JPH03195488A - Process for aerobic culture of living cell, apparatus for blasting aeration gas and apparatus for aerobic culture - Google Patents

Abstract

PURPOSE:To perform the subject culture over a wide range of culture liquid in desirable state without causing the destruction of cell by changing the nozzle diameter of an aeration gas blasting means, thereby adjusting the solubility of the aeration gas in the culture liquid. CONSTITUTION:In the aerobic culture of living cells by passing a prescribed aeration gas through a culture liquid, the bubble diameter of the aeration gas in the culture liquid is varies and the solubility of the aeration gas in the culture liquid is adjusted by varying the nozzle diameter of an aeration gas blasting means for supplying the aeration gas to the culture liquid. The adjustment of the nozzle diameter is preferably carried out in preference to the control of the aeration rate and the composition of the aeration gas.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for aeration culturing biological cells, and an aeration gas blowing device and a culturing device used to carry out the method.

(Prior art) A large number of useful substances such as pharmaceuticals have been industrially produced by culturing biological cells, and in recent years, in particular, the cultivation of biological cells has been carried out in animals for the purpose of producing physiologically active substances such as interferon. Cell culture is being actively carried out.

Currently, in this type of culture, there are two methods: a suspension culture method in which cells, cell aggregates, tissues, etc. themselves, or particles containing these cells supported on a carrier are suspended in a culture solution; There is a known culture method in which the cells are supported on a fixed bed and the culture solution is moved, and the former method has become mainstream because it is relatively easy to scale up.

In order to perform culture with high production efficiency, it is necessary to appropriately adjust the cell concentration that changes over time during culture, and to adjust the dissolved oxygen concentration in the culture medium or It is necessary to adjust the carbon dioxide concentration.

In addition, when culturing biological cells, most biological cultures are mechanically fragile, so it is necessary to supply aeration gas such as oxygen-containing gas or carbon dioxide gas and mix the culture solution at a flow rate that does not destroy the cells. Flowability must also be satisfied.

Typical methods and devices for culturing biological cells are known from the following literature.

JP-A-63-84494, JP-A-64-8686
Publication No. 7, Acta Biotechnologica.

2 (1) p3 (1982) pp. 31-41 (A
ctaBiotechnologica (1) p3
1-41 (1982)), Chemical Journal, Volume 21, No.
, p. 7.21 (1981), Trends in Biotechnology Vol. 3, No. 7゜pp. 162-166 (T
rends in Biotechnology3 (
7) p162-166 (1985) ), Biotechnology and Bioengineering, XI
X, pp. 1503-1522 (Biotechnolo
gy and bi.

engineering, XIX, p1503-1
522 (1977) ), JP-A-62-22919
Publication No. 2.

In the above-mentioned literature, the so-called sparger is a nozzle used as an aeration gas jetting means for supplying aeration gas such as oxygen-containing gas or carbon dioxide into the culture solution in a culture tank. The pore diameter (pore diameter for aeration) is fixed and does not change, and the dissolved gas concentration of the aeration gas in the culture solution is controlled solely by adjusting the amount of aeration gas, or the nozzle pore diameter is This is done by using a plurality of different spargers.

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

To explain the reason in more detail, the dissolution rate of vent gas increases as the aeration rate increases, and even if the aeration rate is the same, the smaller the nozzle hole diameter, the smaller the bubble diameter of the vent gas. It is already known that the rate of dissolution of vent gas increases by increasing the gas-liquid contact area. The amount of aeration gas affects cell culture; if it is too low, the flow of the culture medium is small, causing cells to settle at the bottom of the culture tank, resulting in absorption of dissolved oxygen and nutrients and diffusion of waste components. is reduced and the cells are deactivated. On the other hand, if the amount of aeration is too large, fragile cells will be damaged by shear caused by the flow of the culture medium. That is, there is an appropriate value for the amount of ventilation gas that is determined from the characteristics specific to each type of cell 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 cell physiological activity. It is desired that the oxygen supply rate can be adjusted over a wide range by controlling the oxygen supply rate.

Figure 12 shows the adjustable range of the relationship between ventilation rate and oxygen supply rate when a single sparger with a fixed nozzle diameter is used, and Figure 13 shows the range in which the aeration rate and oxygen supply rate can be adjusted. The figure shows the adjustable range of the aeration rate and oxygen supply rate when two spargers with fixed nozzle hole diameters are used. Note that the ventilation rate corresponds to the ventilation amount, and the oxygen supply rate corresponds to the ventilation gas solubility.

As is clear from the graphs shown in Figures 12 and 13, in the conventional method, the gas dissolution rate is naturally limited to a narrow range corresponding to the range of adjustable aeration amount in terms of cell culture. be done. When two spargers with different nozzle diameters are used, the range in which the gas dissolution rate can be adjusted is expanded compared to the case of a single sparger, and the more spargers with different nozzle diameters are installed, the wider the range. However, the bottom of the culture tank is usually equipped with a draft tube, etc., so the bottom of the tank is narrow. This may not only reduce the effective volume of the culture tank, but also impede the flow of the culture solution, leading to deterioration of culture characteristics. It will also happen. In particular, in order to increase the flow of the culture solution in the culture tank, it is preferable that the lower end of the draft tube be installed as close to the bottom of the tank as possible. If this is not compatible and the lower end of the draft tube is provided close to the bottom of the tank in order to improve the fluidity of the culture solution, it will be extremely difficult to install a plurality of spargers at the bottom of the tank.

The present inventors have improved the shortcomings of the conventional culture methods and culture apparatuses as described above, and have created an environment in which oxygen is supplied in an appropriate amount in response to changes in cell concentration and physiological activity of cells that change over time during culture. We conducted extensive research to propose a method and device for supplying aeration gas into the culture medium and at the same time mixing and flowing the culture medium at an amount of aeration that would not damage the cells.

As a result, we devised a new sparger that can change the nozzle hole diameter with a single unit, and by using this to adjust the nozzle hole diameter with priority over changing the ventilation amount, we can change the bubble diameter of the ventilation gas and increase the ventilation gas. It has been found that the above-mentioned problems can be solved by adjusting the solubility.

As a result of the above-mentioned intensive studies, the present invention provides an aerated culture method for biological cells that allows the solubility of aeration gas to be adjusted over a wide range based on the phenomena discovered, and a method for use in carrying out this aerated culture method. It is an object of the present invention to provide an aeration gas ejecting device in which the aeration gas ejecting device is used, and a culture device using this aeration gas ejecting device.

(Means for Solving the Problems) According to the present invention, one of the objects as described above is to provide a method for culturing biological cells by aerating a predetermined aeration gas into a culture solution. The solubility of the aeration gas in the culture solution is adjusted by changing the nozzle hole diameter of the aeration gas jetting means for supplying the aeration gas into the solution, thereby changing the bubble diameter of the aeration gas in the culture solution. This is achieved by an aerated culture method for biological cells.

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

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

Furthermore, in the aerated culture method of biological cells according to the present invention as described above, the nozzle hole diameter is changed by elastic deformation of a nozzle component made of a rubber-like elastic material, or by aggregation of a plurality of holes with different diameters. This may be done by selectively blocking some of the nozzle holes in the nozzle, or by partially blocking the diameter of each nozzle hole.

Another object of the present invention is to configure a nozzle for ejecting aeration gas in an aeration gas ejection device for supplying aeration gas into a culture solution for aerated culture of biological cells. An aeration gas ejection device characterized by having a nozzle component made of a rubber-like elastic material and an elastic deformation means for elastically deforming the nozzle component, or a plurality of nozzles with different nozzle hole diameters. and a shielding means for selectively shielding a nozzle hole having a predetermined nozzle diameter among the plurality of nozzle holes, or a nozzle for jetting aeration gas. This is achieved by an aeration gas ejecting device characterized by having a nozzle component that constitutes the nozzle hole, and a shielding means that partially shields the nozzle hole.

Another object of the present invention is to provide a culture tank, a variable nozzle diameter sparger provided in the culture tank, and a concentration of dissolved gas in the culture solution of the culture tank. and a control device that changes the nozzle diameter of the variable nozzle diameter sparger based on the dissolved gas concentration detected by the dissolved gas concentration detection means. This is achieved by using an aerated culture device.

The biological cells that can be applied to the method or device according to the present invention are not particularly limited, and may be animal cells, plant cells, or microbial cells, and these cells may be in a single state or in an aggregated state. Good too. A state in which cells are aggregated refers to aggregates or tissues in which cells are attached to each other, and also includes cells attached to other materials such as microbeads, honeycombs, fibers, etc., or cells that are encircled and immobilized. .

The medium used in the aerated culture method or apparatus 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, culture conditions, etc. for example,
As a carbon source, IJ! As nitrogen sources, amino acids, peptides, inorganic nitrogen sources, etc. are used. others,
Depending on the cell type and culture conditions, serum, albumin, vitamins, inorganic salts, organic pH￥! ! Components such as buffering agents, antibiotics, growth-promoting factors, adhesion factors, and differentiation factors are used.

The culturing method applied to the method or device according to the present invention may be a method in which cells or particulate matter containing culture is allowed to flow in a suspended state in a liquid, or a method in which cells are fixed and only the liquid is allowed to flow. There may be. That is, the method and apparatus according to the present invention are suitable for general cultivation methods in which gas components necessary for biological reactions of cells and liquid regulation such as pH are dissolved by aerating the liquid and bringing bubbles into contact with the liquid. It is possible to use

The ventilation gas to be vented may be selected and used as appropriate.

-For boat fishing, use air, oxygen, and carbon dioxide alone or in a mixture of air, oxygen, carbon dioxide, and nitrogen. These gases may be used while the gas components are fixed or changed during the culture period. These ventilation gases are usually sterilized by microbial filter filtration or the like.

The culture tank used is not particularly limited as long as it is of a submerged aeration type. For example, it may have a draft tube or a current plate. The shape of the culture tank is not limited to a cylinder, but may be angular or plate-shaped, or may be a culture tank equipped with a stirring means for stirring the culture solution.

When changing the nozzle diameter by elastically deforming a nozzle component made of a rubber-like elastic material, the external force for elastically deforming the nozzle component may be applied in the horizontal direction of the nozzle component, for example from the outside or the center, or vertically. It may be applied from a direction or from a direction having an angle thereof. The rubber-like elastic material that makes up the nozzle component is suitable for the desired culture conditions, such as heat resistance when steam sterilization is required, water resistance when used for a long period of time, and resistance to elastic fatigue. A durable material may be selected as appropriate. As the rubber-like elastic material constituting this nozzle component, for example, synthetic butadiene rubber, silicone rubber, etc. are suitable.

Furthermore, applying an external force to the nozzle component made of a rubber-like elastic material for elastic deformation may be performed by a fluid pressure method by supplying fluid pressure to a pressure chamber formed around the nozzle component, or by permanently applying an external force to the nozzle component. It may also be performed by electromagnetic force using magnets and electromagnets.

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

Further, in the aeration gas ejection device according to the present invention, by changing the nozzle diameter of the nozzle, the bubble diameter of the aeration gas ejected from the nozzle can be changed over a wide range by a single sparger.

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

FIG. 1 shows one embodiment of the aerated culture apparatus for biological cells according to the present invention. In Figure 1, 1 is a cylindrical culture tank, 2 is a sparger placed at the bottom of the culture tank, and 3 is a sparger placed at the bottom of the culture tank.
4 indicates a draft tube, 4 indicates a constant temperature water jacket, 5 indicates an antifoaming net provided at the top of the culture tank, 7 indicates a dissolved oxygen sensor, and 8 indicates a liquid flow rate sensor.

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

The sparger 2, as shown in FIGS. 2 to 4,
This is a variable nozzle hole diameter type sparger that changes the nozzle hole diameter depending on the fluid pressure, and includes oxygen gas in an oxygen gas cylinder 15, carbon dioxide gas in a carbon dioxide gas cylinder 16, carbon dioxide gas in a carbon dioxide gas cylinder 17, and a near compressor. 14 is supplied at a predetermined mixing ratio while the ventilation amount is adjusted by the ventilation amount adjustment device 18, and the nozzle hole diameter adjustment fluid pressure supply piping 19 is used as the nozzle hole diameter adjustment fluid pressure. Accordingly, the air pressure from the near compressor 14 is supplied while its flow rate is adjusted by the nozzle hole diameter adjustment device 20.

In addition, in FIG. 2, 21 indicates a gas filter.

Next, one embodiment of the aeration gas jetting device according to the present invention will be described using FIGS. 2 to 4.

In these figures, 30 indicates a substantially cylindrical nozzle casing made of a rigid body such as stainless steel, and a disk-shaped nozzle component 32 is arranged concentrically within the nozzle casing 30. . Nozzle component 32
is entirely made of a rubber-like elastic material such as silicone rubber, and has a plurality of nozzles (aeration pores) 34 that are open on the top surface. All of the nozzles 34 are located in the gathering chamber 35.
It communicates with an aeration gas intake port 36 through which a aeration gas such as an oxygen-containing gas is supplied.

An annular space 38 exists between the inner periphery of the nozzle casing 30 and the outer periphery of the nozzle component 32, and a bag 4 made of a rubber-like elastic member is placed in the annular space 38.
0, an annular pressurizing chamber 42 is provided. The pressurizing chamber 42 communicates with an air pressure inlet/outlet 44 for adjusting the nozzle hole diameter,
This allows air pressure to be applied selectively.

The bag body 40 expands according to the air pressure applied to the pressurizing chamber 42, and due to this expansion, the nozzle component 3 made of a rubber-like elastic material
2 is pressurized uniformly in the circumferential direction from its outer circumferential surface in the direction of diameter reduction. Due to this pressure in the diameter reduction direction, the nozzle component 32 is elastically deformed to contract in the diameter reduction direction, and the nozzle hole diameter of the nozzle 34 decreases accordingly.

On the contrary, the bag body 40 contracts as the air pressure in the pressurizing chamber 42 decreases, and the nozzle component 3
2 restores and expands due to its own elastic force, and the nozzle hole diameter of the nozzle 34 increases in accordance with this expansion.

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

The nozzle hole diameter of the sparger nozzle 34 as described above is adjusted according to the dissolved oxygen concentration in the culture solution in the culture tank l detected by the dissolved oxygen concentration sensor 7 in the embodiment shown in FIG. Priority is given to ventilation amount control 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 is detected by the dissolved oxygen concentration sensor 7, and it is determined whether the dissolved oxygen concentration is within a set range. When the dissolved oxygen concentration is outside the set range, sparger nozzle hole diameter adjustment control is performed. This nozzle hole diameter adjustment is performed by reducing the air pressure supplied to the pressurizing chamber 42 and increasing the nozzle hole diameter when the dissolved oxygen concentration is higher than the set range, whereas when the dissolved oxygen concentration is lower than the set range, the nozzle hole diameter is increased. The air pressure supplied to the pressure chamber 42 is increased to reduce the nozzle hole diameter.

In this embodiment, if 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 into the culture tank 1 from the sparger is adjusted as a secondary control means. What is done is done.

FIG. 6 shows the relationship between the ventilation 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 ventilation rate is constant, the oxygen concentration supply rate changes depending on the change in the nozzle hole diameter, and the range in which this can be adjusted is wide.

In the embodiment described above, the pressurizing chamber 42 is connected to the nozzle component 3.
The bag body 40 is made of a rubber-like elastic material and is separate from the nozzle component 32, but as shown in FIG. Good too.

The elastic deformation of the nozzle component 320 made of a rubber-like elastic material is as follows:
It may also be configured to use electromagnetic force instead of fluid pressure, and an embodiment in this case is shown in FIG. In FIG. 8, parts corresponding to FIGS. 2 through 4 are designated by the same reference numerals as those shown in FIGS. 2 through 4.

In this embodiment, a plurality of permanent magnets 46 are embedded near the outer peripheral edge of the nozzle component 32, and electromagnets 48 are fixedly arranged on the nozzle casing 30 side at positions facing each of the permanent magnets 46. ing.

In this embodiment, the permanent magnet 46 is magnetically attracted or repelled by the electromagnetic force of the electromagnet 48, so that the nozzle component 32 is elastically deformed in the direction of diameter expansion or contraction. The nozzle hole diameter of the nozzle 34 comes to change.

FIG. 9 shows another embodiment of the aeration gas blowing device according to the present invention. Furthermore, in Figure 9, Figures 2 to 4 are also shown.
Portions corresponding to the figures are designated by the same reference numerals as those shown in FIGS. 2 to 4. In such embodiments,
The nozzle casing 30 has an inverted cone-shaped nozzle component receiving hole 31, and the nozzle component 32 is formed of a rubber-like elastic material into an inverted cone shape that aligns with the nozzle receiving hole 31.
It is inserted into the receiving hole 31. A bellows-type tensioning device 50 is provided at the lower end of the nozzle component 32 to tension it in the axial direction, and air pressure for nozzle diameter adjustment is supplied to a fluid pressure inlet/outlet 52 of this tensioning device 50. It has become.

In this embodiment, the nozzle component 32 is pulled downward from the nozzle casing 3 by the fluid pressure applied to the tension device 50.
The nozzle 34 is guided to the nozzle receiving hole 31 of No. 0 and undergoes diameter reduction deformation, thereby reducing the nozzle hole diameter of the nozzle 34. On the contrary, as the tensioning device 50 restores and expands due to the reduction in the air pressure applied to the tensioning device 50, the nozzle component 3
After the tension is reduced or released, the nozzle 2 returns to its original diameter and expands due to its own elastic force, increasing the nozzle hole diameter of the nozzle 34.

FIG. 10 shows another embodiment of the aeration gas blowing device according to the present invention. In the embodiment shown in FIG. 10, a nozzle component drum 62 is provided in a nozzle casing 60 having a shape as if a part of a cylindrical body has been removed, and is rotatable about its own central axis with respect to the nozzle casing 60. It is being The nozzle-constituting drum 62 has a plurality of nozzles 64 over the entire outer peripheral surface of the drum, and the nozzles 64 have substantially the same nozzle diameter when arranged along one generatrix. However, the nozzles 64 extending along each generatrix have different nozzle diameters, and the nozzles 64 have different nozzle diameters when viewed in the circumferential direction of the drum. Only the nozzle 64 that is exposed to the outside from the opening 61 of the nozzle casing 60 remains open, and the nozzle 64 located inside the nozzle casing 60 is shielded by the peripheral wall surface of the nozzle casing 60 and substantially ejects gas. It is designed to have no effect. The nozzle component drum 62 is rotatably supported by the nozzle casing 60 by a hollow shaft 66 that also serves as an aeration gas intake port, and a gear 68 is attached to the outer circumference of the hollow shaft 66. The gear 68 is a drive gear 72 of the electric motor 70.
, and is rotationally driven by the electric motor 70.

The nozzle component 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, thereby changing the nozzle 64 exposed to the outside, thereby causing gas to be ejected. The effective nozzle 64 is switched, and the nozzle hole diameter of the nozzle 64 that actually blows out the ventilation gas changes.

Further, FIG. 11 shows another embodiment of the aeration gas blowing device according to the present invention. In this embodiment, a plurality of nozzle holes 82 having a relatively large diameter are provided in a nozzle component 80, and a shutter hole 86 provided in a shutter plate 84 is provided with a plurality of nozzle holes 82 having a relatively large diameter.
Depending on the degree of alignment between the nozzle hole 82 and the nozzle hole 82, the nozzle hole 82 is partially blocked, thereby changing the effective nozzle hole diameter.

In this case, the nozzle hole 82 may have the same width in the direction of movement of the shirt flap plate 84 or may have a different width, and depending on this shape, the effective nozzle diameter changes due to the movement of the shirt flap plate 84. is determined arbitrarily. The shape of the hole is not limited to circular, but may be appropriately selected from elliptical, triangular, quadrangular, polygonal, band-like, etc.

First, use a sparger as shown in Figures 2 to 4.
Suspension culture of animal cells was carried out using a culture apparatus as shown in the figure.

The culture tank used was a cylindrical one with an inner diameter of 120III11, a draft tube with a diameter of 75IllIl and a length of 480s+s, and a 5 liter volume of liquid, and the depth of the liquid level in the tank was 600mm. The temperature inside the tank was adjusted to 37°C using a constant temperature water jacket.

The test cells were rat liver cancer cell line JTC-1 (Japa
nTissue Culture Nol (preserved by the Japanese Society of Tissue Culture) was used. Eagle's MEM medium was used for seed culture, and the culture was carried out in a flat flask and then in a roller bottle. The culture temperature was 37°C. This culture solution was centrifuged to collect cells and used as cells for inoculation.

After introducing 5 liters of Eagle's MEM medium into the culture tank, inoculate the above-mentioned inoculated cells so that the cell concentration becomes I x 10 hcells/ml, and incubate at 36-38°C, pH 68-7.
．． 2 for 10 days. °The ventilation gas used was nitrided oxygen and carbon dioxide gas. The composition of this ventilation gas is 30% oxygen, 5% carbon dioxide, and 65% nitrogen. The set value (setting range) of the dissolved oxygen concentration was 2.0±O, lppm.

The results on the 2nd, 6th, and 100th days after culture are shown in Tables 1, 2, and 3, respectively.

By controlling the nozzle diameter of the nozzle hole according to the dissolved oxygen concentration by giving priority to the ventilation amount according to the flowchart shown in Figure 5, the dissolved oxygen concentration is always maintained within the set value, and the liquid flow rate is also controlled. The cells were kept within a suitable range without deposition or damage, thereby achieving high cell concentrations and high survival rates.

On the other hand, Tables 1 to 3 show the results of Comparative Example 1.2, in which the same specification equipment was used, the same batch of seed cells was used, the sparger nozzle hole diameter was fixed, and the dissolved oxygen concentration was controlled by changing only the aeration rate. Added.

In Example 1 and Comparative Example 1, the cell concentration already reached I x 106 cells/ml on the second and sixth days. In Comparative Example 1, the dissolution of oxygen was 1×10 b
cells If the nozzle diameter is large, oxygen supply will be insufficient even if the aeration rate is increased, and the liquid flow rate will be too low to keep up with the consumption of dissolved oxygen at a cell concentration of 7ml or more. Since this was within the range where damage would occur, it was no longer possible to culture effectively compared to Example 1.

On the other hand, in Comparative Example 2 in which the nozzle hole diameter was fixed small,
In the early stage of culture (2nd day) even if the aeration rate is set to the lower limit that can be operated.
When the cell concentration was low, the dissolved oxygen deviated from the set preferred value and the aeration rate was low, causing the cells to settle at the bottom of the tank, leading to cell death.

As is clear from these results, it will be understood that if the nozzle hole diameter adjustment is performed prior to the aeration rate adjustment as in the present invention, superior culture can be performed compared to the conventional nozzle hole diameter fixed type.

Table 1: Conditions on the 2nd day after the start of culture Table 3: Conditions on the 10th day after the start of culture In this method, the dissolved gas concentration is adjusted by changing the nozzle hole diameter of the aeration gas ejecting means such as a sparger, so that cell destruction is not caused and the liquid is Even a narrow aeration rate range suitable for mixed flow can accommodate a wide range of gas dissolution rates required during culture, allowing good aeration culture to be carried out over a wide area.

Further, in the aeration gas ejection device according to the present invention, by changing the nozzle hole diameter of the nozzle, the bubble diameter of the aeration gas ejected from this can be changed over a wide range by one sparger, There are no installation problems in the culture tank.

[Brief explanation of drawings]

FIG. 1 is a schematic configuration diagram showing one embodiment of the aeration culture device for biological cells according to the present invention, FIG. 2 is a perspective view showing one embodiment of the aeration gas blowing device according to the present invention, and FIG. 2
FIG. 4 is a plan view of FIG. 3, and FIG. 5 is a flowchart showing the method for controlling dissolved oxygen concentration using the aeration gas ejection device according to the present invention. , FIG. 6 is a graph showing the range in which the oxygen supply rate can be adjusted in the relationship between the aeration rate and the oxygen supply rate as the sparger nozzle diameter changes, and FIG. 7 is a graph showing another aeration gas blowing device according to the present invention. 8 to 11 are perspective views showing other embodiments of the aeration gas blowing device according to the present invention, and FIG. 12 shows a case where a sparger whose nozzle hole diameter does not change is used. A graph showing the region where the oxygen supply rate can be tP4fiff in the relationship between the ventilation rate and the oxygen supply rate. Figure 13 shows the ventilation rate and oxygen supply rate when two spargers with different nozzle hole diameters are installed. 2 is a graph showing a region in which the oxygen supply rate can be adjusted in relation to . 1...Culture tank 2...Sparger 19...
Air compressor 20... Nozzle diameter adjustment device 0 2 4 2 8 2 4 2 6... Nozzle casing... Nozzle component... Nozzle 40... Bag body... Pressurizing chamber 46... Permanent magnet...Electromagnet
60... Nozzle casing... Nozzle constituent drum... Nozzle 80... Nozzle constituent member... Nozzle 84... Shutter plate... Shutter hole Fig. 2 34 Fig. 5 Fig. 6 Ventilation speed Figure 7 Figure 8 Figure 9 Figure 13

Claims (10)

[Claims] (1) In the aeration culture method in which biological cells are cultured by aerating a predetermined aeration gas into the culture solution, by changing the hole diameter of the nozzle of the aeration gas blowing means that supplies the aeration gas into the culture solution. 1. A method for aeration culturing biological cells, which comprises adjusting the solubility of the aeration gas in the culture solution by changing the bubble diameter of the aeration gas in the culture solution. (2) In the aerated culture method for biological cells according to claim 1, the aeration is characterized in that the adjustment of the nozzle hole diameter of the aeration gas jetting means is performed with priority over the adjustment of the aeration amount and gas composition of the aeration gas. Culture method. (3) The aerated culture method for biological cells according to claim 1 or 2, wherein the biological cells are animal cells, and the aeration gas is one or a combination of oxygen-containing gas and carbon dioxide gas. Aeration culture method. (4) In the aerated culture method for biological cells according to any one of claims 1 to 3, the nozzle hole diameter is changed by elastic deformation of a nozzle component made of a rubber-like elastic material, and the aeration is ejected from the nozzle hole diameter. An aerated culture method characterized by adjusting the gas bubble diameter. (5) In the aerated culture method for biological cells according to any one of claims 1 to 3, by selectively shielding some nozzle holes in a nozzle having a plurality of holes having different diameters. An aeration culture method characterized by changing the effective nozzle diameter for ejecting aeration gas and adjusting the bubble diameter of the aeration gas ejected from the nozzle diameter. (6) In the aerated culture method for biological cells according to any one of claims 1 to 3, the effective nozzle diameter of the nozzle for ejecting aeration gas is changed by partially blocking the diameter of each hole of the nozzle. An aeration culture method characterized by adjusting the bubble diameter of the aeration gas ejected from the aeration gas. (7) A nozzle component made of a rubber-like elastic material that constitutes a nozzle for ejecting aeration gas in an aeration gas jetting device that supplies aeration gas into a culture solution for aerated culture of biological cells;
An aeration gas ejection device comprising: elastic deformation means for elastically deforming the nozzle component. (8) In an aeration gas blowing device for supplying aeration gas into a culture solution for aeration culture of biological cells, a nozzle component having a plurality of hole collection portions with different nozzle hole diameters, and 1. A ventilation gas ejecting device comprising a shielding means for selectively shielding a hole gathering portion having a predetermined nozzle diameter among the hole gathering portions. (9) In an aeration gas blowout device that supplies aeration gas into a culture solution for aerated culture of biological cells, a nozzle component that constitutes a nozzle for ejecting aeration gas and the nozzle hole are partially shielded. 1. A ventilation gas ejection device characterized by having a shielding means for. (10) A culture tank, a variable nozzle diameter sparger provided in the culture tank, a dissolved gas concentration detection means for detecting the concentration of dissolved gas in the culture solution of the culture tank, and the dissolved gas concentration An aerated culture apparatus comprising: a control device that changes the nozzle hole diameter of the variable nozzle hole diameter sparger based on the dissolved gas concentration detected by the detection means.