JPH0773492B2 - Aeration culture method of biological cells, aeration gas ejection device and aeration culture device - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for aeration culture of biological cells, and an aeration gas ejection device and a culture device used for carrying out the method.

(Prior Art) A large number of useful substances such as pharmaceuticals have been industrially produced by culturing biological cells, and in particular, animals aiming at production of physiologically active substances represented by interferon and the like in recent years. Cell culture is being actively carried out.

Currently, in this type of culture, cells, cell clumps, tissues, etc. themselves, or a suspension culture method in which particles having these in the form of being supported on a carrier are suspended in a culture solution, and cells are used. Is known as a culture system in which the culture medium is carried on a fixed bed and the culture solution is moved. The former is the mainstream because it is relatively easy to scale up.

In order to perform culture with high production efficiency, the concentration of cells that changes over time during culture should be appropriately adjusted, and the dissolved oxygen concentration or dissolved concentration in the culture medium should be adjusted to suit the changes in physiological activity. It is necessary to adjust the carbon dioxide concentration.

In addition, in culturing biological cells, most of the biological culture is mechanically fragile, so at the same time as supplying the aeration gas such as oxygen-containing gas or carbon dioxide gas, the culture solution is mixed at a flow rate that does not destroy the cells. Flowing must also be satisfied.

As typical methods or devices for culturing biological cells, those shown in the following documents are known.

JP-A-63-84494, JP-A-64-86867, Acta Biotechnologica, 2 (1) p3 (1982), pages 31-41 (Acta Biotechnologica (1) p31-41 (1982)), Chemistry Magazine, Volume 21, No. 7, 21 (Showa 58), Torrens in Biotechnology Volume 3, No. 7, 162-166 (Trends
in Biotechnology 3 (7) p162-166 (1985)), Biotechnology & Bioengineering, XI
X, pp. 1503-1522 (Biotechnology and Bioengineerin
g, XIX, p1503-1522 (1977)), JP-A-62-229192.

In what is shown in the literature as described above, a so-called loose sparger as an aeration gas jetting means for supplying an aeration gas such as oxygen-containing gas and carbon dioxide gas into the culture solution in the culture tank, The nozzle hole diameter (pore diameter for air diffusion) is fixed and does not change. Therefore, the dissolved gas concentration of the aeration gas in the culture solution is mainly adjusted by adjusting the aeration amount of the aeration gas, or the nozzle hole diameter is This is done by properly using a plurality of different spargers.

(Problems to be solved by the invention) In the culture using a sparger in which the nozzle hole diameter is fixed and does not change, the range in which both the aeration gas solubility and the culture solution flow rate can be satisfied is relatively narrow. Limited, this may make it impossible to cover the required area.

Explaining the reason further in detail, the dissolution rate of the ventilation gas increases with the increase of the ventilation amount, and even if the ventilation amount is the same, the bubble diameter of the ventilation gas is reduced by decreasing the nozzle hole diameter. It is already known that the dissolution rate of aeration gas is increased by increasing the gas-liquid contact area. The amount of aeration gas affects the culture of cells, and if it is too small, the flow of the culture solution is so small that the cells are deposited at the bottom of the culture tank, which absorbs dissolved enzymes and nutrients and diffuses waste components. Is reduced and cells are inactivated. On the other hand, if the amount of aeration is too large, fragile cells will be damaged by shearing due to the flow of the culture solution. That is, the ventilation amount of the ventilation gas naturally has an appropriate value determined by the characteristics peculiar to various cells and the structure of the device.

On the other hand, during culture, cells change their oxygen demand in response to changes in cell concentration due to proliferation and changes in physiological activity of cells. It is desirable to have a wide adjustable range of the enzyme supply rate by controlling the.

FIG. 12 shows the adjustable range of the aeration rate and the oxygen supply rate when a single sparger with a fixed nozzle diameter is used, and FIG. 13 shows different nozzle hole diameters. The adjustable range of the sparger with a fixed nozzle hole diameter is shown in the relationship between the aeration rate and the oxygen supply rate when two spargers are used. The aeration rate corresponds to the air flow rate, and the oxygen supply rate corresponds to the aeration gas solubility.

As is clear from the graph shown in FIG. 12 or FIG. 13, in the conventional case, the gas lysis rate is naturally limited to a narrow range in the cell culture in accordance with the adjustable aeration rate range. To be done. When two spargers with different nozzle diameters are used, the adjustable range of the gas dissolution rate is expanded compared to the case of one sparger, and the more spargers with different nozzle diameters are installed, the more However, since a draft tube or the like is usually arranged at the bottom of the culture tank, the bottom of the tank is narrow, and a plurality of spargers are provided at the bottom of the tank, especially 3 or more units are arranged side by side. It may be difficult to install in space, and this not only reduces the effective volume of the culture tank, but also acts in the direction of impeding the flow of the culture solution, which may cause deterioration of the culture characteristics. Also becomes. In particular, the draft tube is preferably provided with its lower end as close as possible to the bottom of the tank in order to increase the flow of the culture solution in the culture tank. Incompatible, if the lower end of the draft tube is provided close to the inner bottom of the tank in order to improve the fluidity of the culture solution by the draft tube, it becomes extremely difficult to install a plurality of spargers on the inner bottom of the tank.

The present inventors have improved the drawbacks of the conventional culture method and culture device as described above, and have sufficient oxygen and the like in response to changes in cell concentration and changes in physiological activity of cells that change over time during culture. The present inventors have intensively studied to propose a method and an apparatus for supplying the aeration gas of (3) to the culture solution and at the same time mixing and flowing the culture solution with an aeration amount that does not damage the cells.

As a result, a new sparger that can change the nozzle hole diameter by a single group was created, and by using this, the bubble diameter of the ventilation gas is changed by changing the bubble diameter of the ventilation gas by adjusting the nozzle hole diameter in preference to the change of the ventilation amount. It has been found that the problems as described above can be solved by adjusting the solubility.

The present invention, as a result of earnest studies as described above, is used for carrying out the aeration culture method of biological cells capable of adjusting the solubility of aeration gas over a wide range on the basis of the event found, and the implementation of this aeration culture method. It is an object of the present invention to provide an aeration gas ejecting device, and a culture device using the aeration gas ejecting device.

(Means for Solving the Problems) One of the above-mentioned objects is, according to the present invention, in the aeration culture method of aerating a predetermined aeration gas in a culture medium to culture biological cells, It is characterized in that the bubble diameter of the aeration gas in the culture solution is changed by changing the nozzle hole diameter of the aeration gas jetting means for supplying the aeration gas into the solution to adjust the solubility of the aeration gas in the culture solution. This is achieved by the aeration culture method of biological cells.

In the aeration culture method for biological cells according to the present invention as described above, the adjustment of the nozzle hole diameter of the aeration gas ejection means may be performed prior to the adjustment of the aeration amount and gas composition of the aeration gas.

In the aeration culture method for biological cells according to the present invention as described above, the biological cells may be animal cells, and the aeration gas may be any of oxygen-containing gas and carbon dioxide gas, or a combination thereof.

Further, in the aeration culture method for biological cells according to the present invention as described above, the change in the nozzle hole diameter is caused by the elastic deformation of the nozzle constituent member made of a rubber-like elastic material, or by collecting a plurality of holes having different diameters. This may be performed by selectively blocking some of the nozzle holes in the nozzle or partially blocking the hole diameter of each nozzle hole.

According to the present invention, another one of the above-mentioned objects is to construct a nozzle for jetting aeration gas in a device for jetting aeration gas for supplying aeration gas into a culture solution for aeration culture of biological cells. A rubber-like elastic nozzle forming member and elastic deformation means for elastically deforming the nozzle forming member, or a plurality of nozzles having different nozzle hole diameters. A vent gas ejecting apparatus, or a nozzle for ejecting a vent gas, comprising: a nozzle constituent member having the nozzle member; and a shielding means for selectively shielding a nozzle hole having a predetermined nozzle diameter among the plurality of nozzle holes. It is achieved by a vent gas ejecting device characterized in that it has a nozzle constituting member that constitutes the above and a shielding means for partially shielding the nozzle hole.

According to the present invention, the other one of the above-mentioned objects is a culture tank, a variable nozzle hole type sparger provided in the culture tank, and a concentration of dissolved gas in a culture solution of the culture tank. And a controller for changing the nozzle diameter of the variable nozzle diameter type sparger based on the dissolved gas concentration detected by the dissolved gas concentration detecting means. It is achieved by the aeration culture device.

Biological cells applicable to the system or device according to the present invention are not particularly limited, and may be animal cells or plant cells, and these cells may be in a single state or an aggregated state. The aggregated state of cells refers to a clump or tissue in which cells are attached to each other, and also includes cells in which cells are attached to other materials such as microbeads, honeycombs, fibers, or cells in which cells are entrapped and immobilized. .

The medium used in the aeration culture method or device according to the present invention is not particularly limited as long as it is a liquid medium, and may be appropriately selected depending on the test cells or culture conditions. For example,
As the carbon source, sugars ranging from low to high molecular weights, organic acids, alcohols, etc. are used, and as the nitrogen source, amino acids, peptides, inorganic nitrogen sources, etc. are used. In addition, components such as serum, albumin, vitamins, inorganic salts, organic pH buffers, anti-substances, growth promoting factors, adhesion factors, and differentiation factors are used depending on the cell type and culture conditions.

The culturing method applied by the method or device according to the present invention can be a method of culturing cells or a granular material containing the culture in a liquid by suspending them in a liquid, or a method of fixing cells and flowing only the liquid. It may be. That is, the method and apparatus according to the present invention is used for general culture of a system in which a gas component necessary for adjusting liquid properties such as biological reaction of cells and pH is brought into contact with air bubbles or liquid by aeration in the liquid. It is possible.

The aeration gas to be aerated may be appropriately selected and used. Generally, air, oxygen, and carbon dioxide gas are used alone, or one of air, oxygen, carbon dioxide gas, and nitrogen is mixed and used. These gases may be used by fixing or changing the gas components during the culture period. As these aeration gases, those that have been sterilized by filtration with a microbial filter or the like are usually used.

The culture tank to be used is not particularly limited as long as it is a submerged aeration system. For example, it may have a draft tube or a current plate. The shape of the culture tank is not limited to a cylinder, and may be a square shape or a plate shape. Further, it may be a culture tank provided with a stirring means for stirring the culture solution.

When the nozzle diameter is changed by elastically deforming the nozzle constituent member made of rubber-like elastic material, the external force for elastically deforming this is the horizontal direction of the nozzle constituent member, for example, from the outside or the center, or vertically. It may be operated from the direction or from a direction having an angle with them. The rubber-like elastic material constituting the nozzle constituent member has heat resistance, for example, when steam sterilization is required, has water resistance when used for a long time, and is suitable for elastic fatigue, so as to be suitable for intended culture conditions. A material that can endure may be appropriately selected. As the rubber-like elastic material forming the nozzle constituent member, for example, synthetic butadiene rubber, silicon rubber or the like is suitable.

Further, the external force for elastically deforming the nozzle component made of a rubber-like elastic material may be fluid-pressured by supplying fluid pressure to a pressure chamber formed around the nozzle component, or permanently. It may be performed by an electromagnetic force using a magnet and an electromagnet.

(Operation) As described above, in the method for culturing biological tissue according to the present invention, the dissolved gas concentration is adjusted by changing the nozzle diameter of the aeration gas jetting means such as the sparger.

Further, in the vent gas ejecting apparatus according to the present invention, since the nozzle diameter of the nozzle changes, the bubble diameter of the vent gas ejected thereby changes over a wide range by one sparger.

(Example) Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

FIG. 1 shows one embodiment of the aeration culture apparatus for biological cells according to the present invention. In FIG. 1, 1 is a cylindrical culture tank, 2 is a sparger located at the bottom of the culture tank, 3
Is a draft tube, 4 is a constant temperature water jacket, 5 is a defoaming net provided on the upper part of the culture tank, 7 is a dissolved oxygen sensor, and 8 is a liquid flow rate sensor.

The liquid culture medium is supplied from the liquid culture medium storage tank 10 into the culture tank 1 through the liquid culture medium transfer pipe 9, and the culture solution in the culture tank 1 is sent out to the culture solution storage tank 12 through the culture solution transfer pipe 11.

The sparger 2 is, as shown in FIGS. 2 to 4,
It is a variable nozzle hole diameter type sparger that changes the nozzle hole diameter according to the fluid pressure. Ventilation control device with a predetermined mixing ratio with 14 air
The air flow rate is adjusted by 18 so that the air is supplied, and the air pressure is adjusted by the nozzle hole diameter adjusting fluid pressure supply pipe 19 as the nozzle hole diameter adjusting fluid pressure.
The air pressure from 14 is supplied while the flow rate is adjusted by the nozzle hole diameter adjusting device 20.

In FIG. 2, reference numeral 21 denotes a gas filter.

Next, one embodiment of the vent gas ejection device according to the present invention will be described with reference to FIGS.

In these figures, reference numeral 30 denotes a substantially cylindrical nozzle casing formed of a rigid body such as stainless steel, and a disk-shaped nozzle component member is provided in the nozzle casing 30.
32 are arranged concentrically. The nozzle constituting member 32 is entirely made of a rubber-like elastic material such as silicon rubber, and has a plurality of nozzles (air diffuser pores) 34 opened on the upper surface. All of the nozzles 34 communicate with a ventilation gas inlet 36 through a collecting chamber 35, and a ventilation gas such as an oxygen-containing gas is supplied from this.

An annular space 38 exists between the inner peripheral portion of the nozzle casing 30 and the outer peripheral portion of the nozzle component member 32, and the annular space
A ring-shaped pressurizing chamber 42 is provided at 38 by a bag body 40 made of a rubber-like elastic member. The pressurizing chamber 42 communicates with the nozzle hole diameter adjusting air pressure inlet / outlet 44 so that the air pressure can be selectively applied thereto.

The bag body 40 expands according to the air pressure applied to the pressurizing chamber 42,
By this expansion, the nozzle component member 32 made of rubber-like elastic material is uniformly pressed in the circumferential direction from the outer peripheral surface thereof in the radial direction. By the pressurization in the diameter reducing direction, the nozzle constituent member 32 is elastically deformed so as to contract in the diameter reducing direction, and accordingly, the nozzle hole diameter of the nozzle 34 is reduced.

In contrast to this, the bag body 40 contracts in response to a decrease in the air pressure of the pressurizing chamber 42, and in response thereto, the nozzle constituent member 32 is restored and expanded by its own elastic force, and in response to this expansion, the nozzle
The nozzle hole diameter of 34 increases.

Therefore, the nozzle hole diameter of the nozzle 33 is continuously changed by increasing / decreasing the air pressure supplied to the pressurizing chamber 42.

The adjustment of the nozzle hole diameter of the sparger nozzle 34 as described above depends on the dissolved oxygen concentration in the culture solution in the culture tank 1 detected by the dissolved oxygen concentration sensor 7 in the embodiment shown in FIG. The ventilation is controlled with priority so that the dissolved oxygen concentration of the lever is within the set range.

FIG. 5 shows an example of the control process for controlling the dissolved oxygen concentration as described above. In the flowchart shown in FIG. 5, the dissolved oxygen concentration sensor 7 detects the dissolved oxygen concentration, and it is determined whether or not the dissolved oxygen concentration is within the set range. When the dissolved oxygen concentration is outside the set range, the sparger nozzle hole diameter adjustment control is performed. This adjustment of the nozzle hole diameter is performed when the dissolved oxygen concentration is higher than the set range so that the air pressure supplied to the pressurizing chamber 42 is reduced to increase the nozzle hole size, whereas when the dissolved oxygen concentration is smaller than the set range, it is added. The air pressure supplied to the pressure chamber 42 is increased to reduce the nozzle hole diameter.

In this embodiment, when the dissolved oxygen concentration cannot be adjusted within the set range by adjusting the nozzle hole diameter of the sparger, the amount of aeration gas supplied from the sparger into the culture tank 1 is adjusted as a secondary control means. Is done.

FIG. 6 shows the relationship between the aeration rate and the oxygen supply rate for each nozzle hole diameter of the sparger. From this graph,
It will be understood that even if the aeration rate is constant, the oxygen concentration supply rate changes with a change width according to the change width of the nozzle hole diameter due to the change of the nozzle hole diameter, and it is understood that the adjustable range of this is wide.

In the above-described embodiment, the pressurizing chamber 42 is constituted by the bag body 40 made of a rubber-like elastic member which is separate from the nozzle constituting member 32. This is as shown in FIG. It may be formed integrally with the outer peripheral portion of the nozzle constituting member 32.

The elastic deformation of the nozzle-constituting member 32 made of a rubber-like elastic member may be performed by electromagnetic force instead of fluid pressure, in which case an embodiment is shown in FIG. still,
In FIG. 8, the portion corresponding to FIGS. 2 to 4 is the second portion.
It is designated by the same reference numeral as the reference numerals given in FIGS. In this embodiment, a plurality of permanent magnets 46 are embedded near the outer peripheral edge of the nozzle constituent member 32, and electromagnets 48 are fixedly arranged at positions facing the permanent magnets 46 on the nozzle casing 30 side. Has been done.

In this embodiment, the electromagnetic force of the electromagnet 48 causes permanent magnets.
The nozzle component member 32 is elastically deformed in the radial expansion direction or the radial contraction direction by the magnetic attraction or repulsion of 46, and the nozzle hole diameter of the nozzle 34 is changed in accordance with this elastic deformation.

FIG. 9 shows another embodiment of the ventilation gas ejection device according to the present invention. Incidentally, in FIG. 9 as well, FIG.
Portions corresponding to the drawings are denoted by the same reference numerals as those given in FIGS. 2 to 4. In such an embodiment,
The nozzle casing 30 is an inverted conical nozzle component member receiving hole.
The nozzle component member 32 has an inverted conical shape that is aligned with the nozzle receiving hole 31 by a rubber-like elastic material.
Has been inserted inside. A bellows type tension device 50 for pulling the nozzle component member 32 in the axial direction is provided at the lower end portion of the nozzle component member 32, and a nozzle diameter adjusting air pressure is supplied to a fluid pressure inlet / outlet 52 of the tension device 50. Has become.

In this embodiment, the nozzle component 32 is guided to the nozzle receiving hole 31 of the nozzle casing 30 by pulling the nozzle component 32 downward in the drawing by the fluid pressure applied to the tension device 50. The diameter of the nozzle 34 is reduced and the diameter of the nozzle hole of the nozzle 34 is reduced. On the contrary, the tension device
As the tension device 50 restores and expands due to the reduction of the air pressure applied to the nozzle 50, the nozzle component member 32 reduces or releases the tension and restores and expands its diameter by its own elastic force.
Increase the nozzle hole diameter of 34.

FIG. 10 shows another embodiment of the ventilation gas ejection device according to the present invention. In the embodiment shown in FIG. 10,
A nozzle-constituting drum 62 is provided in a nozzle casing 60 having a shape in which a part of a cylindrical body is removed so as to be rotatable with respect to the nozzle casing 60 about its own central axis.
The nozzle forming drum 62 has a plurality of nozzles 64 over the entire outer peripheral surface of the drum, and the nozzles 64 arranged along one generatrix have substantially the same nozzle diameter. Nozzles 64 extending along each busbar
The respective nozzle diameters are different from each other, and the nozzles 64 have different nozzle diameters when viewed in the circumferential direction of the drum. Only the nozzles 64 that are exposed to the outside from the opening 61 of the nozzle casing 60 maintain the open state, and the nozzles 64 located inside the nozzle casing 60 are shielded by the peripheral wall surface of the nozzle casing 60 and a substantial gas is ejected. It has no effect. The nozzle-constituting drum 62 is rotatably supported by the nozzle casing 60 by a hollow shaft 66 that also serves as a ventilation gas intake, and a gear is provided on the outer peripheral portion of the hollow shaft 66.
68 is installed. The gear 68 is a drive gear of the electric motor 70.
It engages with 72 and is rotated by an electric motor 70.

The nozzle-forming drum 62 is rotationally driven by the electric motor 70, and the portion exposed to the outside from the opening 61 of the nozzle casing 60 changes in the circumferential direction, so that the nozzle 64 exposed to the outside changes, which causes gas ejection. The effective nozzle 64 is switched to the nozzle that actually ejects the ventilation gas.
64 nozzle hole diameter will change.

FIG. 11 shows another embodiment of the vent gas ejection device according to the present invention. In this embodiment, the nozzle constituent member 80 is provided with a plurality of nozzle holes 82 having a relatively large diameter, and the shutter holes 86 and the nozzle holes provided in the shutter plate 84 are provided.
The nozzle hole 82 is partially shielded by the degree of matching with 82,
As a result, the effective nozzle hole diameter is changed.

In this case, the nozzle holes 82 may have the same width with respect to the moving direction of the shutter plate 84, but the width thereof may change. Due to this shape, the effective nozzle diameter changes due to the movement of the shutter plate 84. The characteristics are set arbitrarily. The shape of the hole is not limited to a circle, and may be appropriately selected from an ellipse, a triangle, a quadrangle, a polygon, a band, and the like.

Embodiment 1 A sparger as shown in FIG. 2 to FIG.
Suspension culture of animal cells was carried out by incorporating it into a culture device as shown in the figure.

The culture tank has an inner diameter of 120 mm and a diameter of 75 mm inside.
It has a draft tube with a length of 480 mm and the amount of filling liquid is 5
Using a liter cylindrical type, the liquid level in the tank is 600 mm
The temperature inside the tank was adjusted to 37 ° C with a constant temperature water jacket.

The test cells were rat liver cancer cell line JTC-1 (Japan Tissue Cu
lture No. 1, preserved by Japan Tissue Culture Society) was used. Seed culture using Eagle MEM medium, after culturing in a flat flask,
Cultured in roller bottles. The culture temperature was 37 ° C. This culture solution was centrifuged to collect the cells, which were used as cells for inoculation.

After introducing 5 liters of Eagle MEM medium into the culture tank, the inoculated cells were inoculated so that the cell concentration became 1 × 10 ⁶ cells / ml, and cultured at 36 to 38 ° C. and pH 6.8 to 7.2 for 10 days. A gas obtained by nitriding oxygen and carbon dioxide was used. The composition of this gas is 30% oxygen, 5% carbon dioxide and 65% nitrogen.
The set value (set range) of the dissolved oxygen concentration was 2.0 ± 0.1 ppm.

Table 1 shows the results on the 2nd, 6th and 10th days after the culture.
The results are shown in Tables 2 and 3.

By controlling the nozzle hole diameter of the nozzle holes in accordance with the dissolved oxygen concentration by giving priority to the aeration amount according to the flow chart shown in FIG. 5, the dissolved oxygen concentration is always kept within the set value, and the liquid flow velocity is also the cell flow rate. The cells were kept within a suitable range where no sedimentation or damage occurred, which resulted in a high cell concentration and a high survival rate.

On the other hand, Tables 1 to 3 show the results of Comparative Examples 1 and 2 in which the nozzle diameter of the sparger was fixed by using the same batch of seed cells by the same specification device and the dissolved oxygen concentration was controlled only by changing the air flow rate. I added it.

In Example 1 and Comparative Example 1, the cell concentration reached 1 × 10 ⁶ cells / ml on the second and sixth days. Comparative Example 1
, The oxygen dissolution cannot catch up with the consumption of dissolved oxygen at the cell concentration of 1 × 10 ⁶ cells / ml or more, and if the nozzle diameter is large, the air flow rate increases on the 10th day when the oxygen concentration becomes high. Even so, the oxygen supply was insufficient, and the liquid flow rate was in the range where cell damage occurred, so that effective culture could not be performed as compared with Example 1.

On the other hand, in Comparative Example 2 in which the nozzle hole diameter was fixed to be small,
Even when the aeration rate is at the lower limit that can be controlled, the initial stage of culture (2nd day)
At the time when the cell concentration was low, the dissolved oxygen deviated from the preferable value which was the set value and the low aeration amount caused the cells to deposit on the bottom of the tank, resulting in the death of the cells.

As is clear from this result, if the nozzle hole diameter adjustment is performed prior to the air flow rate adjustment according to the present invention, it is understood that the culture is superior to the conventional fixed nozzle hole diameter type.

[Effect of the invention] As can be understood from the above description, in the method for culturing biological tissue according to the present invention, since the dissolved gas concentration is adjusted by the change in the nozzle hole diameter of the aeration gas ejection means such as the sparger, Compared to the case where this is done by changing the aeration rate, it does not cause cell destruction, and it can support a wide range of gas dissolution rates necessary for culturing even in a narrow aeration rate range suitable for mixed flow of liquids. Good aeration culture can be performed over the entire period.

Further, in the vent gas ejecting apparatus according to the present invention, the nozzle hole diameter of the nozzle is changed, the bubble diameter of the vent gas ejected from the nozzle is changed over a wide range by one sparger, There is no problem of installation in the culture tank.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram showing one embodiment of an aeration culture device for biological cells according to the present invention, FIG. 2 is a perspective view showing one embodiment of an aeration gas ejection device according to the present invention, and FIG. Two
FIG. 4 is a vertical cross-sectional view of the ventilation gas injection device shown in FIG. 4, FIG. 4 is a plan view of FIG. 3, and FIG. 5 is a flow chart showing a dissolved oxygen concentration control procedure performed by using the ventilation gas injection device according to the present invention. FIG. 6 is a graph showing a region where the oxygen supply rate can be adjusted in the relationship between the air supply rate and the oxygen supply rate according to the change in the nozzle diameter of the sparger, and FIG. 8 is a vertical cross-sectional view showing an embodiment, FIG. 8 to FIG. 11 are perspective views showing another embodiment of the ventilation gas ejection device according to the present invention, and FIG. 12 is a case where a sparger whose nozzle hole diameter does not change is used. A graph showing a region where the oxygen supply rate can be adjusted in the relationship between the aeration rate and the oxygen supply rate, and FIG. 13 shows the aeration rate and the oxygen supply rate when two spargers having different nozzle hole diameters are provided. In relation to It is a graph showing a region with adjustable speed. 1 ... Culture tank, 2 ... Sparger 19 ... Air compressor 20 ... Nozzle diameter adjusting device 30 ... Nozzle casing 32 ... Nozzle component 34 ... Nozzle, 40 ... Bag 42 ... Pressurization chamber, 46 …… Permanent magnet 48 …… Electromagnet, 60 …… Nozzle casing 62 …… Nozzle component drum 64 …… Nozzle, 80 …… Nozzle component member 82 …… Nozzle, 84 …… Shutter plate 86 …… Shutter hole

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Harumi Matsuzaki, Inventor Harumi 4026, Kuji Town, Hitachi City, Ibaraki Prefecture, Hitachi Research Institute, Ltd. (72) Fumio Maruhashi, 4026, Kuji Town, Hitachi City, Ibaraki Prefecture Hitachi Research Laboratory (72) Inventor Takamori Nakano 794, Higashitoyoi, Shimomatsu City, Yamaguchi Prefecture Inside the Kasado Plant, Hiritsu Seisakusho Co., Ltd. (56) Reference JP-A 64-86867 (JP, A)

Claims (12)

[Claims] 1. An aeration culture method for culturing biological cells by aerating a predetermined aeration gas into a culture solution in a culture tank, comprising one aeration gas ejecting means having a plurality of nozzle holes in the culture tank. When only the base is placed and the solubility of the aeration gas in the culture solution is adjusted, the bubble diameter of the aeration gas in the culture solution is continuously changed by continuously changing the hole diameter of the nozzle hole of the aeration gas ejection means. A method for aeration culture of biological cells, which comprises varying the solubility of aeration gas in a culture solution. 2. The aeration culture method for biological cells according to claim 1, wherein the adjustment of the nozzle hole diameter of the aeration gas ejection means is performed prior to the adjustment of the aeration amount and gas composition of the aeration gas. Aeration culture method. 3. The aeration culture method for biological cells according to claim 1 or 2, wherein the biological cells are animal cells and the aeration gas is any of oxygen-containing gas and carbon dioxide gas or a combination thereof. Aeration culture method. 4. The aeration culture method for biological cells according to any one of claims 1 to 3, wherein the nozzle hole diameter is continuously changed by elastic deformation of the nozzle component member made of a rubber-like elastic material, The aeration culture method is characterized in that the bubble diameter of the aeration gas ejected by this is adjusted. 5. The method for aeration culture of biological cells according to any one of claims 1 to 3, wherein some nozzle holes are selectively shielded in a nozzle in which a plurality of holes having different diameters are assembled. Thus, the aeration culture method is characterized in that the effective nozzle diameter for ejecting the aeration gas is continuously changed, and thereby the bubble diameter of the aeration gas ejected is adjusted. 6. The method for aeration culture of biological cells according to any one of claims 1 to 3, wherein the effective nozzle diameter is continuous by partially blocking each hole of the nozzle for ejecting aeration gas. The aeration culture method is characterized in that the bubble diameter of the aeration gas ejected is adjusted by changing the bubble diameter. 7. An aeration gas ejecting device arranged in a culture tank for supplying aeration gas into a culture solution for aeration culture of biological cells, the rubber-like elastic material constituting a nozzle for aeration gas ejection. A vent gas ejecting apparatus comprising: a nozzle constituent member made of elastic material, and elastic deformation means for elastically deforming the nozzle constituent member so as to continuously change the hole diameter of the nozzle for ejecting the vent gas. 8. The vent gas ejection apparatus according to claim 7, wherein the elastic deformation means is means for elastically deforming the nozzle constituent member by supplying fluid pressure. 9. The vent gas ejecting apparatus according to claim 7, wherein the elastic deformation means is means for elastically deforming the nozzle component member by magnetic attraction or repulsion of a permanent magnet by electromagnetic force. A ventilation gas injection device. 10. An aeration gas jetting device arranged in a culture tank for supplying aeration gas into a culture solution for aeration culture of biological cells, comprising a plurality of hole assembly portions having different nozzle hole diameters. A drum-shaped nozzle constituent member and a shielding member which is fitted onto the nozzle-shaped nozzle constituent member and can selectively shield a hole assembly portion having a predetermined nozzle diameter of the plurality of hole assembly portions. And a drive means for relatively rotating the nozzle constituent member and the casing, and the effective gas diameter is continuously changed by the relative rotation between the nozzle constituent member and the casing, and the ventilation gas is ejected. A vent gas ejector characterized by adjusting the bubble diameter of the. 11. An aeration gas ejecting device arranged in a culture tank for supplying aeration gas into a culture solution for aeration culture of biological cells, comprising a plurality of nozzle holes for aeration gas ejection. A nozzle component member and a shield member having a shutter hole aligned with the nozzle hole and capable of sliding the nozzle component member, and the nozzle component member and the shield member are moved relative to each other to form the nozzle hole. Vent gas characterized in that the nozzle hole can be partially shielded by changing the degree of alignment between the shutter gas and the shutter hole, thereby continuously changing the effective nozzle diameter and adjusting the bubble diameter of the vented gas. Ejection device. 12. A culture tank, a variable nozzle diameter type ventilation gas jetting device according to claim 7, which is provided in the culture tank, and a concentration of dissolved gas in a culture solution of the culture tank. And a controller for changing the nozzle hole diameter of the variable nozzle hole diameter type vent gas ejector based on the dissolved gas concentration detected by the dissolved gas concentration detector. Aeration culture device characterized by.