. In addition, according to the stirring blade of the above shape, even if the rotation speed of the stirring blade is low, it is possible to generate the flow of the culture medium as described above, so cells can be stirred into the culture medium with very slow stirring. Can be uniformly dispersed.

〔Example) Hereinafter, one embodiment of the present invention will be described based on the drawings.

Figure 1 shows the overall configuration of cell culture device d, with cells i
8. The strawberry device 1 includes a supply section 2 having a liquid system for supplying a culture solution and a gas system G for supplying oxygen, etc., a culture section 3 for culturing cells, and a filter for removing waste materials, etc. It is partly composed of 4 parts.

The culture section 3 includes a culture tank 5 and the culture! ! It consists of stirring blades 6 placed on the top of the culture tank 5.
Dissolved oxygen sensor 7, DI (sensor 8) for various controls
A gas supply tube 10 is provided in addition to the level sensor +J9.

As shown in FIG. 2, the upper 2 stirring W6 includes a boss member 12 fixed to the drive shaft 11, four plate-shaped stirrers 13 provided on the outer periphery of the lower surface of the boss member 12, The stirrer 1
3 and a ring 14 connecting the lower outer edges of the culture tank 5 and the ring 14, which is formed to an appropriate size depending on the size of the culture tank 5 and the strength of the beaten culture solution.

The boss member 12 [Yo, -F downward through hole 12 in the center]
A is formed, and the drive shaft 11 is inserted through the through hole 12a, and a screw is screwed into a screw hole 12b formed in the side wall to be fixed to the drive shaft 11. The stirring blades 13 are arranged in the normal direction of a circle centered on the drive shaft 11, and form a space at the center of rotation of the stirring blades 6 inside each stirrer 13. . Further, the ring 14 acts as a rectifying plate when the culture solution Wt is itchy, and connects the lower part of the outer edge of the stirrer 13 and is installed around the lower part of the stirrer 'lJ6 at an appropriate height.

The stirring m6 formed as described above is located at the top of the culture tank 5,
That is, it is placed on top of the culture solution, and it is used! l! ll@1
1 is rotated by being rotationally driven by a motor (not shown).

When the culture solution and cells are put into the culture tank 5 and the one-party stirring bacteria 6 is rotated, the culture solution in the center of the stirring blade 6 is spread onto the wall of the culture tank 5 due to the action of centrifugal force accompanying the rotation of the stirrer 13. It flows almost horizontally towards the side.

When this flow of liquid 18a reaches the wall surface of the culture tank 5, it settles on the wall surface, flows down toward the bottom of the culture tank 5, and flows toward the center of the bottom of the culture tank 5. - The culture solution inside the ring 14 rises to the center of the stirring blade 6 as the culture solution flows toward the wall, and sequentially flows to the outer circumferential side of the stirrer 16 as described above. That is, the culture solution in the center of the culture tank 5 rises within the ring 14 as the stirring blade 6 rotates and is sucked up to the center of the stirring blade 6. At this time, even if the culture solution at the bottom of the stirring blade 6 is stirred by the stirring blade 13, the ring 14 can stop it from flowing out to the outer circumferential side of the itching blade 6 by 1 ft. The liquid in the culture medium 1 at the bottom of the stirring blade 6 smoothly flows upward to the center of the stirring blade 6.

In this way, by placing the stirring m6 at the top of the culture tank 5 and rotating it, the culture! ! A downward flow near the wall surface of the culture tank 5 and an upward flow in the center of the culture tank 5 can be generated in the culture solution in the culture tank 115, and a circulating flow can be created that flows inside the culture tank 5 without any grooves. Therefore, with the flow of the above culture solution, I
Cells can be uniformly dispersed in the culture medium, and sufficient nutrients and oxygen can be supplied to the cells.

In addition, by forming the stirring blade 6 as described above and placing it in the corner of the culture tank 5, it is possible to maintain the flow of the culture solution as described above even at a very slow rotation speed of 10 rotations per minute. Therefore, the cells can be uniformly dispersed in the culture solution without damaging them, and the cells can be cultured efficiently. In particular, when culturing easily damaged animal cells such as Moe's, or cells supported on a microparticle carrier (Microcarrier A7), the cells can be placed in the culture solution without damaging the cells or causing them to fall off the carrier. can be uniformly dispersed.

On the other hand, a supply section 2 that supplies culture solution, oxygen, etc. to the culture section 3
The liquid system consists of a culture solution tank 21 and a culture solution pump 22 for supplying fresh culture N solution, and oxygen gas (0?), nitrogen gas (N2), and air (air) in the culture solution. and a gas system G that supplies carbon dioxide gas (CO2) for DH adjustment, and supplies culture solution and various gases according to the conditions of the culture section 3.

The gas system G has valves 23 provided in each circuit.
The supply amount of various gases is adjusted by adjusting the opening degree of the
The R cable gas circuit Go and the carbon dioxide gas circuit GC are provided with replenishment circuits 24 and 25 each including controllers 24a and 25a and timers 24b and 25b. The replenishment circuit 24 of the oxygen gas circuit Go is the dissolved oxygen sensor! When the dissolved oxygen l in the culture solution detected by the cell 7 becomes less than a predetermined amount, the controller 24a is activated and opens the valve 24C for a predetermined time set in the timer 24b to replenish oxygen. Similarly, in the replenishment circuit 25 of the carbon dioxide circuit Gc, the controller 25a is activated when the pH of the culture solution detected by the pH sensor 8 becomes high, and the replenishment circuit 25 listens to the valve 25c for a predetermined time set in the timer 25b. Replenish carbon dioxide.

Further, as a supply source of oxygen gas and nitrogen gas, for example, an oxygen gas supply device 1t26 using an adsorbent filled with an adsorbent that selectively adsorbs nitrogen gas is used, and oxygen gas that is richer than the atmosphere (for example, 0290 % to 93%, with the remainder being N2). Nitrogen gas can also be supplied by using a nitrogen gas supply device 27 that uses an oxygen gas adsorbent in the same way as the oxygen gas supply device 26 described above. can do.

The various gases mentioned above are the culture! It is supplied into the culture solution through a tube 10 disposed in the I tank 5.

The tube 10 is made of gas-permeable silicone resin, and can dissolve the various gases mentioned above into the culture solution without causing foaming.

Further, this tube 10 is arranged in a spiral manner in the center of the culture tank 5, and is provided in the shape of a drum 71-tube that is open in the vertical direction.

In this way, the tube 10 for supplying various gases is connected to the driver 7.
By arranging the tube 10 in a tube shape, the upward flow and downward flow of the J8 nutrient solution in the #i culture tank 5 can be separated into the inner and outer peripheries of the tube 10, resulting in a more circular circulation flow. can be formed, and the state of dispersion of cells in the culture solution can be further improved.

Further, the filter part 4 removes products and wastes produced during the culture process C of the staff member along with a part of the culture solution, and maintains the culture solution in the culture tank 5 in a predetermined state. The culture tank 71R circulation pump 32 is composed of a handling pump 33 and a collection tank 34, and is connected to a level sensor 9 installed in the culture tank 5. 33 is under Ll control, and the regeneration operation of the Celsius noiler 31 is automatically carried out).

A predetermined amount of the culture solution in the culture N tank 5 is supplied to the culture solution circulation pump 32.
The water is sucked in, introduced into the lower part of the external chamber 31a of the filter 31, where products and wastes are removed, and circulated through the route returning to the culture tank 5 from the upper part of the external chamber 31a. The igniter pump 33 normally sucks the culture solution from the internal chamber 31E of the filter 31 together with products and wastes into the collection tank 34.
However, depending on the bottle of culture solution in culture tank 5 (-
When the controller 35 operates, this drawing pump 33 is reversed to avoid backflowing the C culture solution from the internal chamber 31b of the C filter 31 to the outside, and
Perform 1 +Tj raw.

By configuring the cell culture device 1 in this way, it is possible to always supply fresh culture fluid and necessary oxygen etc. into the culture tank 5. It can be extracted.

Therefore, by the action of the agitating blades 6, it is possible to constantly supply the cells uniformly dispersed in the culture solution with a portion of fresh culture solution Al1, etc., and also to continuously remove products and wastes. Since an optimal culture environment can be created, cell proliferation rate and culture efficiency can be improved.

[Effects of the Investigation] As explained above, the present invention stirs the culture solution by disposing stirring blades of a specific shape in one part of the soil of the culture tank.
The 18-nutrient solution in the culture tank can be sufficiently stirred by slow rotation. Therefore, even when culturing easily damaged animal cells supported on particulate carriers, it is important to avoid damaging the cells, dropping them from the particulate carriers, and ensuring that they are uniformly dispersed in the culture solution. As a result, sufficient nutrients can be given to the cells, and the cell culture efficiency can be improved.Additionally, a simple stirring knife can be rotated in the center line to provide sufficient agitation. Since the action is reduced, the structure of the culture tank, including the rotation mechanism, etc. can be downsized, and the cell culture device can be manufactured at low cost.In addition to manufacturing and assembling the stirring blades, maintenance such as cleaning and replacement can be done. It is also easier to handle and easier to handle.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram showing the overall structure of the cell culture apparatus, and FIG. 2 is a perspective view of a stirring blade. 1...Cell culture device 3...Culture section 5...
18 Nutrient vI 6... Stirring blade 11... Drive shaft
12... Boss member 13... Stirring blade 14.
...Ring Patent Applicant Tokyo Rikakikai Co., Ltd. Agent Patent Attorney Denichi Kido
Kido - Stupid question
Ogawa Masuji 2 Indan stirring wing

Claims (1)

[Claims] 1. Stir the culture solution by rotating the stirring blade with the drive shaft,
In a cell culture device that performs tissue culture by dispersing cells in a culture solution, a stirring blade for stirring the culture solution is attached to a boss member fixed to the drive shaft and a rotating part on the outer periphery of the lower surface of the boss member. It is characterized by being formed by a plurality of plate-shaped stirring blades spaced apart from the center and a ring connecting the lower outer edges of the stirring blades, and the stirring blades are arranged above the culture solution. A cell culture device for