JP2983384B2 - Method and apparatus for suspension culture of biological cells - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for culturing biological cells, and more particularly to a method and apparatus for automatically subculturing biological cells in suspension.

[0002]

2. Description of the Related Art Subculture is a method in which a part of a current culture solution is used as a seed cell solution and superimposed on a culture cycle used for the next batch culture. Has been performed for the purpose of mutagenesis and the like. Usually, static culture using a flat flask, a petri dish, a test tube, or the like, or a flask capable of stirring the inside has been performed, and the pipette has been operated in a sterile atmosphere of a clean bench or a clean room. It is not uncommon for the passages to last for several months to several years at regular intervals over several tens to several hundreds of generations.

The following are typical known techniques of the conventional culture method. 1) Satoko Takaoka "Introduction to Tissue Culture" p33-38 (1986) Societies Publishing Center 2) Satoko Takaoka "Introduction to Tissue Culture" p53-56
(1986) Gakkai Shuppan Center, 3) Yukiaki Kuroda, "Tissue Culture Techniques", p.172-174 (1984) New Science Company. In addition, as a general technique for culturing animal cells, in order to efficiently supply a fresh medium to animal cells and remove by-products, and perform high-scale cell culture on a large scale, the culture solution in a culture tank is used. A method in which cells are supplemented to a filter aid through a filter installed separately, and the supplemented cells are returned to the culture monster together with the culture solution (Japanese Patent Application Laid-Open No. Sho 64-5486). In order to carry out the method, after the cells and the culture solution are separated from the culture solution, a predetermined component in the culture solution is separated and sterilized, and the culture solution is returned to the culture tank again (Japanese Patent Laid-Open No. 3-98573). Publication).

[0004]

The above-mentioned conventional subculturing requires manual repetition many times and for a long time in a microorganism-free atmosphere, requiring not only a great deal of labor and time, but also There is a high risk of contamination with various bacteria in the culture system. As a solution to these problems, it is possible to connect a plurality of conventionally known culture tanks and perform the work in combination with an advanced robot.
Not only does the development and production of the system require a large investment, but of course the system becomes cumbersome and bulky.

[0005] Also, even in the above-mentioned general technique for culturing animal cells, there is no particular disclosure of an effective technique for suspending some cells for the next subculture. SUMMARY OF THE INVENTION An object of the present invention is to provide a method and an apparatus capable of performing automatic or semi-automatic subculturing with a simple mechanism that does not cause the risk of bacterial contamination.

[0006]

In order to achieve the object of the present invention, it is necessary to solve at least the following requirements. 1) The whole subculture apparatus is configured as a closed system at least for the entry of microorganisms, and the culture solution in the culture tank constituting the apparatus is indirectly operated from outside the system without destroying the closed system. In other words, culture without invasion of microorganisms from outside the system
The amount of the culture solution to be used for the seed cells of the next culture is set in advance in the culture tank. 2) Similarly, the culture solution is set in the culture tank without the invasion of microorganisms from outside the system by the above-mentioned operation from outside the system. 3) Similarly, the extra culture solution is discharged from the culture tank without the invasion of microorganisms from outside the system by the above-mentioned operation from outside the system, and a fresh liquid medium is further placed in the culture tank . Introducing and mixing with the seed culture.

[0007] Culture without invasion of microorganisms from outside the system
There are many methods for operating the inside of the culture tank, for example, a method of performing a mechanical operation from the outside in a state in which the inside of the culture tank and the outside are completely airtightly closed, or a culture tank. The inside and the outside can be partially ventilated, but the state is microbiologically blocked by a sterile filter or the like, and a method of performing a treatment by generating a pressure difference between the inside and the outside of the tank can be effectively used.

In the case where a method is employed in which the inside and the outside of the culture tank are completely airtightly closed by a mechanical operation from the outside, a mechanical operation relatively movable with respect to the culture tank is required. The means is connected to the culture tank side by a sealing means having no relatively moving surface, such as a sealing bellows or a diaphragm, not by a sealing means having a sliding portion such as a conventional mechanical seal or packing. Further, the sealing means having no relatively moving surface can be effectively used in a method of performing treatment by generating a pressure difference between the inside and outside of the tank.

The feature of the present invention for solving the above-mentioned problem is that, as described above, a part of the culture solution in the culture tank is continuously set within a limited range in a non-invasive state, , The remaining culture solution is discharged out of the system, a fresh medium is introduced into the culture tank, and the reserved culture solution is mixed with the fresh medium. It is effective to take any one of the following means to store the set amount of the culture solution in the culture tank. That is, 1) a small chamber for storing a required amount of culture solution is separately arranged in the culture tank, and the inside of the small chamber is communicated with the culture solution in the culture tank by an S-shaped tube, and the S-shaped tube is sealed. It is moved up and down through sealing means such as bellows or diaphragms.

2) A small chamber for storing a required amount of the culture solution in the culture tank is supported via a means capable of forming a gas-permeable but microbial shut-off state such as a sterile filter, One end of the small chamber is opened in the liquid, and the other end is connected to a decompression mechanism. 3) A small chamber is formed at the bottom of the culture tank, and a base extending from the wall of the culture tank into the small chamber through a sealing means such as a sealing bellows or a diaphragm has a base portion extending and retractable. Is arranged, and the degree of expansion and contraction of the base of the tube member is adjusted and set from outside the system.

Regarding the above 1), the small chamber may be fixed to the wall of the culture tank, and may be configured to be indirectly movable from the outside up and down so as to correspond to the liquid level in the next culture. Good. In addition, the small chamber is usually located inside the culture tank, but in special cases, a communication hole is formed in the peripheral wall located outside the peripheral wall of the culture tank to communicate the small chamber with the culture solution. You may do so.

The system (apparatus) having the function of subculturing suspension culture according to the present invention comprises, in addition to the above-described structure of the culture tank, gas dissolving means, culture medium extracting means, culture medium storage means necessary for the culture tank. Means, medium storage means, medium supply means, temperature adjustment means, and program adjustment means. Further, a plurality of culture tanks are connected by a liquid communication pipe, and the culture liquid withdrawal pipes, culture medium supply pipes, and gas supply pipes coming out of the respective culture tanks are assembled via automatic valves, respectively, for each type of the pipes. A system that can connect to storage tanks, medium storage tanks, aseptic processing gas supply sources, and further connect to automatic valves, liquid transfer drive sources, and process sequencers to automatically continue passage multiple times.
(Apparatus) .

Although the size of the culture tank is not particularly limited, the present invention can be sufficiently applied to a mini-scale culture in which the effective volume of the culture tank is about 20 ml. Although the material is not particularly limited, a disposable type tank can be used by molding the tank with plastic and sterilizing the inside of the tank with γ-ray or ethylene oxide gas.

[0014]

In the method and apparatus for continuously culturing and subculturing living cells according to the present invention, it is required for the next culture by an operation from outside the system that is at least aseptically isolated from the inside of the culture tank. The volume of the culture solution is set steplessly. Next, the set amount of the culture solution is retained in the small chamber formed in the culture layer, and at the same time, the remaining culture solution is discharged out of the culture tank, and a new medium is introduced into the culture tank. The culture solution held in the cell compartment is mixed with the introduced medium, and the inoculation to the next culture is completed. All these operations can be performed automatically or semi-automatically in a simple configuration of the cultivation device, at least in an environment that is isolated aseptically from the outside of the system and without any risk of contamination within the culture system. It can be carried out.

[0015]

EXAMPLES Hereinafter, be described in more detail on the basis of the present invention embodiment. Embodiment 1 FIG. 1 shows an embodiment of a culture tank which is a main configuration of a culture apparatus used in the method for subculture and suspension culture of biological cells according to the present invention.

A support plate 7 for supporting the small chamber 3 described later is fixed to the inner surface of the side wall of the culture tank 1. The small chamber 3 is a container having a substantially triangular cross section that opens upward and tapers downward, and is slidably mounted on the support plate 7 in the vertical direction. Further, the small chamber 3 is suspended from the upper portion of the culture tank 1 by a small chamber support cord 5. Reference numeral 4 denotes an S-shaped tube, one end of which is disposed in the small chamber 3 and the other end thereof is located in the space inside the culture tank outside the small chamber 3, and the middle part is supported by the support cord 6 from the top of the culture tank. Be suspended.

Bellows 9, 9 are integrally formed near the upper ends of the small chamber supporting rope 5 and the S-shaped pipe supporting rope 6, as shown in the figure, and the lower ends of the bellows 9, 9 are bellows. 9,
9 is hermetically connected to the lower part of the bellows casings 11 and 11 by appropriate means. Bellows casing 1
A female screw for up / down adjustment is formed at the upper end of 1 and 11, and a screw is also formed at the lower outer peripheral portion. The screw is fixed to the culture tank lid 2 by screw engagement.

The female screws of the bellows casings 11, 11 are rotatably screwed with male screws 10, 10 having a through hole at the center for vertical adjustment. Both upper ends of the cable 6 pass through the through holes of the male screws 10 for vertical adjustment. Further, the small chamber support cable 5 and the S-shaped pipe support cable 6 have flanges extending in the horizontal direction in the vicinity of the distal ends, and the flanges rotate into concave grooves formed inside the male screws 10 for vertical adjustment. Freely engaged. Therefore, by rotating the male screw 10 for vertical adjustment with respect to the bellows casing 11, the small chamber support cable 5 and the S-shaped pipe support cable 6 move in the vertical direction.

Further, a magnet rotor 13 having a stirring blade 12 at a substantially central position in the culture tank 1 is rotatably supported by a bearing 15 via a stirring shaft 14. The liquid in the tank is rotated by the action of a rotating magnet at the bottom of the tank (not shown). In this embodiment, the culture tank 1 further has a culture liquid extracting pipe 16 extending from the bottom of the culture tank 1 through the culture tank lid 2 and extending out of the tank. The culture medium supply pipe 19 penetrates the culture tank lid 2 from outside the culture tank and opens at the upper part in the culture tank. Further, an air supply pipe 20 penetrating the culture tank lid 2 and connecting the filter 22 and an exhaust pipe 24 connecting the exhaust filter 26 and the drain trap 25 are provided.

Hereinafter, a method of using the apparatus for continuously culturing and subculturing living cells having this culture tank will be described with reference to FIG.
First, prior to the secondary culture, that is, any time from the start of the primary culture to the start of the secondary culture, the vertical adjusting male screw 10 is turned, and the small chamber 13 connected to the small chamber support cord 5 is rotated.
Is immersed below the liquid level of the current culture (FIG. 2a). Although the small chamber support cord 5 and the bellows casing 11 which is a part of the culture tank move relatively, as described above, both are not a conventional mechanical seal, a sealing means having a sliding portion such as packing, but a sealing means. Are hermetically separated by a bellows 9 which is a sealing means having no relatively moving surface. Therefore, even when the small-chamber support line 5 moves up and down, the atmosphere in the culture tank (atmosphere in the system) can be kept completely isolated from the atmosphere outside the system. Mixing of various bacteria and the like into the culture system can be completely prevented.

Next, the support cable 6 is moved up and down by turning the vertical adjusting screw 10 connected to the S-shaped tube to move the small room 3 of the S-shaped tube 4.
The position of the end opening inside is set (FIG. 2b). Thereby, the amount of the culture solution that remains in the small chamber 3 when the culture solution in the culture tank 1 is extracted is set. It can be easily understood that even when the support rope 6 moves, no germs or the like enter the culture system at all due to the movement for the same reason as in the case of the small chamber support rope 5 described above.

Next, the culture solution 17 in the culture tank 1 is stirred by the action of the magnet rotor 13 so that the cells are uniformly suspended in the culture solution, and the culture solution 17 is withdrawn through the culture solution extraction tube 16. The small chamber 13 appears on the liquid surface. Further, when the level of the culture solution 17 in the tank becomes equal to or lower than the level of the open end of the S-shaped tube 4 on the side located in the culture tank, the culture solution in the small room 3 is reduced by a set amount, that is, the small room 3 It flows down to the inside of the culture tank 1 while leaving an amount equal to or less than the level of the open end located inside (FIG. 2c). The flowing culture solution is discharged out of the culture tank together with the culture solution 17 in the culture tank 1.

Next, the culture medium 19 is introduced into the culture tank 1 through the medium introduction tube 18 (FIG. 2d). Medium into which chamber 3 is introduced
The medium in the small chamber 3 and the new medium are mixed until the liquid level drops below the level of 19, and the inoculation to the secondary culture is completed, and the secondary culture starts (FIG. 2d). Hereinafter, the above operation is repeated as many times as necessary. During the culture, the air supply filter 22
Then, air may be supplied to the gas phase portion of the culture tank through the air supply pipe 20 and then exhausted to the outside of the system via the drain trap 25 and the exhaust filter 26.

In this embodiment, the bellows 9 is shown as being formed integrally with the small chamber supporting cable 5 and the S-shaped pipe supporting cable 6, but it is constructed separately, and is provided with an adhesive, a heat seal or a fastening tool. It is also possible to airtightly join by appropriate means such as. The shape of the small chamber or the S-shaped tube is also arbitrary, but making the shape of the small chamber tapered as shown in the figure is particularly effective for accurately quantifying a small amount of culture solution.

Embodiment 2 FIG. 3 shows another embodiment, in which the small chamber is fixed to the outside of the culture tank 1. Except for the points described below, the other configuration of the culture tank is the same as that shown in Example 1, and therefore, illustration and detailed description will not be made. In this embodiment, the small chamber 303 is attached to a screw hole opened in the side wall of the culture tank 1 by screw engagement from outside the culture tank 1.
The S-shaped tube 304 has one end opened in the small chamber 303 and the other end opened in the culture tank 1. The S-shaped pipe 304 is supported by an S-shaped pipe support cord 306. A bellows 309 is integrally formed near the upper end of the S-shaped pipe support cord 306 as shown in FIG.
The thread is cut above 309. In addition, the lower end of the bellows 309 is hermetically connected to a flange formed below the bellows casing 311 formed integrally with the small chamber 303 in the figure by appropriate means.

In the vicinity of the upper end of the bellows casing 311, a vertically adjustable female screw 328 having a screw hole formed at the center is supported rotatably in the horizontal direction.
A screw formed near the upper end of the pipe support cable 306 is engaged. Therefore, the S-shaped pipe support cable 306 and the S-shaped pipe 304 move up and down by rotating the vertical adjusting female screw 328. At the time of the movement, the culture tank 1
It will be easily understood that the inside atmosphere can be kept completely isolated from the outside atmosphere, and the contamination of the culture system with various bacteria and the like due to the movement can be completely prevented.

The method for using the culture tank may be the same as that in Example 1, and the description is omitted. Also bellow 3
09 is separate from the S-shaped pipe support cable 306 and may be hermetically joined by appropriate means as in the case of the first embodiment. Further, the small chamber 303 and the bellows casing 311 are separately provided. And may be assembled integrally by screw engagement or the like.

In the culturing tank of this embodiment, since the small chamber is located outside the culturing tank, there is an effect that the reserve culture solution can be easily observed and the amount can be easily set. Embodiment 3 FIG. 4 shows still another embodiment, in which a dropper-shaped small chamber is arranged in the culture tank 1. Except for the points described later, the other configurations of the culture tank in this embodiment are the same as those shown in Embodiment 1, and therefore are not shown or described in detail.

In this embodiment, the small chamber 403 has a tubular shape, the upper end of which is fixed by penetrating the culture vessel lid 2.
The lower end extends into the culture tank 1. In addition, Komuro 4
A bellows 409 is connected to the upper end of 03 by an appropriate means. The lid 2 is formed with a bellows casing 411 with a vertically adjustable female thread, and a male thread 410 with a vertically adjustable male thread is engaged with the female thread of the bellows casing 411. Further, a pipe 420 is integrally formed above the bellows 409, and a flange extending in the horizontal direction is formed near the distal end of the pipe 420. The flange is rotatably engaged with a groove formed inside the male screw 410 for vertical adjustment. The distal end of the pipe 420 is further connected to a sterile filter 430, a small chamber decompression pipe 429 equipped with a solenoid valve 432 and a pressure balancing solenoid valve 431. Both solenoid valves are connected to the process sequencer 433 by signal lines.

The operation method will be described below. Before the end of the primary culture, the amount of the culture solution held in the small chamber 403 is set and adjusted. When the pressure-balancing solenoid valve 431 is opened, the culture solution level in the small chamber 403 becomes the same as the liquid level in the culture tank 1. Next, the pressure-balancing solenoid valve 431 is closed, and the bellows 409 is expanded and contracted by turning the male screw 410 to adjust the liquid level in the small chamber 403 to the amount of culture solution used for inoculation in the secondary culture. The solenoid valves 431 and 432 are closed. As a result, a predetermined amount of the culture solution is held in the small chamber 403. The primary culture is completed, and the culture solution is withdrawn from the culture tank 1 to hold a set amount of the culture solution in the small chamber 3.

The operation of setting and controlling the amount of culture solution holding and holding the culture solution in the small chamber 3 is performed by the presence of a means including a sterile filter. Specifically, this is achieved by creating a pressure difference between the inside and outside of the tank in a shut-off state, and it is possible to completely prevent entry of various bacteria and the like into the culture system during operation.

Next, when the medium required for the secondary culture is introduced into the culture tank 1, the cells in the small chamber 3 are mixed with the medium and the inoculation is completed. The time for setting the inoculum volume can be arbitrarily set in the process sequencer 433, that is, by the timer. In this embodiment, if the amount of the culture solution held in the small chamber 403 is within a certain distance from the lower end of the small chamber, for example, within a range of 2 to 3 cm from the lower end of the small chamber, the cells in the culture solution held in the small chamber are small. It has been demonstrated that it can pass outside and does not make much difference in its activity from cells outside the small room. Also, the shape of the tip of the cell compartment may be vertical downward when the amount of the retentate is very small (0.3 ml or less). As shown in the figure, the angle between the vertical part and the vertical part is 80 to prevent air bubbles from flowing back and to allow cells inside the cell to communicate with cells outside the cell.
Preferably, it is set to ~ 98 degrees.

Embodiment 4 FIG. 5 shows another modification in which a dropper-shaped chamber is disposed in the culture tank 1. Except for the points described later, the other configurations of the culture tank in this embodiment are the same as those shown in Embodiment 1, and therefore are not shown or described in detail. In this embodiment, the tubular small chamber 503 has an upper end fixed through the culture tank lid 2, and a lower end having a thin tubular shape extends inside the culture tank 1. The upper end of the small chamber 503 passing through the lid 2 is further connected to a sterile filter 530, a suitable sealable joint 535, and a syringe barrel 536 in this order. Syringe barrel 536 inside the syringe barrel 536
A seal tube 540 with an O-ring 539 supported on the bottom is located. A piston rod 538a having a piston 538 at the tip is provided on the inner surface of the seal tube 540 and the O-ring.
It is inserted so that it can slide up and down with 539. Between the lower end of the piston 538 and the lower end of the syringe barrel 536, a spring 537 biased in a direction to constantly push up the piston 538 is arranged.

The outer cylinder 536 of the syringe has a solenoid casing 543 at the upper part, and the solenoid 541 is fixedly disposed on the inner periphery in the solenoid casing 543. In addition,
In the figure, the solenoid casing 543 is a syringe barrel
Although shown as being integral with 536, it is clear that it may be configured as a separate member. Piston rod 538a
Penetrates a piston up / down adjusting screw 548 located below the solenoid casing 543, and a magnetic body 542 is provided at a position corresponding to the solenoid 541 on the inner periphery of the solenoid casing 543. Further, a female screw is cut inside the syringe outer cylinder 536, and the female screw and a screw cut on the outer periphery of the vertical adjustment screw 548 are configured to engage with each other. A solenoid electrical wiring 534 extends from the solenoid 541 and is connected to the process sequencer 533.

The operation method will be described below. Before the end of the primary culture, the amount of culture solution held in the small chamber is set and adjusted. First, the piston upper / lower adjustment screw 548 is turned to set the upper limit of the piston. During the primary culture, at the time set in the process sequencer 533, the solenoid 541 is actuated, the magnetic body 542 descends, and the air in the small chamber 3 is released from the lower end of the small chamber 3. Solenoid 541 by the following command from process sequencer 533
When the current is cut off, the piston 538 rises to the previously set level due to the stress of the spring 537, and the culture solution is held in the small chamber 3 by the set amount. Next, the culture solution is withdrawn, and a medium for secondary culture is introduced into the culture tank. The solenoid 541 is actuated again by the last command, the culture solution held in the small chamber 3 is released from the small chamber 3, and the medium is mixed to complete the inoculation.

In this embodiment, as in the case of the third embodiment, the setting of the amount of the culture solution held and the operation of holding the culture solution in the small chamber 3 are controlled by the presence of the means including the sterile filter 530 and the solenoid. This is achieved by creating a pressure difference between the inside and outside of the culture tank under a state in which the inside and outside of the culture tank is partially ventilated but is microbiologically shut off. It will be easily understood that it is possible to completely prevent the mixing of the like. In addition, by making the configuration of the tip portion that opens into the culture solution in the small chamber into a thin tube, it is possible to prevent the ingress of bubbles when the culture solution is retained in the small room, and the retention amount is more accurate. Is secured.

Embodiment 5 FIG. 6 shows still another embodiment, in which a small chamber is provided at the bottom of the culture tank 1. Except for the points described later, the other configurations of the culture tank in this embodiment are the same as those shown in the first embodiment, so that illustration and detailed description are not made. A recess 603 that functions as a small chamber is formed at the bottom of the culture tank 1. In addition, the vicinity of the tip is supported by the culture tank lid 2 via a bellows 609, and the other end reaches the recess 603.
Place. A male screw is formed on the outer periphery of the upper end portion of the culture solution discharge pipe 616. The bellows casing 627 is attached to the lid 2.
A vertically adjustable female screw 628 having a female screw at the center is rotatably locked at an upper portion thereof. A male screw formed on the upper part of the culture solution extraction pipe 616 is screw-engaged with a female screw formed on the vertical adjustment female screw 628. Therefore, the height of the lower end of the culture solution extracting pipe 616 in the concave portion 603 can be adjusted by turning the vertical adjusting female screw 628. The upper end of the culture solution extraction pipe 616 is connected to a culture solution extraction pump 644 via an electromagnetic valve 632.

The following operation method will be described. Before the end of the primary culture, turn the female screw 628 up and down to turn the culture medium extraction pipe 6
Set the height in the recess 603 at the lower end of 16
Set and adjust the amount of culture solution retained in Next, the solenoid valve 632
Is opened, and the culture liquid extracting pump 644 is operated to discharge the culture liquid at the lower end of the culture liquid extracting pipe 616 to the outside of the system. Thereby, as in the other examples, the culture tank 1
A predetermined amount of the culture solution can be held and maintained in the small chamber in a state where the inside atmosphere is completely isolated from the outside atmosphere. Next, when a culture medium required for the secondary culture is introduced into the culture tank 1, the culture medium in the small chamber, that is, the recess is mixed with the culture medium, and the inoculation is completed.

It will be easily understood that, in this embodiment as well, the incorporation of various bacteria and the like into the culture system during the operation can be completely prevented. Example 6 Next, FIG. 7 shows an example of a culture system in which a single culture tank is used to carry out subculture of living cells.

The same culture tank as that shown in Example 1 was used. The tank wall of the culture tank 1 is in contact with a heater 47 supported by a gantry 48 in order to keep the inside of the culture tank at a constant temperature. The stirring in the culture tank is performed by rotating the magnet rotor 49 provided outside the tank bottom by the stirring motor 50 to rotate the stirring blade 12. The medium 19 and the culture solution 17 are stored in a medium storage tank 51 provided with a filter 52 and a culture solution storage tank 53 provided with a filter 54, respectively. The withdrawal of the culture solution 17 and the supply of the culture medium 17 are performed by operating the corresponding electromagnetic valve 46, culture solution withdrawal pump 44, and culture medium supply pump 45 in accordance with a command from the process sequencer 33.

Using the present system, the culture can be successively subcultured by successively repeating the introduction of a medium for the next culture after extracting the current culture solution. Example 7 An example of a system having a plurality of culture vessels is shown in FIG. The same culture tank 1 as that shown in Example 3 is used. Culture liquid extraction pipe 16, medium supply pipe 18, air supply pipe
20 are assembled with a valve interposed between each tank. The air supply system is an air compressor, as shown in the example of animal cell culture.
55 and a carbon dioxide gas cylinder 56 are connected, and connected to each tank via a gas mixer 35 and a filter 22. Exhaust is performed by an exhaust pipe 57 for each tank. The temperature of the heater 47 is controlled by a temperature controller 58, and the operation of each valve and pump is controlled by a process sequencer 33.
Performed by

By using this system, Embodiment 6
As in the case of the above, the culture can be successively subcultured by sequentially repeating the introduction of the medium for the next culture after extracting the current culture solution. This system is particularly useful when the culture vessel is small and is made of plastic as a disposable type. Example 8 Subculture was actually performed using the system shown in FIG. The culture tank 1 (inner diameter 25 mm × height 20 mm) shown in FIG. 1 was made of glass, and the lid 2 and various lid accessories were made of polypropylene. The piping used was a silicone rubber tube. Medium storage tank 75
1. The system including the culture tank 1 including the culture solution storage tank 753 was heat-sterilized in an autoclave.

In the primary culture, the Dulbecco's modified Eagle MEM medium was injected from the medium storage tank 1 into a 17 ml culture tank with a pump 745. Then, mouse-mouse hybridoma ST which is a floating animal cell as a seed cell in a clean bench
The K-1 strain was inoculated at 1 × 10 ⁵ cells / ml, and 5% CO ₂ -enriched air was passed through the gas phase in the tank through a sterile filter to start culturing at 37 ° C. Prior to the start, an adjusting screw was used so that the residual liquid volume in the small chamber 3 was 0.2 ml as a seed cell liquid for secondary culture.
Turn 10 to set. The sequencer 33 is set so that the start of the secondary culture can be performed 3 days later, and the culture can be subcultured in the same cycle every 3 days after the tertiary culture. Was observed.

On the other hand, as a comparative example, a conventional subculture method in which inoculation was carried out manually with a sterilized pipette in a clean bench using the same culture tank 1 and lid 2 was carried out.
Workers are required to test three highly skilled workers who have at least 3 years of experience, two months of experienced workers, and one less than one week of unskilled workers. Was experimented. The results are shown in Table 1.

[0045]

[Table 1]

The conventional method requires a great deal of labor for inoculation,
In addition, even a skilled person can cause biological contamination of the culture. On the other hand, in the present invention, the inoculation amount can be set in advance without invasion, so that long-term subculture can be automatically continued without microbial contamination.

[0047]

According to the present invention, the subculture of cells can be automatically repeated without using complicated and expensive robots and using a simple mechanism without causing contamination of various bacteria.

[Brief description of the drawings]

FIG. 1 is a cross-sectional view showing an embodiment of a culture tank according to the present invention, showing an example in which a small chamber is provided in the culture tank.

FIG. 2 is a diagram schematically showing a typical flow of a subculture method according to the present invention.

FIG. 3 is a cross-sectional view showing another embodiment of the culture tank according to the present invention, showing an example in which a small chamber is connected to the outside of the culture tank.

FIG. 4 is a cross-sectional view showing still another embodiment of the culture tank according to the present invention, showing an example having a small chamber connected to a decompression mechanism.

FIG. 5 is a sectional view showing still another embodiment of the culture tank according to the present invention, showing another example having a small chamber connected to a decompression mechanism.

FIG. 6 is a cross-sectional view showing still another embodiment of the culture tank according to the present invention, showing an example in which a small chamber is formed at the bottom of the culture tank.

FIG. 7 is a diagram showing an example of a culture system (apparatus) according to the present invention.

FIG. 8 is a diagram showing another example of the culture system (apparatus) according to the present invention.

[Description of Signs] 1 Culture tank 2 Lid 3 Small chamber 4 S-shaped tube 5 Small chamber supporting line 6 S-shaped tube supporting line 9 Bellows 10 Bellows casing

Claims (13)

(57) [Claims] 1. A subculture suspension culture method biological cells, characterized by culturing in the next step, (1) constructed in a closed system with respect to entry of at least microorganism entire culture device, the device Indirectly operate the culture solution in the constituting culture tank without destroying the closed system from outside the system, that is, seeds of the next culture in the culture tank without invasion of microorganisms from outside the system. A step of steplessly setting the amount of the culture solution to be used for the cells in advance, (2) In a state where the cells are uniformly suspended in the solution, the culture solution of the set amount is placed in a small chamber provided in the culture tank. (3) extracting the remaining culture solution out of the culture tank, (4) introducing a fresh medium into the culture tank, (5) mixing the reserved culture solution with the fresh medium, and ( 6) The above method comprising the step of performing the next culture. 2. The upper part of the small chamber communicates with the space in the culture tank. The S-shaped tube communicating the inside of the small chamber and the space in the culture tank moves up and down to adjust and set the amount of the culture solution held in the small chamber. The method for subculture of biological cells according to claim 1, wherein the method is carried out. 3. The biological cell according to claim 2 , wherein the small chamber can be moved up and down in the culture tank, and the vertical position of the small chamber is also adjusted and set when adjusting and setting the culture solution holding amount. Subculture method for suspension culture. Wherein chamber is only the end in contact with both cultures having extensible part is opened, and adjusting sets the culture hold volume by the expansion and contraction of about Komuro, claim 1, wherein Method for subculture of biological cells in suspension. 5. Komuro and adjusting and setting a hold volume of culture by adjusting the degree of pressure reduction in the gas phase has a gas phase, the following biological cell of claim 1, wherein Suspension culture method. 6. A culturing tank having a means for stirring the culture solution, a small container having an open upper part, and an S-shaped tube connecting the inside of the small container with the inside of the culturing tank. The S-shaped tube is mounted so as to be able to move up and down through the inner space of the culture tank and sealing means, and further has means for operating the S-shaped tube from outside the culture tank. Subculture suspension culture tank. 7. A means for stirring the culture solution in the culture tank, and a small container is fixed outside the side wall of the culture tank, and the inside of the small container communicates with the inside of the culture tank. Yes,
An S-shaped tube communicating with the small container and the inside of the culture tank is disposed across the side wall portion, and the S-shaped tube is mounted so as to be able to move up and down through an inner space of the culture tank and sealing means. And a means for operating the S-shaped tube from outside of the culture tank. 8. A small container is characterized in that it further comprises means for vertically moving operation from the outside of the culture tank independently of the S-shaped tube, passage suspension according to claim 6 or 7, wherein the biological cells Culture tank. 9. A means for stirring the culture solution in the culture tank, one end of which is open in the culture tank, the other end is fixed to the wall of the culture tank, and a part thereof expands and contracts. It has a flexible chamber, a small chamber having a means for adjusting and setting the degree of expansion and contraction from the outside of the culture tank, and further has, at its base, a branch pipe into which an automatic valve connected to a timer via a filter is inserted. A passage of a biological cell, wherein a predetermined amount of culture solution can be held in the compartment within a limit range by manual operation of an automatic valve connected to the timer.
Suspended culture tank . 10. A means for agitating a culture solution in a culture tank, wherein one end is fixed to a culture tank wall, the other end is opened in the liquid, and a path extending outside the culture tank wall is provided. Has a filter, has a piston whose volume can be set next to the filter,
Next, a means for moving the axis of the piston in the reciprocating direction is provided, and by moving the piston axis by the means for moving in the reciprocating direction, a predetermined amount of the culture solution within the limit range is introduced into the small chamber. A subculture suspension cell culture tank for biological cells, which can be stored . 11. A culture vessel having a small recess at the bottom thereof for forming a small chamber, extending from the culture vessel wall through the wall toward the recess, and having a base joined to the vessel wall at the base thereof. It is connected to the wall via a sealing means of a stretchable structure that can constitute a non-invasive state with the internal space of the inside of the tank, and the means for adjusting and setting the degree of expansion and contraction from outside the tank is within the limit range. in passaging suspension culture vessel organism cells, characterized in that the predetermined amount of the culture solution can be held in the small chamber. 12. The subculture suspension culture tank for biological cells according to any one of claims 6 to 11, oxygen supply means for the culture tank, culture liquid withdrawal means in the culture tank, culture liquid storage means, and culture medium. A subculture apparatus for subcultured biological cells, further comprising a storage unit, a medium supply unit, a temperature control unit, and a program control unit. 13. A plurality of subculture tanks for biological cells according to any one of claims 6 to 11, which are connected in series by a liquid communication pipe, and a culture liquid extraction pipe from each culture tank, and a medium supply. The pipes and the gas supply pipes are assembled for each type of pipe via an automatic valve, and a culture solution storage tank, a medium storage tank, and a sterile processing gas supply source are connected to each other, and further, each automatic valve and a liquid transfer drive source are connected. Subculture of biological cells connected to a process sequencer
Equipment .