JPH0380071A - Submerged culture under aeration and apparatus therefor - Google Patents

Abstract

PURPOSE:To carry out the culture of cells in a culture tank containing a draft tube or a built-in straightening vane in high efficiency by adjusting the gap between the lower end of the draft tube or the straightening vane and the bottom of the tank in such a manner as to get the cell concentration difference between the bottom and the inside of the tank to be fallen within a prescribed range. CONSTITUTION:Living cells are cultured in a liquid in suspended state under aeration using a culture tank containing a draft tube or a built-in straightening vane. In the above process, the gap between the lower end of the draft tube or the straightening vane and the bottom of the tank is adjusted in such a manner as to get the cell concentration difference between the bottom and the inside of the tank to be fallen within a prescribed range. The cells are smoothly fluidized and cultured without causing the precipitation of the cells even under low rate of aeration.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a submerged aerated suspension culture method for biological cells, a culture device, and a culture system.

[Conventional technology]

In recent years, suspension culture of animal, plant, and microbial cells has become a mainstream technology for producing useful substances such as pharmaceuticals.

An air lift system with a draft tube is known as a typical conventional method for culturing these cells in suspension. Examples of documents describing this method include the following.

(1) Japanese Patent Application Laid-Open No. 63-84492 (Publication 1986)
Year) (2) Journal of Chemical Technology, Volume 21, No. 7, p.
, 21 (1982) (3) Acta, Biotechnology Power, Hi, (1), 3 (1982) 3rd to 4th
1 page (8cta Biotecl+nologica
2 + (1) p, 31-41 (1982) ) (4
) Torrens, in, Biotechnology, Volume 3 No. 7
, pp. 162-166 (Trends i
nBi.

technology, 3. (7) p. 162
~166 (1985)) (5) Biotechnology, and Bioengineering, XIX, 1
Pages 503 to 1522 (Biotechnology
and Bioengineering,
X, p., 1503-1522 (1977)) These methods: 1. Dissolve gases necessary for biological reactions in the culture solution by aerating gas into the solution; 2. Mix the culture solution. It has the advantage of allowing the cells to be suspended in the liquid, and also has the advantage of being able to cope with larger scales because it does not have a complicated structure unlike mechanical agitation.

However, these methods have the following problems when culturing cells.

■As the gas to be vented, not only air but also a mixed gas enriched with oxygen, carbon dioxide, nitrogen, etc., which is more expensive than air, is often used.

■Vent gas must be sterilized by microbial filter filtration, etc.

■Continuous aeration is required during the culture period, which lasts from several days to several months, consuming a large amount of aeration gas.

■During the culture period, not only the concentration of cells and the viscosity of the solution, but also the ease with which cells can be separated as a cell population during the process in which each cell in the culture solution divides from mother cells to daughter cells and proliferates. Cell characteristics, such as the ease with which cells adhere when they come into contact with each other, change from moment to moment, and the specific gravity of cells and cell aggregates, that is, the sedimentation characteristics, change accordingly.

■Compared to the amount of ventilation required to dissolve gas, the amount of ventilation required for liquid flow is usually larger.

■Even if it is temporary, the amount of aeration can be adjusted to changes in cell concentration, cell specific gravity, and liquid properties. Even if the amount is increased, resuspension becomes difficult. When deposited, the cells in the deposited layer are starved of oxygen and nutrients, leading to growth inhibition or death.

■Excessive aeration actually inhibits cell growth.

This has a particularly large effect on fragile animal cells.

■ When adding proteins such as serum or aldagon to the culture medium or secreting proteinaceous products, as in the case of culturing animal cells, it is necessary to provide an antifoaming mechanism that does not destroy the cells. As the amount of ventilation increases, the load on the defoaming mechanism increases accordingly, and the capacity of the defoaming mechanism increases.

In this way, under certain fixed conditions, the conventional trough and tube-equipped air lift type culture tank described above
For example, they are fixed in a structure that optimizes flow under a certain cell concentration, cell specific gravity, and liquid properties, and if conditions change, the only way to do so is to regulate the amount of ventilation.

Therefore, depending on changes in conditions, the amount of ventilation becomes larger than when using the optimal structure, which not only reduces economic efficiency but also has many drawbacks such as making it difficult to widen the operational range of operation. .

[Problem to be solved by the invention]

The purpose of the present invention is to improve the drawbacks of the conventional draft tube or air lift type culture device with a baffle plate, and to adapt to cell characteristics such as cell concentration, cell specific gravity, and cell adhesion, and liquid properties such as viscosity. It is an object of the present invention to provide a culture device, a culture method, and a culture system that can smoothly fluidize cells without depositing them even at a low air flow rate.

(Means for Solving the Problems) 0 The inventor of the present invention first investigated the causes of cell sedimentation and found that when cells come into contact with each other at rest or at a low flow rate, they tend to adhere to each other and sediment. , - It was found that redispersion of the sedimentary layer becomes difficult once it has been deposited.We found that this tendency is particularly pronounced in animal cells.The cells inside the sedimentary layer provide oxygen and nutrients to the cells in the surface layer. Dissemination is impeded, leading to stunted growth and death.

The inventor further focused on the flow of cells near the bottom of the culture tank and the draft tube or the lower part of the rectifying plate, and observed the flow state while changing the cell concentration and liquid properties.

As a result, by adjusting the gap between the bottom of the tank and the bottom of the draft tube or current plate based on the contrast between the cell concentration at the bottom of the tank and the bottom of the draft tube or current plate, and the cell concentration at other locations, We have discovered that even if the cell concentration changes over time, cells can be smoothly fluidized without sedimentation with a much lower aeration rate than when using a conventional draft tube or a culture tank with a fixed rectifier plate.

1 The present invention was made based on the results obtained from the above-mentioned observations.

The present invention provides a culture method using a culture tank with a built-in draft tube or baffle plate for supplying gas to biological cells in a suspended state by aeration into the liquid, by reducing the gap between the lower end of the draft tube or baffle plate and the bottom of the tank. A method for culturing biological cells characterized by changing and regulating cell flow,
The difference in cell concentration between the bottom of the tank and the inside of the other tanks is detected, and the draft tube or current plate is moved so that the concentration is within a preset concentration difference range. and the bottom of the tank, and also detects the concentration of one or both of oxygen and carbon dioxide in the culture solution in the tank, so that the concentration is within a preset range of dissolved gas concentration. By adjusting the gas concentration in the ventilation gas and also detecting the pH in the culture solution in the tank,
Adjusting the carbon dioxide concentration in the ventilation gas so that the pH is within a preset pH range, and adjusting the concentration gives top priority to the cell concentration difference, followed by dissolved oxygen concentration 2 degrees, pH When adjusting the dissolved oxygen concentration, if the adjustment cannot be achieved by increasing the oxygen concentration in the raw material gas mixture, the dissolved oxygen concentration should be adjusted by increasing the aeration rate. In addition, changing the distance between the upper end of the draft tube or the current plate and the liquid level within a range where the upper end of the draft tube or the current plate does not protrude above the liquid level is a method of controlling the biological cells of the present invention. This is a specific or more preferred embodiment for carrying out the culture method.

In addition, in the present invention, the upper limit of the linear velocity of the test cells in the liquid flow in the gap between the draft tube or the lower end of the rectifying plate and the bottom of the tank, which is caused by ventilation, is 3 m/s.

A method for culturing biological cells characterized by limiting the amount of ventilation so that the flow rate of the liquid is preferably 3 m/s.
If the difference exceeds the set value, increase the gap, then detect the difference in cell concentration, and if the difference in concentration exceeds the set value, increase the ventilation amount so that the difference in cell concentration falls within the set range. , dissolved gas concentration, and normal velocity are adjusted so as to satisfy them.

3 The present invention also provides a draft that is movable in the vertical direction within a range where the upper end of the draft tube or the rectifier plate does not protrude above the liquid surface in a culture tank that supplies oxygen to biological cells in a suspended state by ventilating the liquid. A culture tank for biological cells characterized by having a built-in tube or a rectifying plate, the draft tube or the lower part of the rectifying plate, the draft tube or the upper part of the rectifying plate, or both being able to move up and down; trough] - The tube is composed of an inner cylinder and an outer cylinder, a part of the draft tube or rectifier plate is fixed to the tank, and the other parts are movable, and a mechanism for moving the draft tube or rectifier plate. A specific or more preferable example of the present invention is that the mechanism is one or a combination of a mechanism for introducing fluid into and out of a bellows or piston, a drive mechanism using a geared motor, an expansion/contraction mechanism for thermal expansion material, and a magnet drive mechanism. It is a mode.

Furthermore, the present invention is a biological cell culture system characterized in that a plurality of the above-mentioned culture vessels are stacked in series or in a mixed state of serial and parallel culture vessels in terms of liquid flow, and is an object of the present invention. As a specific or more preferable embodiment to achieve this, in the culture tank,
Means for controlling the movement of draft tubes or rectifying plates and other means for controlling cell culture conditions, such as D○
It includes a control system, a pH control system, and a medium supply system, and allows the movement of the trough L-tube or rectifier plate and other cell culture conditions in each of the plurality of stacked culture tanks to be individually controlled. It is.

In addition, in the aspect of the present invention, stacking the culture tanks in series in terms of liquid flow refers to a state in which the liquid flowing sequentially in the stacked culture tanks flows in one direction as shown in H in FIG. 11. This means that the culture tanks are stacked in parallel in terms of liquid flow, as shown in J in Figure 11. This means that they are stacked in such a way that they flow. Therefore, stacking multiple layers in a state where the liquid flow is mixed in series and in parallel means that the first
It means the state shown in Figure 1 (■).

The biological cells applicable to the present invention are not particularly limited. That is, it can be applied to animal cells, plant cells, and microbial cells. These cells may be in a solitary state or in an aggregated state. Aggregated state refers to flocs, organs, and tissues in which cells are attached to each other. Furthermore, granules obtained by using other materials as carriers can also be included. For example, microbeads to which cells are attached and cell-encompassing gel particles that enclose cells are also included.

The medium is also not particularly limited. That is, the test cells or the culture medium passed through the culture conditions can be appropriately selected and used. For example, as a carbon source, saccharides ranging from low molecules to polymers, organic acids, alcohols, etc. are used, and as a nitrogen source, amino acids, peptides, inorganic nitrogen sources, etc. are used. Other components such as serum, albumin, vitamins, inorganic salts, buffering agents, antibiotics, growth-promoting factors, adhesion factors, differentiation factors, etc. may also be used depending on the cell type and culture conditions.

The culture of the present invention is limited to a method in which cells or particulate matter containing cells are cultured by flowing them in a suspended state in a liquid. In other words, by injecting gas into the liquid and bringing the bubbles into contact with the liquid, gas components necessary for biological reactions in cells and adjustment of liquid properties such as pH are dissolved, and at the same time, particulate matter is removed using an air lift. Limited to culture suspended in liquid. At this time, the temperature, pH 1 substance concentration, dissolved gas concentration, ionic strength, etc. are appropriately adjusted according to the type of cells and set culture conditions, as in general culture.

The gas to be vented may also be appropriately selected and used.

For boat fishing, air, oxygen, and carbon dioxide are used alone or in a mixture of air, oxygen, carbon dioxide, and nitrogen.

These gases may be used either with the gas composition fixed or with the gas composition changed during the culture period.

Note that all of these gases are used after being sterilized by microbial filter filtration or the like.

As a culture tank, among the culture tanks suitable for the above-mentioned culture,
It is not particularly limited as long as it has a draft tube or a current plate. For example, shapes such as a cylinder, a prism, a plate, etc. can be used. Particularly, prismatic or plate-shaped ones are preferable because they can be used even if a plurality of them are laminated.

The draft tube may be a right cylinder or one whose diameter changes in the vertical direction, and is appropriately selected depending on the culture tank shape and culture conditions. As for the latter, for example, a conical shape, or a shape in which the lower part, the upper part, or both parts are expanded into a funnel shape is used. If the culture tank is prismatic or plate-shaped,
A two-dimensional rectifier plate is used.

Depending on whether the flow of the liquid in the draft tube or the current plate is upward or downward, the shape of the draft tube or the current plate is appropriately selected. For example, a draft tube with an enlarged lower part or a baffle plate is suitable for upward flow.

In the present invention, as described above, the difference in cell concentration between the bottom of the tank and other parts is detected, and if the cell concentration exceeds the set range, that is, if the cells tend to settle, the lower end of the draft tube or current plate is detected. By adjusting the gap with the bottom of the tank, the linear velocity of the liquid in the gap area can be changed.

At least two detection points for cell concentration are required.

That is, the bottom of the tank or its vicinity, and other locations, such as the center of the tank or the upper half of the tank.

The method for measuring cell concentration is not particularly limited. For example, there are particle concentration measuring devices such as a turbidity meter that uses transmitted light, transmitted waves, reflected light reflected waves, or scattered light, and particle concentration measuring devices that use image measurement.

The vertical movement and expansion/contraction of the draft tube or current plate may be any mechanism as long as it can withstand steam sterilization in the tank and can maintain the inside of the tank in a sterile state during the culture period. Specific examples include the expansion and contraction of a heros operated by the inflow and outflow of fluid from outside the tank, a sealed electric motor, and an electromagnetic piston.

In this case, the part that generates power and the power transmission mechanism may be located inside or outside the tank.

The draft tube or baffle plate is not only the lower part, but also the upper part, which can move and expand and contract within a limited range at the end of the liquid level, making the flow of the liquid more consistent. is also possible.

The draft tube or the current plate can be expanded or contracted by combining the divided draft tubes or the current plate in double or multiple layers, or by making them made of a stretchable material. In the case of draft tubes, the divided tubes are formed into double tubes or multiple tubes, which may not move horizontally but only in the vertical direction, or may mutually rotate using a screw mechanism. It may be expanded and contracted by a combination of these methods.

The structure of the bottom of the tank may have any shape as long as it allows smooth flow of the liquid. For example, the concave surface may be gently curved, or only the center portion of the concave surface may be convex.

The ventilation nozzle is also appropriately selected depending on the culture conditions.

Usually, it is provided in the middle between the bottom of the tank and the lower end of the draft tube or current plate. In order to smoothly fluidize the bottom of the tank, an auxiliary ventilation nozzle may be provided that opens at the bottom of the tank.

0 In addition to fluidizing the liquid, culture requires the supply of dissolved gas.

In the present invention, the cell concentration difference is given first priority, and at least two of the dissolved concentration of foreign gas and pH are adjusted in this order of priority. For example, in the case of animal cell culture, the amount of aeration gas due to the difference in cell concentration,
The ventilation gas composition is controlled according to the dissolved oxygen concentration, and the carbon dioxide concentration in the ventilation gas is controlled according to the pH.

When adjusting the dissolved oxygen concentration, if the concentration of the raw material gas in the mixed gas for aeration exceeds the adjustment limit, the adjustment of the dissolved oxygen concentration will be prioritized over the adjustment of the cell concentration difference. This increases the amount of ventilation beyond the amount suitable for regulation.

By adopting the operating method described above, it is possible to ensure smooth flow of the liquid and to satisfy other requirements such as dissolved oxygen concentration.

Examples of the present invention will be described in detail below.

<Example ■> The culture system of the present invention shown in FIG. 1 is a flowchart of one such embodiment.

The culture tank 1 is a cylindrical draft consisting of a trough L-tube outer cylinder 3 fixed on the inner wall surface of the tank by a draft duplex outer cylinder support 5, and a draft tube inner cylinder 4 which is movable downward by a draft duplex inner cylinder vertical drive unit 6. It has a built-in tube.

A liquid medium 27 is introduced into the culture tank 1 from a liquid medium storage tank 29, and a culture solution 28 can be extracted into a culture solution storage tank 30 as needed.

A gas containing air, oxygen, nitrogen, and carbon dioxide is transferred from the gas collection pipe 18 and diffused into the culture solution from the sparger 7 at the bottom of the draft tube.

In order to monitor the deposition of cells at the bottom of the culture tank, cell concentration sensors 10, specifically, for example, turbidity sensors, are installed at two locations. One is installed in or near the gap between the bottom of the culture tank and the lower end of the draft tube, and the other is installed at a position away from the former, for example, in the upper part of the tank for comparison. The sensor and the cell concentration detection device 16 measure the difference in cell concentration, and the gap controller 15
This sends a signal to the draft tube inner cylinder up and down drive section 6 to move the cylinder up and down, thereby increasing the gap by 8jI.

If it is necessary to eliminate foam on the upper surface of the liquid, a net 2 for defoaming can be provided above the liquid surface.

Dissolved oxygen is regulated by a Do sensor 2 and a D○ controller 13, and pH is regulated by a pH sensor 11 and a pH controller 14. By adjusting the composition and amount of ventilation gas, DO can adjust the pH by, for example, changing the concentration of carbon dioxide in the ventilation gas and the amount of ventilation. In addition, a liquid flow rate sensor 31 is installed in the gap 4=J
The linear velocity of the liquid in the gap is measured by a liquid flow rate detector 32, and the amount of aeration gas and the gap are adjusted so that the linear velocity falls within a flow rate range in which shearing of the cells does not occur.

The culture apparatus used for culturing biological cells in the first embodiment of the present invention is as follows.

◎Culture tank shape Bicylindrical 3 Amount of liquid filled = 51 Tank inner diameter: 120 mm Tank inner depth 7600 mm ◎Draft tube outer cylinder upper part: diameter 50 mm, length 33 mm Lower outer cylinder: diameter 80 mm, length Within 50 mm Tube: Diameter 7
5 mm, length 90 mm The vertical mechanism of the inner cylinder in the draft tube of the above-mentioned culture device is shown in FIG.

A metal bellows 36 is arranged near the outer cylinder 3 and the outer cylinder support 5, and the lower part of the bellows and the draft tube inner cylinder are connected via a bellows expansion/contraction transmission limb 38.

Compressed air is introduced and discharged from the air-driven bellows supply/exhaust pipe 37 located above the bellows, thereby expanding and contracting the bellows to move the inner cylinder 4 up and down.

The temperature inside the tank is controlled to be constant by a constant temperature water circulation jacket 33.

What is shown in FIG. 3 is a flowchart in this embodiment.

When culturing was carried out using the present culture method and culture apparatus while adjusting the cell concentration, pH, Do, the gap between the lower end of the draft tube or rectifier plate and the bottom of the tank, etc.
The results of comparison with other examples are described below.

○Culture conditions The test cells were rat liver cancer cell line JTC,-1' (Ja
pan Ti5sue Culture No. 1
Stock) is used.

Seed culture uses Eagle's MEM medium, and is cultured in a flat flask and then in a roller bottle.

The culture temperature is 37°C.

The above culture solution is centrifuged to collect cells and used as cells for inoculation.

After introducing Eagle's MEM medium into the culture tank, the above seed cells were inoculated so that the cell concentration became IX106 cells/mR, and the mixture was incubated at 36-38°C, pH 6,8-7.2.
Cultivate in batch mode for 10 days while adjusting DO2 to 5 to 3.0 ppm.

Culture was carried out under the above conditions, and 34 days after the start of culture.
The results of the adjustment up to the time are shown in FIG.

5 ○Adjustment process■ After seed cells were inoculated, aeration was started and after 10 minutes, measurement of cell concentration difference, DO, and liquid flow rate in the pH1 gap was started.

(2) DO and pH gradually decreased, and after 5 hours both reached the set lower limit (indicated by the dotted line in Figure 4).

(2) For D○, the oxygen gas concentration was increased, and the pH was adjusted by decreasing the carbon dioxide concentration.

Similar adjustments were made 18 hours after the start.

■After 21 hours, the cell concentration difference rate, i.e., exceeded the upper limit of 10%, and sedimentation at the bottom of the tank was detected, so the gap was reduced from 5cn+ to 4c+n, and as a result, the normal velocity in the gap became 0. .5m/s, to 0.7m
/s, and the cell concentration difference rate also became below the set upper limit, making it possible to prevent cell deposition.

■After 32 hours, the DO reached the set lower limit again, but the oxygen concentration in the aeration gas had already reached 40% of the adjustment upper limit and it was not possible to increase the oxygen concentration, so the aeration rate was reduced to 0.3 ff. /min, to 0.3! M! /min,
increased to As a result of this operation, D○ returned to within the set range. At this time, the linear velocity in the gap was 1 m/s, which was within the set upper limit (3 m/s), so the culture was continued without changing the gap.

■If, in the case of (■) above, the normal velocity reaches the set upper limit or higher, as shown in the flowchart in Figure 3, open the gap to 3 m/s or less and reduce the cell concentration difference again. Measure. At this time, if the cell concentration difference is within the set range, the culture is continued; if it exceeds the set range, the linear gas velocity is increased until it falls within the set range.

O Comparative Example 1 The same culture apparatus and the same cells as in Example 1 were used, except that the gap between the lower end of the draft tube or rectifier plate and the bottom of the tank was not adjusted. The cells were cultured for 10 days using the following procedures.

7 ○Comparative Example 2 The same culture apparatus and the same cells as in Example 1 were used, except that the gap between the lower end of the draft tube or rectifier plate and the bottom of the tank was fixed at 0.4 crn without adjustment. The cells were cultured for 10 days using the following procedures.

○Culture results A summary of the culture results of the above three cases is shown in Table 1.

It can be seen that in the case of Example 1, the achieved cell concentration and survival rate were higher than in Comparative Examples 1 and 2, and culture could be performed with a small aeration amount.

In the case of Comparative Example 1, by increasing the amount of aeration, cells can be cultured while keeping them uniformly suspended in the same way as in Example 1, but the amount of aeration required is three times that of Example 1, and The cell concentration and survival rate are also lower than in Example 1.

In Comparative Example 2, since the gap was narrow and fixed, an equilibrium state was maintained in which the cells were deposited at the bottom of the tank and were sequentially resuspended. Since the gap is narrow, the normal velocity in the gap approaches 3 m/s, which causes shearing to the cells, and the supply of oxygen to the deposited cells and the diffusion of nutrients become insufficient, causing the cells to The concentration and survival rate were significantly inferior to the results of Example 1.

(Margins below this page) 9 0 <Example 2> The top of the culture tank similar to Example 1 was made of transparent glass, the illuminance of the liquid surface was maintained at 1000 lux using a white light source, and Chlorella ellipsoidea was grown in a palindromic manner. Culture in suspension.

The conditions used for culturing biological cells in the second embodiment of the present invention are as follows.

Filling liquid amount: 5I! .

Medium Nibrisdol medium inoculation concentration: 5 x 10' cells/mRPH
: 6.6 to 7.5 Temperature: 27 to 29°C Aeration gas: Mixed gas of air, carbon dioxide gas (upper limit 10%), and nitrogen Next, the culture method and method described in detail later as a second embodiment of the present invention. Using a culture device, batchwise process 2 times while adjusting the gap between the lower end of the draft tube or rectifier plate and the bottom of the tank.
The results of comparing the case of suspension culture for 0 days and other examples will be described below.

○Comparative Example 3 1 The same culture apparatus and the same cells as in Example 2 were used, except that the gap between the lower end of the draft tube or rectifying plate and the bottom of the tank was fixed at 8 cm, and the same operation was performed for 20 days. Cultured.

Comparative Example 4 The same culture apparatus and cells were used as in Example 2, except that the gap between the lower end of the draft tube or rectifying plate and the bottom of the tank was fixed at 0.4 cTrl, and the cells were cultured for 20 days in the same manner.

The results of the above three cases are summarized in Table 2.

As is clear from Table 2, culturing according to Example 2 of the present invention is different from the conventional method in which the gap between the lower end of the draft tube or rectifying plate and the bottom of the tank is fixed as shown in Comparative Example 3.4. Compared to the case of using an apparatus, the culture efficiency is far superior, and the amount of aeration gas can be significantly reduced.

(Margins below this page) 2 3 [Function] As described above, according to the biological cell culture method, culture device, and culture system of the present invention, the gap between the lower end of the draft tube or the rectifier plate and the bottom of the tank can be changed. It not only adjusts the flow of cells and changes the gap by detecting the difference in cell concentration between the bottom of the culture tank and the other parts of the tank, but also changes the gap between oxygen and carbon dioxide in the culture solution in the tank. It also detects the concentration of both, and also detects the pH in the culture solution in the tank, and adjusts the gas concentration or pH in the ventilation gas so that it is within the set dissolved gas concentration range or within the set pH range. Since it also adjusts the aeration rate, it can respond well to changes in cell concentration, liquid viscosity, and changes in the specific gravity of cells and cell aggregates, that is, changes in sedimentation characteristics due to changes in cell properties, preventing cells from settling at the bottom of the tank, and Since growth inhibition or cell death due to lack of oxygen and nutrients can be eliminated, biological cells can always be cultured with a certain degree of precision.

<Example 3> 4 As shown in FIG. 5, this is a cylindrical draft tube used in the biological cell culturing apparatus of the present invention.

In FIG. 5, A: A type in which the entire draft tube 8 placed in the culture tank 1 is moved up and down by a vertical drive device 7.

B: A type having a vertical drive device outside the culture tank 1.

A type in which the draft tube C is divided into upper and lower parts, the upper draft tube inner cylinder 9 is fixed, and the lower draft tube outer cylinder 6 moves up and down.

D1 is divided into upper, middle and lower parts, with only the middle outer cylinder being fixed, and both the upper and lower inner cylinders 5 and 6 moving up and down.

The later ones are shown.

<Example 4> As shown in FIG. 6, the culture tank shares a culture tank wall with a draft tube or has a rectifying plate.

5 In Fig. 6, E: The horizontal cross section of the tank is rectangular, and the draft tubes 4 and 5 are formed by the rectifying plate and the tank side wall.The second part 4 of the draft tube is fixed, and the lower part 5 of the draft tube is A format that moves up and down.

Type F has a rectifier plate parallel to the side surface, the rectifier plate 5 is fixed, and the rectifier plate 8 moves up and down.

G: A type in which the tank is divided into a culture tank 2 and a draft tube 4 by a cavity extending through it in the lateral direction, and a rectifying plate 8 provided at the bottom of the draft tube 4 is moved up and down.

are shown respectively.

<Example 5> As shown in Fig. 7, in addition to adjusting the gap by raising and lowering the inner cylinder of the draft tube, the bottom of the culture tank opposite to the bottom of the draft tube was made concave, and the bottom of the tank near the sparger was made convex. This is a culture tank in which the settling cells can easily ride the upward flow of the liquid.

<Example 6> 6 As shown in Fig. 8, in addition to adjusting the gap by raising and lowering the inner cylinder of the draft tube, a sub-sparger separate from the main sparger at the top of the bottom is provided on the bottom to remove cells that tend to stay on the bottom. This is a culture tank that makes it easy to carry the liquid upstream.

<Example 7> As shown in FIG. 9, this is a culture tank in which the intermediate part of the draft tube is fixed in E of Example 4, and the upper rectifying plate is moved up and down.

The type of this embodiment can be used depending on the amount of liquid to be filled. For example, it can be used for so-called fed-batch culture, in which a small amount of liquid is used at the start of culture, and the culture medium is added sequentially without the need for the progress of culture.

<Example 8> As shown in FIG. 10, this is a culture tank in which a plurality of unit culture tanks of Example 7 were stacked.

The example of the combination of unit culture tanks in this example is the 11th example.
As shown in the figure. That is, H: simple series system.

7 ■: A group of unit culture tanks connected in parallel are piped in series.

J: Stacked culture tanks are divided into unit culture tank groups and each is operated separately. In the figure, two
Flowchart 1, which operates separately in two unit culture tanks.
is exemplified.

〔effect〕

As described above, the submerged aeration culture method of the present invention is a culture method using a draft tube or a culture tank with a built-in rectifier plate that aerates the liquid and supplies gas to biological cells in a suspended state. This device adjusts the flow of cells by changing the gap between the lower end of the tube or rectifying plate and the bottom of the tank, and detects the difference in cell concentration between the bottom of the tank and the rest of the tank, and sets this difference in advance. The draft tube or current plate is moved to adjust the gap between the lower end of the draft tube or current plate and the bottom of the tank so that the concentration difference is within the range of the oxygen in the culture solution in the tank,
It detects carbon dioxide gas and pH as well, and adjusts the gap between the draft tube or the lower end of the rectifying plate and the bottom of the tank so that they are within a preset range, so the flow of the culture solution in the tank is controlled. It has the great effect of always being smooth and not requiring a large amount of ventilation gas.

In the culture of biological cells, during the culture period, not only the concentration of cells and the viscosity of the liquid, but also the separation of cells during the process of each cell in the culture medium dividing from a mother cell to a daughter cell and multiplying. Cell characteristics such as ease of handling and adhesion when cells come into contact with each other change from time to time, and the specific gravity of cells and cell aggregates, that is, sedimentation characteristics, change accordingly.
According to the culture method of the present invention, not only the cell concentration difference but also the concentration of oxygen and carbon dioxide in the culture solution, as well as the pH, are detected, and these concentrations are adjusted, and the lower end of the draft tube or rectifier plate and the bottom of the tank are detected. Since it adjusts the gap between the two, it has the excellent effect of satisfying the conditions necessary for culturing efficiently and with high precision.

Furthermore, in the culture method of the present invention, cell deposition 9 is not prevented only by the amount of aeration, so that excessive aeration does not actually inhibit cell proliferation.
It has an excellent effect in that it can give good results especially in culturing fragile animal cells.

The present invention also provides a draft tube that is movable in the vertical direction within a range where the upper end of the draft tube or current plate does not protrude above the liquid surface in a culture tank that supplies oxygen to biological cells in a suspended state by aerating the liquid. Or a biological cell culture tank with a built-in current plate, and an organism with a built-in draft tube or current plate in which the draft tube or the lower part of the current plate, the draft tube or the upper part of the current plate, or both can move up and down. Since this is a cell culture tank, if the culture conditions (for example, cell concentration, cell specific gravity, liquid properties, etc.) change during the culture period of biological cells, the amount of ventilation can be changed in the conventional tank. The only means of control was to increase or decrease, which lowered economic efficiency and made it difficult to widen the range of operation.However, by using the culture tank of the present invention, the trough 0 tube or the lower end of the rectifier plate and the bottom of the tank By changing the gap, it is possible to quickly respond to changes in culture conditions as described above, and even at low aeration rates, cells can flow smoothly without sedimentation, and the operating range is wide. It is possible to obtain an outstanding effect of increasing the economic effect.

Furthermore, as described in the examples of the present invention, by using a plurality of stacked culture tanks of the present invention, parallel operation of different batch cultures can be performed under conditions where heat, ventilation gas, and culture solution are reduced. This method has a great economical effect and is also extremely effective in scaling up culture.

In addition, in the conventional culture method, culture device, and culture system in which a draft tube is fixed, it is virtually impossible to change the amount of solution in the tank during the culture period, but the culture method, culture device, or culture system of the present invention According to the system, culture can be performed while changing the amount of solution in the tank during the culture period. For example, it is also possible to carry out everything from seed culture to main culture continuously in the same tank. In other words, it is possible to always perform culture with an appropriate volume of liquid depending on the culture scale and changes in the characteristics of cells in the culture medium, to maintain a constant culture density, to meet needs, and to maintain high Economic effects can be obtained.

[Brief explanation of drawings]

FIG. 1 and FIGS. 5 to 10 are explanatory diagrams showing the culture system and culture apparatus of the present invention, FIG. 2 is an enlarged view of the vertical driving portion of the draft tube inner cylinder of FIG. 1, and FIG. FIG. 4 is an explanatory diagram of the cultivation method of the invention, FIG. 4 is an explanatory diagram of the progress of culture according to the invention, and FIG. 11 is an explanatory diagram of the operation of the culture tank shown in FIG. 10. FIG. 12 is an explanatory diagram showing the cell distribution within the tank during the period of culturing biological cells. 0: Culture tank, 1 Nidraft tube, 2: Antifoam net +-
, a draft tube outer cylinder, 4; draft tube inner cylinder, 5 draft tube outer cylinder supporting pair, 6: draft tube inner cylinder vertical drive section, 7: sparger, 8: air filter, 9: exhaust 2 piping , 10: cell concentration sensor, 11: pH sensor 12: DO total sensor 13: DO controller, 14
: pH controller, 15: Gap controller 1
6: Cell concentration detection device, 17: Gas composition, flow rate controller, 18: Gas collection pipe, 19: Oxygen gas storage tank, 2
0: Nitrogen gas storage tank, 21: Carbon dioxide gas storage tank, 22: Air compressor, 23.24.25.26: Flow rate adjustment valve, 27: Liquid medium, 28: Culture solution, 29: Liquid medium storage tank, 30: Culture solution storage tank , 31: Liquid flow rate sensor, 32:
Liquid flow rate detector, 33: Constant temperature water jacket), 34 Nidraft tube inner cylinder vertical drive section casing, 35: Air drive bellows, 36: Supply and exhaust piping for air drive bellows, 3
7: Helows telescopic transmission limb, 38: Hanging arm. 39: rectifier plate, 40: rectifier plate vertical drive device, 41; upper rectifier plate, 42: middle part of rectifier plate, 43: lower part of rectifier plate, 44:
Tank bottom hill, 45: Sparger for tank bottom stirring 246: Sparger air transfer piping, 47: Exhaust pipe, 48: Constant temperature water extraction piping, 49: Culture solution extraction piping, 50: Culture medium introduction piping,
51: Constant temperature water introduction piping, 52.53: Air transfer piping,
54: Gas transfer piping, 55 Two-liquid inflow piping 3 A: Unit culture tank. : Stacked culture tank C: Liquid transfer piping

Claims (1)

[Scope of Claims] 1. In a culture method using a culture tank with a built-in draft tube or baffle plate for supplying gas to biological cells in a suspended state by aerating the liquid, the lower end of the draft tube or baffle plate and A method for culturing biological cells characterized by adjusting the flow of cells by changing the gap between the tank and the bottom. 2. Detect the difference in cell concentration between the bottom of the tank and the inside of the other tanks, and move the draft tube or rectifier plate so that the difference is within a preset concentration difference range. 2. The method for culturing biological cells according to claim 1, wherein the gap between the lower end of the plate and the bottom of the tank is adjusted. 3. Detect the concentration of one or both of oxygen and carbon dioxide in the culture solution in the tank, and adjust the gas concentration in the ventilation gas so that the concentration is within a preset dissolved gas concentration range. The method for culturing biological cells according to claim 2, characterized in that: 4. The pH of the culture solution in the tank is also detected, and the carbon dioxide concentration in the ventilation gas is adjusted so that the pH is within a preset pH range. 3. The method for culturing biological cells according to 3. 5. Give top priority to the cell concentration difference, followed by dissolved oxygen concentration and pH.
The gap between the lower end of the draft tube or the rectifier plate and the bottom of the tank is adjusted to give priority to either of the above.
The method for culturing biological cells according to any one of claims 2 to 4. 6. When adjusting the dissolved oxygen concentration, if the adjustment cannot be achieved by increasing the oxygen concentration in the mixed gas of the raw material gas, the dissolved oxygen concentration is adjusted by increasing the ventilation amount. The method for culturing biological cells according to claim 5. 7. Claims 1 to 6, characterized in that the gap between the upper end of the draft tube or the rectifying plate and the liquid level is also changed.
The method for culturing biological cells according to any one of the above. 8. The biological cell according to any one of claims 1, 2, or 6, wherein the adjustment of the draft tube or the current plate is performed within a range where the upper end of the draft tube or the current plate does not protrude above the liquid surface. Cultivation method. 9. The upper limit of the linear velocity of the test cells in the liquid flow in the gap between the draft tube or the lower end of the rectifying plate and the tank bottom caused by ventilation is 3 m/s. A method for culturing biological cells, characterized by limiting the amount of aeration so that. 10. The liquid flow velocity in the gap between the lower end of the draft tube or rectifying plate and the bottom of the tank is 3 m/s. If the difference exceeds the set range, first increase the gap, then detect the difference in cell concentration, and if the difference exceeds the set range, increase the ventilation amount so that the difference in cell concentration falls within the set range. , dissolved gas concentration,
10. The method for culturing biological cells according to claim 9, wherein the liquid linear velocity is adjusted to satisfy the liquid linear velocity. 11. The gap with the bottom of the tank or the gap with the liquid level is changed by moving, expanding and contracting the draft tube or the current plate, according to any one of claims 1 to 9 or 10. Method for culturing biological cells. 12. In a culture tank that supplies oxygen to biological cells in suspension by aerating the liquid, it has a built-in draft tube or rectifier plate that can be moved vertically within the range where the upper end of the tube or plate does not protrude above the liquid surface. A culture tank for biological cells characterized by: 13. A draft in which the draft tube or the lower part of the current plate, or the draft tube or the upper part of the current plate, or both, are movable up and down in a culture tank that supplies oxygen to biological cells in a suspended state by ventilating the liquid. A biological cell culture tank characterized by having a built-in tube or rectifying plate. 14. The biological cell culture tank according to claim 12 or 13, wherein the draft tube is composed of an inner tube and an outer tube. 15. Part of the draft tube or current plate is fixed to the tank, and the other part is movable.
The biological cell culture tank according to any one of claims 12 to 14. 16. The mechanism for moving the draft tube or rectifying plate is one of a bellows or piston fluid input/output mechanism, a drive mechanism using a geared motor, a thermal expansion material expansion/contraction mechanism, a magnet drive mechanism, or a combination of these. The biological cell culture tank according to any one of claims 12 to 15, characterized in that: 17. A system for culturing biological cells, characterized in that a plurality of culture vessels each having the culture vessel according to any one of claims 12 to 16 are stacked in series. 18. A system for culturing biological cells, characterized in that a plurality of culture vessels according to any one of claims 12 to 16 are stacked in a state in which the culture vessels are mixed in series and in parallel in terms of liquid flow. 19. In a biological cell culture system that supplies oxygen to biological cells in a suspended state by aerating the liquid, the movement of a draft tube or a rectifier plate is controlled in the culture tank according to any one of claims 12 to 18. 1. A system for culturing biological cells, comprising a means for controlling cell culture conditions and a means for controlling other cell culture conditions. 20. In a culture system that supplies oxygen to biological cells in a suspended state by aerating the liquid, the culture tank according to any one of claims 12 to 16 is provided with either a DO control system or a pH control system, or both. A biological cell culture system comprising: 21. The culture system for biological cells according to claim 20, further comprising a culture medium supply system. 22. Claim 1, characterized in that the movement of draft tubes or rectifying plates in a plurality of stacked culture tanks and other cell culture conditions can be individually controlled.
22. The biological cell culture system according to any one of 7 to 21.