JPH04197170A - Stepwise aerobic culture and apparatus therefor - Google Patents

Abstract

PURPOSE:To carry out the subject high-density aeration culture without damage to cells by changing the method for supplying oxygen to a culture tank from the liquid surface aeration method through the membrane surface aeration method to the submerged aeration method in order in accordance with the stage of the culture. CONSTITUTION:In an aeration culture, oxygen is supplied to a culture tank while changing the aeration method from the liquid surface aeration method through the membrane surface aeration method to the submerged aeration method in order in accordance with the stage of the culture. Specifically, oxygen is supplied by the liquid surface aeration method at the initial stage of the culture. In case the consumption of oxygen is increased following the growth of the cells, the aeration method is changed to the membrane surface aeration method capable of supplying more oxygen than in the liquid surface aeration method without generation of air bubbles. At the stationary state of the culture, the aeration method is further changed from the membrane surface aeration method to the submerged aeration method.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method and device for supplying oxygen to a culture tank, and more particularly, to a method and apparatus for supplying oxygen to a culture tank, and more specifically, for culturing at high density without damaging the culture. The present invention relates to an oxygen supply method and device.

[Conventional technology]

Various culturing methods and devices have been proposed for producing useful substances on an industrial scale by culturing animal cells, plant cells, microorganisms, viruses, etc. (hereinafter collectively referred to simply as cells).

In cell culture, in order to efficiently produce useful substances, it is necessary to culture cells at high density.

To this end, perfusion methods and the like have been proposed for supplying nutritional components and removing waste components.

On the other hand, regarding the supply of oxygen, there are two methods: a liquid level aeration method that aerates the top of the culture medium, and oxygen that is diffused into the culture medium through an air-liquid contact interface formed on a hydrophobic membrane surface or an oxygen permeable membrane surface. Two methods have been proposed: a membrane aeration method that supplies air bubbles, and a submerged aeration method that blows air bubbles into the culture solution.

[Problem to be solved by the invention]

Regarding the above-mentioned oxygen supply methods, the liquid surface and membrane surface aeration methods cannot sufficiently supply oxygen necessary for high-density culture of cells, and cannot support scale-up of culture vessels.

The submerged aeration method can supply sufficient oxygen for high-density culture, and can also be used to scale up the culture tank.
It has the advantage and disadvantage of causing significant damage, especially to cells that have low resistance to shearing force.

The present invention has been made in view of the above points, and its purpose is to provide a method and device that can supply oxygen for culturing cells at high density without damaging cells. It is in.

[Means to solve the problem]

The inventors repeated culture experiments with cells that have particularly low resistance to shear force, and found that the resistance to shear force gradually changes (increases) depending on the culture period of the cultured cells, that is, the cell density. This led to the invention.

Basically, we propose the following problem-solving methods.

That is, when culturing cells, depending on the stage of culture, either (a) liquid surface aeration, membrane surface aeration, and submerged aeration are sequentially switched to supply oxygen, or (b) membrane surface aeration is performed after liquid surface aeration. (c) Depending on the stage of culture, first perform at least one of liquid level aeration and membrane surface aeration, and then switch to submerged aeration. supplying oxygen, or (d) initially performing at least one of liquid surface aeration and membrane surface aeration depending on the stage of culture;
We propose a stepwise aerated culture method in which submerged aeration is then added to supply oxygen.

In addition, when performing aerated culture as described above, it is proposed that the aeration method be changed or added in stages based on the state of dissolved oxygen concentration in the culture medium and the state of cell density in the culture. do.

Furthermore, as an apparatus for carrying out the various stepwise aerated culture from (a) to (d) above, basically, at least one of a liquid surface aeration means and a membrane surface aeration means is provided in the culture tank,
We propose a method that includes a submerged aeration means and a means for operating these liquid surface aeration means, membrane surface aeration means, and submerged aeration means selectively or in combination depending on the stage of culture.

In addition, as a problem-solving means for automating such a stepwise aerated culture device, in addition to the above-mentioned liquid level aeration means, membrane surface aeration means, submerged aeration means, etc., the dissolved oxygen concentration in the culture solution of the culture tank is and ventilation control means for controlling the operation of the liquid level ventilation means, membrane surface ventilation means, and submerged ventilation means by switching or combining them based on a set pattern using the detected value of the dissolved oxygen concentration as an index. suggest something.

Furthermore, as an alternative automated means, a means for detecting the cell density of the cultured material in the culture tank, and a means for detecting the cell density of the cultured material in the culture tank, and controlling the liquid level ventilation means, membrane surface ventilation means, and submerged ventilation means using the detected value of the cell density as an index. We propose a device equipped with ventilation control means that controls the operation by switching or combining them based on the setting pattern.

Note that the present invention is applicable to, for example, culturing animal cells, plant cells, microorganisms, and viruses.

Furthermore, in the case where at least membrane surface ventilation and submerged ventilation are performed sequentially, the following membrane is proposed.

In other words, the material of the membrane is not particularly limited, as long as it is harmless to cells and can supply oxygen to the culture solution, but it is preferable that the material has a large air-liquid contact area (it is made of a material that can be obtained). , one with high oxygen supply efficiency is preferable.This membrane is formed to have pores that change from membrane surface ventilation to liquid ventilation when the pressure of the gas to be vented is increased.For example,
The membrane has fine pores for ventilation on the membrane surface, and loose pores that allow ventilation on the membrane surface under normal gas pressure, but when the pressure of the ventilation gas exceeds a certain threshold, bubbles are generated and ventilation in the liquid becomes possible. form a large number of The overall area ratio of the fine pores and the loose pores can be set appropriately, but normally, the oxygen supply capacity of submerged aeration is much larger than that of membrane surface aeration, so the area of the sparse pores is quite large. Small (can be held down)

Then, such an oxygen-permeable membrane is immersed in the culture solution.

[Effect]

When comparing liquid surface aeration, membrane surface aeration, and submerged aeration, the amount of damage to cultured cells is small for liquid surface aeration and membrane surface aeration, and is greatest for submerged aeration. - On the other hand, the oxygen supply capacity is greatest for submerged aeration, followed by membrane surface aeration and then liquid surface aeration.

By the way, when culturing cells, in the early stage of cell culture, that is, when the cell density is low, the resistance of the cells to shear force is extremely low, and in this case, the amount of oxygen consumed by the culture is also small. As cultured cells proliferate, their oxygen consumption also increases, but their resistance to shear forces also gradually increases.

Therefore, if, for example, the above-mentioned methods (a) to (d) are appropriately adopted in accordance with such culture stages, high-density culture can be achieved without damaging the cultured cells.

Specifically, for example, liquid surface aeration is performed at the initial stage of cells. As cells proliferate, oxygen consumption increases,
Liquid surface aeration is not sufficient (in the case of animal cells,
The cell density at this time was 106/m from the latter half of 10
(often in the first half of 6/mE), switch the ventilation method to membrane ventilation, or add membrane ventilation to liquid surface ventilation.

Membrane surface aeration allows for a larger air-liquid contact area than liquid surface aeration, so more oxygen can be supplied and cells proliferate further.

In addition, since membrane surface aeration reduces the generation of bubbles (air bubbles) in the liquid, even cells with low resistance to shearing force can be cultured without being damaged.

Many animal cells have a cell density of 107 due to this membrane surface ventilation.
High densities of 1/m lb can be reached. but,
Cells often consume more oxygen per cell in the stationary phase than in the growth phase, and there is a limit to the membrane area per culture tank volume for membrane aeration. It becomes impossible to supply enough oxygen to maintain density.

Therefore, either switch from membrane surface ventilation to submerged ventilation, or add this. Cell density is high (for many animal cells 10
7 cells/ml level), the resistance to shear force has increased to a sufficient extent for continued culture, and the cells can be grown for 10' without being damaged by submerged aeration.
The oxygen supply will be sufficient to maintain the oxygen concentration at or above the 2000 ml/ml level.

If this aeration method is selected based on cell density, dissolved oxygen concentration, or both, accurate stepwise aeration culture can be achieved.

In addition, which of the stepwise aerated culture methods (a) to (d) above should be adopted depends on the degree of resistance of cells to shear force during the initial stage of culture, cell proliferation stage, and subsequent steady stage. What is necessary is to determine the type of oxygen supply required and the amount of oxygen supply required, and then adopt the most appropriate method.

Note that the operation of the apparatus for implementing these methods has been described in detail in the Examples section, and therefore will not be described here.

Next, the operation of a ventilation system using a membrane having fine pores and sparse pores as described above will be explained.

For example, when culturing cells, aeration is carried out at a normal gas pressure, in most cases around 0-1 atm, in the early stage of the culture, and the membrane surface aeration is also used in the fine and sparse pore areas of the membrane.

When cells proliferate and sufficient oxygen cannot be supplied through membrane ventilation, the pressure of the ventilation gas is increased. The pressure at which bubbles are generated from the sparse pores depends on the pore diameter.

This adds pressure to generate bubbles from the loose pores, allowing cells to grow at high density and supplying enough oxygen to maintain this state.

〔Example〕

Embodiments of the present invention will be described based on the drawings.

FIG. 1 is a schematic diagram of a culture apparatus according to a first embodiment of the present invention.

The culture volume of culture tank 1 is approximately 1.22. Here, a nozzle 3 for aeration of the liquid surface, a tube 4 for aeration of the membrane surface, an aeration filter 5 for aeration of the liquid, and a dissolved oxygen meter 1O are installed in the culture tank 1. The culture tank 1 is also equipped with means for performing perfusion and devices such as various sensors, but these are omitted in the figure.

A culture solution 2 is stored in the culture tank 1 .

6 is a dissolved oxygen concentration controller (hereinafter referred to as a DO controller). A is a gas supply line such as oxygen,
Line A branches into line B, line C, and line D along the way. Of these, line B is for liquid surface ventilation and is connected to the nozzle 3, line C is for membrane surface ventilation and is connected to the tube 4, and line D is for submerged ventilation and is connected to the aeration filter 5.

A valve 7, a flow meter 8, and a sterilization filter 9 are arranged in line B, line C, and line D, respectively.

E is an exhaust line.

The Do controller 6 has a function of taking in an appropriate amount of oxygen gas, air, nitrogen gas, etc. and sending these to the supply line A, and also has a function of dissolving oxygen meter (dissolved oxygen concentration detection means) 1.
It has a function of inputting detection data from 0 and using the detected value as an index to make a determination for sequentially switching between liquid surface ventilation, membrane surface ventilation, and submerged ventilation. This switching judgment signal is sent to the valve control circuit 16, and the valve control circuit 16
The valves of the liquid level ventilation line B, the membrane surface ventilation line C1, and the submerged ventilation line D are switched by this.

Since the cell density is low in the early stage of culture, it is detected that the dissolved oxygen concentration is relatively high, and in this case, the Do controller 6 controls the valve 7 of line B via the valve control circuit 16.
, and leave the other valves in line C1D closed.

In this case, the gas controlled by the Do controller 6 is vented from line A to line B into the culture tank by liquid level ventilation.

Evacuation takes place via line E.

As the cells proliferate, the amount of oxygen consumed increases, and surface aeration is no longer sufficient to supply oxygen. In this case, the line B is closed and the line C is opened according to commands from the Do controller 6 and the valve control circuit I6 using the detected dissolved oxygen concentration value as a guide, and the system is switched to membrane surface ventilation.

If the cells further proliferate, oxygen cannot be supplied sufficiently by membrane surface aeration, so line C is closed and line D is opened to switch to submerged aeration.

The results of culture using the stepwise aerated culture method of the present invention are shown in FIG. The broken line is the result of this example.

The test cells were mouse-human hybridomas, and the medium used was a serum-free medium. At the initial stage of culture, liquid surface aeration was performed, and when the cell density reached I x 10' cells/me, it was switched to membrane surface aeration. After that, the cell density is lXl0' cells/m
When the temperature reached 1, it was switched to submerged aeration.

As a result, it was possible to culture for a long period of time while maintaining the cell density at around 10 cells/ml.

FIG. 2 is a schematic diagram of a second embodiment of the culture apparatus according to the present invention. In the figure, the same reference numerals as in the first embodiment indicate the same or common elements.

The culture volume of the culture tank 1 in this example is approximately 1°2. Inside the culture tank 1, there are provided a nozzle 3 for aeration of the liquid surface, a tube 4 for aeration of the membrane surface, an aeration filter 5 for aeration of the liquid, and a dissolved oxygen meter 1O, as in the first embodiment. .

In this embodiment, in addition to a ventilation system that selectively connects the DO controller 6 to the lines B, C, and D via the line A via the valve 7, the ventilation system connects the DO controller 6 to the lines B, C, and D via a separate line F. , D through a valve 7'.

In addition to this, the culture tank 1 is equipped with means for performing perfusion and devices such as various sensors.
These are omitted in the figure.

A culture solution 2 is contained within the culture tank 1 .

Since the cell density is low in the early stage of culture, the same valve control as in the first embodiment is performed, and the gas controlled by the DO controller 6 is supplied to the culture tank 1 by surface ventilation from line A to line B ( Line C and Line D are in a closed state). Evacuation takes place via line E.

As the cells proliferate, the amount of oxygen consumed increases, and surface aeration is no longer sufficient to supply oxygen. In this case, by selectively opening and closing the valves 7 and 7', the base gas from the line F flows through the line B, and the gas from the Do controller 6 flows through the line C, thereby achieving liquid level ventilation. Perform membrane ventilation.

If the cells further proliferate, it will no longer be possible to supply sufficient oxygen even with liquid surface ventilation and membrane surface ventilation, so the base gas from line F is flowed into line B and line C, and the line D is
Valve 7.7' to flow gas from controller 6.
In addition to the 1M membrane surface ventilation, submerged ventilation is performed.

The results of the culture using the stepwise aerated culture method of this example are shown in the fifth column.
Indicated by solid lines in the figure. The test cells were mouse-human hybridomas, and the medium used was a serum-free medium. Liquid surface aeration was performed at the initial stage of culture, and membrane surface aeration was added when the cell density reached 1 x 106 cells/ml. Then the cell density is l
When the number of particles reached 10'/me, submerged aeration was added.

As a result, the cell density was 10? pieces/mJ! It was possible to perform long-term culture while maintaining the surrounding area.

Also, compared to the first example, the damage at the time of adding submerged aeration was reduced, and the subsequent progress was smooth.

FIG. 3 is a schematic diagram of a culture apparatus in a third embodiment of the present invention. In this embodiment, ventilation is performed by a combination of membrane surface ventilation and submerged ventilation.

The culture volume of the culture tank 1 in this example is also about 1°21. Inside the culture tank 1, a ventilation tube 4 having a hole area O for aeration in the liquid and a dissolved oxygen meter 1O are installed. That is, the tube 4 is formed of an oxygen-permeable membrane, and this membrane 4 has micropores that exclusively perform membrane surface ventilation;
At normal gas pressure, membrane surface ventilation is performed, and when the pressure of the ventilation gas exceeds a certain threshold value, bubbles are generated to enable submerged ventilation.

Although not installed in this embodiment, a means for venting the liquid surface may also be provided.

In this embodiment, the flow rates of oxygen and nitrogen to be supplied to the culture tank 1 are controlled by operating the flow control valves 11 provided in the lines J and L in response to a command signal from the Do controller 6.

Further, a valve 12 for controlling the gas pressure inside the ventilation tube 4 is attached on the exhaust line E of the ventilation tube 4, and this gas pressure is controlled by the DO controller 6.

In addition to this, the culture tank 1 is equipped with means for performing burfusion and devices such as various sensors.
These are omitted in the figure.

A culture solution 2 is contained in the culture tank 1.

Since the cell density is low in the early stage of culture, this state is detected from the detected value of dissolved oxygen concentration, and gas controlled by the Do controller 6 is passed through the ventilation tube 4 from the line A to ventilate the membrane surface. When cells proliferate, sufficient oxygen cannot be supplied through membrane ventilation (this is why the valve 12 on line E is controlled by the DO controller 6 and the ventilation tube 4 is
The gas pressure inside is increased to generate air bubbles from the kneading hole area O, and the air bubbles are used to vent the liquid.

However, in this embodiment as well, the above-mentioned 1. The same effects as in the second embodiment can be achieved.

FIG. 4 is a schematic configuration diagram showing a fourth embodiment of the present invention.

The culture volume of culture tank 1 is approximately 1.22. Here, a nozzle 3 for aeration of the liquid surface, a tube 4 for aeration of the membrane surface, an aeration filter 5 for aeration of the liquid, and a dissolved oxygen meter 10 are installed in the culture tank l. In addition, in the culture tank L,
A sterile sampling system 13, a microscope 14, and an image processing device 15 are connected as means for detecting cell density.

The culture tank 1 is also equipped with means for performing perfusion and devices such as various sensors, but these are omitted in the figure.

In this example, the culture solution 2 is sampled over time throughout the entire culture period, and the cell density is monitored. Based on the detected value of cell density and the detected value from the dissolved oxygen meter 10, the Do controller 6 selectively controls which of the lines B, C, and D to open. It is similar to

[Comparative example]

Describing a comparative example of the present invention 6 In the first comparative example, the structure of the culture device, cell line, and medium used were the same as in the first example, except that the culture tank was not equipped with a membrane ventilation means. is the same as

Liquid level aeration was performed at the beginning of the culture, and when the cells proliferated and sufficient oxygen could no longer be supplied by liquid level aeration, it was switched to submerged aeration.

The results are shown by the dotted line in FIG. Since the cell concentration at the time of switching to submerged aeration was relatively low at 10 cells/me, the cells still did not have sufficient resistance to shear force (due to damage from air bubbles caused by submerged aeration, cells After that, they were unable to reproduce and died.

In the second comparative example, the structure of the culture apparatus used was
The cell line and culture medium are the same as in the first example.

Surface aeration was performed throughout the culture period. The results are shown by the dashed line in FIG. After the cells proliferated and sufficient oxygen could no longer be supplied by aeration at the liquid level, the cells gradually died.

In the third comparative example, the structure of the culture apparatus, cell line, and medium used were the same as in the first example, except that the culture tank was not equipped with a means for submerged aeration.

Liquid surface aeration was performed at the beginning of the culture, and when the cells proliferated and oxygen could no longer be sufficiently supplied by liquid surface aeration, it was switched to membrane surface aeration. The results are shown by the two-dot chain line in FIG. Cells proliferated to around 107 cells/me by membrane surface aeration, but after that, membrane surface aeration was no longer able to provide sufficient oxygen supply and the cells gradually died.

〔Effect of the invention〕

As described above, by adopting the stepwise aerated culture method according to the present invention, sufficient oxygen can be supplied according to the cell growth state of the culture without damaging the culture, and high-density culture can be achieved. enable.

[Brief explanation of the drawing]

FIG. 1 is a block diagram of a culture device used in the first embodiment of the present invention, FIG. 2 is a block diagram of the culture device used in the second embodiment of the present invention, and FIG. 3 is a block diagram of the culture device used in the third embodiment of the present invention. FIG. 4 is a diagram showing the configuration of the culture device used in the fourth example of the present invention. FIG. 5 is an explanation of the culture results using the stepwise aerated culture method of the present invention and the culture results of a comparative example It is a diagram. DESCRIPTION OF SYMBOLS 1...Culture tank, 2...Culture solution, 3...Nozzle for liquid surface ventilation, 4. Tube for membrane surface ventilation (membrane body), 5..Filter for submerged ventilation, 6.Do controller (ventilation) control means), 7...valve, 8.flow meter, 9. sterilization filter,
10-Dissolved oxygen meter (dissolved oxygen concentration detection means), 11...
Flow rate control valve, 12... Pressure control valve, 13... Sterile sampling system, 14... Image processing device, 16... Compiler (valve control means). Agent Patent attorney Akio Takahashi -m-ゝ, :・) (2 others) Preliminary judgment-゛' 1...Cultivation tank, 2.III nutrient solution, 3.Liquid level aeration nozzle, 4.1 side Ventilation tube (membrane body), 5... Filter for submerged ventilation, 6... DO controller (ventilation control means), 7 Valve, 8... Flow rate needle, 9 Divided by 1 filter, 1o... Cumulative total of dissolved M (Dissolved oxygen-degree detection hand &), I
B... Valve # control means

Claims (1)

[Claims] 1. A method for supplying oxygen to a culture tank, characterized in that oxygen is supplied by sequentially switching liquid surface ventilation, membrane surface ventilation, and submerged ventilation according to the stage of culture. Aerated culture method. 2. A method for supplying oxygen to a culture tank, which is a stepwise aerated culture characterized by adding membrane surface aeration after liquid surface aeration and then adding submerged aeration to supply oxygen depending on the stage of culture. Method. 3. The method for supplying oxygen to a culture tank is characterized in that at least one of liquid surface aeration and membrane surface aeration is performed first depending on the stage of culture, and then switching to submerged aeration to supply oxygen. A stepwise aerated culture method. 4. The method for supplying oxygen to a culture tank is characterized in that at least one of liquid surface aeration and membrane surface aeration is first performed depending on the stage of culture, and then submerged aeration is added to supply oxygen. A stepwise aerated culture method. 5. In any one of claims 1 to 4, stepwise switching or addition of ventilation methods such as liquid surface ventilation, membrane surface ventilation, and submerged ventilation to the culture tank may include A stepwise aerated culture method that is characterized by being carried out by monitoring the state of oxygen concentration. 6. In any one of claims 1 to 4, stepwise switching or addition of ventilation methods such as liquid surface ventilation, membrane surface ventilation, and submerged ventilation to the culture tank may A stepwise aerated culture method that is characterized by being carried out by monitoring the state of density. 7. The stepwise aerated culture method according to any one of claims 1 to 6, wherein the cultured material is any one of animal cells, plant cells, microorganisms, and viruses. 8. The culture tank is provided with at least one of liquid level ventilation means and membrane surface ventilation means, and submerged ventilation means, and these liquid level ventilation means, membrane surface ventilation means, and submerged ventilation means are adjusted according to the stage of culture. 1. A stepwise aerated culture device characterized by comprising: means for switching or combining the two methods. 9. The culture tank is provided with at least one of a liquid surface ventilation means and a membrane surface ventilation means, and a submerged ventilation means, and a means for detecting the dissolved oxygen concentration in the culture solution of the culture tank, and a means for detecting the dissolved oxygen concentration in the culture solution of the culture tank. A stepwise aerated culture characterized by comprising: an aeration control means for controlling the operation of the liquid level aeration means, membrane surface aeration means, and submerged aeration means based on a set pattern by switching or combining them based on a detected value as an index. Device. 10. The culture tank is provided with at least one of a liquid surface ventilation means and a membrane surface ventilation means, and a submerged ventilation means, and a means for detecting the cell density of the culture in the culture tank, and a detected value of the cell density. A stepwise aerated culture apparatus characterized by comprising: an aeration control means for controlling the operation of the liquid level aeration means, membrane surface aeration means, and submerged aeration means based on a set pattern by switching or combining them based on a set pattern. 11. In any one of claims 9 to 11, an oxygen-permeable membrane is immersed in the culture solution of the culture tank, and the membrane changes when the pressure of the gas to be aerated is increased. A stepwise aeration culture device characterized by having pores that change from surface aeration to submerged aeration.