JPH06178684A - Method for culturing virus-infected cell - Google Patents

Abstract

PURPOSE:To provide a method for efficiently increasing the yield of a virus by devising the oxygen feed into a culture medium for the liquid culture of a virus-infected cell. CONSTITUTION:The liquid culture of a virus-infected cell is carried out. In the process, pure oxygen gas is solely fed from an oxygen feed pipe into a space above the liquid surface of a culture solution in a culture vessel. Thereby, an atmosphere consisting wholly of the pure oxygen gas or consisting essentially of the oxygen gas is formed to cover the liquid surface of the culture solution therewith. At this time, the feed of the pure oxygen gas from the oxygen feed pipe into the atmosphere is controlled to thereby maintain the content of the dissolved oxygen in the culture medium at a value of <=20% based on the saturation content of the dissolved oxygen in the culture medium during the initial culture period from the time of inoculating the virus into the cell to the time of starting logarithmic growth phase of the virus with the proviso that the content of the dissolved oxygen is maintained at a constant value or a nearly constant value satisfying the oxygen demand of the cell. Furthermore, the value is kept at >=50% based on the saturation content of the dissolved oxygen in the culture medium with the proviso that the content of the dissolved oxygen is maintained at a constant value or a nearly constant value of lower than 100% during the subsequent logarithmic growth phase or thereafter.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to virus infection using animal, plant or microbial cells, and in particular organism cells such as insect cells as host cells for producing viruses by culturing virus infected cells. The present invention relates to a liquid culture method for cells, and more specifically, to inoculate the above-mentioned biological cells with a virus to infect them, and rapidly supply an energy source and a nutrient source to host cells whose metabolic activity is increased by the virus infection. The present invention relates to an improved method for culturing virus-infected cells, which enables the metabolic system of the virus-infected cells in culture to be maintained for a suitably long time, thereby increasing virus production. In the method of the present invention, in order to measure the rapid supply of oxygen required for respiration of virus-infected cells in culture, the supply of oxygen gas to the culture solution is controlled to control the liquid phase of the culture solution, that is, in the medium. A means for adjusting the dissolved oxygen concentration over time is used.

[0002]

2. Description of the Related Art In the culture of animal cells and plant cells (hereinafter sometimes simply referred to as animal and plant cells), organ or tissue cells of animals and plants, and cells of microorganisms, especially insect cells, they have been infected with a virus. Also in the culture of animal and plant cells, the necessary supply of oxygen to the liquid medium is often carried out by aerating a gas containing oxygen into the liquid medium and mixing them in the form of bubbles. However, in this aeration culture method, it is known that contact between the cells and the oxygen-containing gas added as bubbles causes the cells to be inhibited from developing due to oxygen poisoning or mechanical shear stress. In the method of increasing the aeration amount of a gas containing oxygen into the liquid medium in the culture tank and diffusing oxygen into the medium by the stirring action of the bubbles to make the oxygen concentration uniform in the medium, Air bubbles often significantly inhibit cell growth and proliferation.

As another method, a method has been proposed in which a gas containing oxygen is blown into the liquid medium in the culture tank without being ventilated into the liquid medium, but in this method, effective oxygen in the medium is In order to increase the supply amount, the shape of the blowing nozzle is improved (Japanese Patent Laid-Open No. 62-195276) to increase the amount of gas blown onto the medium surface, or the medium surface is waved by additional mechanical stirring. It is necessary to make the liquid surface area wider by standing it and increase the amount of oxygen dissolved from the gas containing oxygen into the medium body. Even with this method of blowing an oxygen-containing gas onto the surface of the liquid medium, if stirring is performed vigorously to increase the oxygen supply efficiency, the shearing force due to stirring acts on the cells in culture, and the growth and proliferation of the cells are significantly hindered. Therefore, as long as the above-mentioned oxygen supply method according to the prior art is adopted, the efficiency of virus production is low when culturing virus-infected cells for the purpose of virus production.

Since the conventional method has disadvantages as described above, it is desired to develop a method for culturing virus-infected cells which does not have such drawbacks.

[0005]

The present invention is advantageous in that the supply of oxygen is controlled so that the growth of virus-infected cells can be well maintained, the survival time of virus-infected cells is prolonged, and the yield of virus is high. An object of the present invention is to provide a method for liquid culture of animal and plant cells infected with a virus having a bacterium.

[0006]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies in order to achieve the above object. First, a method of aerating oxygen-containing gas in a medium known as a method of supplying oxygen to a medium in a liquid culture method for animals and plants in a culture tank, a method of spraying the medium surface, and a method of covering the medium surface with an atmosphere containing high concentration oxygen. In order to investigate the relative strengths and weaknesses of the method, various amounts of dissolved oxygen in the culture medium at the time of culturing cells in the absence of virus infection using a liquid medium with a constant composition, Experiments were carried out by culturing cells under conditions kept constant, and measuring the time-dependent change in the number of viable cells in the culture with the lapse of the culture time. And a method of coating the medium surface with an oxygen-containing gas.

Further, the amount of dissolved oxygen in the medium at the time of cell culture is maintained at a relatively low or high value during the initial period of the culture, and increased or decreased to a relatively high or low value after the initial stage of the culture. The cells were cultured under such varied conditions, and an experiment was also conducted to measure the temporal change in the number of living cells in the culture with the passage of the culture time. Moreover, in these experiments, pure oxygen gas, air, and oxygen-nitrogen mixed gas were supplied.

When a large number of experimental results obtained in this way are comprehensively examined, a method of forming an atmosphere for covering the surface of the medium using pure oxygen gas supplies a mixed gas of oxygen and other gas. It has been found that it can give better results overall than the method.

Therefore, in the case of a method of forming an oxygen-containing atmosphere for covering the surface of the medium by supplying pure oxygen gas, the supply rate (volume / hour) of the oxygen gas flow discharged from the pure oxygen gas supply nozzle, Direction, the distance between the gas outlet of the supply nozzle and the surface of the culture medium, etc., while varying the various conditions such as the time-dependent change in the dissolved oxygen concentration in the culture medium and the time-dependent change in the number of cultured cells. An experiment to measure was conducted.

When the results of a large number of experiments are compared and examined, it is generally confirmed that the surface of the liquid culture medium does not undulate due to the collision of the pure oxygen gas flow supplied from the nozzle. It was found that the method of supplying pure oxygen gas gives excellent results compared with other cases.

Even more importantly, through these numerous experiments, the unit number of cells required by the cells inoculated into the new liquid medium during the early stage of the culture from the time of inoculation to the start of the cell logarithmic growth phase. Oxygen consumption per cell (oxygen demand of cells, O ₂ , μ1 / hour) is the oxygen consumption per unit number of cells required by cells in the logarithmic growth phase after entering the logarithmic growth phase (cells Oxygen demand, O ₂ , μ1 /
It was found that it was only about half of the time). Through this finding, during the logarithmic growth phase of cells, it is necessary to maintain the dissolved oxygen concentration in the medium to satisfy the oxygen demand of the cells in the culture in this period, but to the extent that oxygen poisoning does not occur. During the early stages, it was found that lower levels of dissolved oxygen concentration need to be maintained in the medium, which rather helps prevent cell planting pain.

On the basis of these findings, roughly speaking, if the cells are not infected with the virus, animal and plant cells,
In particular, when performing liquid culture of insect cells, an atmosphere for covering the surface of the medium in the culture tank containing the liquid medium is formed by supplying oxygen gas alone, in other words, supplying pure oxygen gas. In addition, the pure oxygen gas is supplied under the condition that the surface of the liquid medium does not wavy due to the collision or the disturbing action of the supplied oxygen gas flow, and the supply of the pure oxygen gas is adjusted and / or adjusted. By intermittently, the dissolved oxygen content in the medium is maintained at a certain low value from the time of inoculation of the cells to the logarithmic growth phase of the cells, but a certain high value is maintained in the logarithmic growth phase of the cells and thereafter. It was found that by maintaining the value, the logarithmic growth phase of cells can be started earlier and the logarithmic growth phase can be kept longer, and thus the yield of cultured cells can be increased.

Therefore, the present inventors have previously used pure oxygen gas alone in the space above the liquid surface of the culture solution in the culture tank containing the liquid medium inoculated with the cells to be cultured. By blowing and supplying from an oxygen gas supply pipe, an atmosphere consisting exclusively of pure oxygen gas or an atmosphere consisting mainly of oxygen gas and a small amount of water vapor that spontaneously evaporates from the culture solution, a small amount of carbon dioxide gas and a small amount of other species gas. Is formed to cover the liquid surface of the culture solution with the atmosphere, and at the same time, the atmosphere of the culture solution is prevented while preventing the liquid surface of the culture solution from undulating due to the disturbing action of the supply flow of pure oxygen gas. Keep it above the surface of the culture,
Further, adjusting the supply amount of pure oxygen gas and / or
By controlling the supply of pure oxygen gas from the oxygen gas supply pipe to the above atmosphere by intermittently supplying pure oxygen gas,
By controlling the supply of this pure oxygen gas, the amount of dissolved oxygen in the medium is adjusted to 20% of the saturated amount of dissolved oxygen in the medium at the initial stage of culture from the time of inoculation of cells to the start of the logarithmic growth phase of cells.
The following value, which is maintained at a value almost or almost constant to meet the oxygen demand of the cells, and the dissolved oxygen content of the medium is 50% of the saturated dissolved oxygen content during and after the cell logarithmic growth phase. Above, however, there has been proposed a deep liquid culturing method for biological cells, which is characterized by maintaining a value lower than 100% constant or almost constant (see Japanese Patent Application No. 3-279627).

Next, for the purpose of virus production, the present inventors have taken into consideration various findings obtained in the invention relating to the liquid cell culture method previously proposed.
When the virus-infected cells were cultured in a liquid medium, the time course of the oxygen consumption (oxygen demand) of the cells infected with the virus was studied. In that research, for example, using a cultured cell line derived from Spodoptera frugiperda as a host cell, and using a nuclear polyhedrosis virus as a virus to be infected and increased, cultivated Spodoptera frugiperda-derived cells infected with the nuclear polyhedrosis virus The time-dependent changes in oxygen consumption of infected cells and the time-dependent changes in oxygen consumption of non-virus-infected cells were compared.

That is, in the process of culturing cultured cells derived from Spodoptera frugiperda infected with nuclear polyhedrosis virus, the oxygen consumption of virus-infected cells in the culture was measured using a recipe rometer, and as a result, when the virus was inoculated into the host cells, In addition, the oxygen consumption of virus-infected cells on the 3rd day of culture, which is in the logarithmic growth phase of the virus, is 2.5 times that of the cells immediately after virus infection on the 1st day of culture, which is the initial stage of virus infection. Furthermore, it was found that the oxygen consumption of non-virus-infected cells was increased by about 50% over the cell logarithmic growth period.

From these findings, the condition for supplying oxygen to cells during the culture of virus-infected cells is that the period from inoculation of the virus to the logarithmic growth phase of the virus is small, and a large amount of oxygen is supplied to the cells after the logarithmic growth phase of the virus. It was found that it is necessary to supply oxygen to the cells in a higher amount than the oxygen demand in the cell logarithmic growth phase in the culture of non-virus-infected cells. As a result of examining the oxygen supply method that meets these requirements, oxygen gas to the virus-infected cells in the culture was obtained by the method of supplying oxygen gas alone to the gas portion in the culture tank, as in the previously proposed invention. It was found that the harmful effects of the above can be extremely reduced, the culture solution can be stirred without causing foaming, and oxygen can be rapidly supplied to the required oxygen concentration of the medium.

Thus, in short, according to the present invention, on the liquid surface of the culture solution of virus-infected cells in a culture tank containing a liquid medium containing host cells infected with virus after virus inoculation. By supplying pure oxygen gas by blowing it alone into the space from the oxygen gas supply pipe, the oxygen gas mainly composed of pure oxygen gas or the main oxygen gas and a small amount of water vapor and a small amount of carbon dioxide gas spontaneously evaporated from the culture solution And an atmosphere consisting of a small amount of another kind of gas is formed to cover the liquid surface of the culture solution with the atmosphere, and at this time, the liquid surface of the culture solution may be undulated by the disturbing action of the supply flow of pure oxygen gas. Oxygen gas supply by maintaining the above-mentioned atmosphere on the liquid surface of the culture solution while preventing it from occurring and further adjusting the supply amount of pure oxygen gas and / or intermittently supplying pure oxygen gas. From the tube The supply of pure oxygen gas to the atmosphere described above is controlled, and by controlling the supply of this pure oxygen gas, the liquid phase of the culture medium, that is, the amount of dissolved oxygen in the medium, is determined when the virus is inoculated into the host cells. To 20% or less of the saturated dissolved oxygen content of the medium at the initial stage of virus culture from the start of the viral logarithmic growth phase to a value that satisfies the oxygen demand of the host cell, or is maintained almost constant. During the logarithmic growth phase of the virus thereafter and thereafter, the dissolved oxygen content of the medium is 50% or more of the saturated dissolved oxygen content, but 100%.
There is now provided a liquid culture method for virus-infected cells, characterized in that it is kept constant or almost constant at a lower value.

The method for carrying out the method of the present invention will be described in detail below.

The culture tank used in the method of the present invention may be any conventionally used vertical type or horizontal type as long as it is suitable for stirring such as a cylindrical shape or a spherical shape. Further, in this method, the liquid medium may be stirred by a stirrer during the culturing process, and the stirring method may be a propeller method having a rotating shaft in the center of the culture tank or a pump if the stirring means is suitable for culturing cells. Any method of circulating the medium may be used.

The virus-infected cells cultured by the method of the present invention may be animal cells, plant cells, plant organs or microbial cells. Moreover, the cells may be either floating or adhesion-dependent. For example, when insect cells are cultivated by the method of the present invention, the insect cells to be inoculated into the medium should be cut off from any part of the body of any insect and treated with protease to isolate the cells one by one. Can be obtained. However, since the availability of cells and the property thereof are fixed and publicly known, the handling is simple, and a known cell line (cell line) capable of continuing subculture is easier to use.

For this purpose, conventionally known cell lines of insect cells, for example, cell lines of Spodoptera frugiperda-derived cells, SES-MaBr-1 strain, SES-MaBr-.
2 strains, SES-MaBr-3 strain, SES-MaBr-4
Strain, a cell line of cells derived from the same blood cell, NIAS-Ma
Br-32 strain, NIAS-MaBr-92 strain, NIAS
Using the MaBr-93 strain or the like is also advantageous in terms of reproducibility of the culture results.

The Spodoptera frugiperda-derived cell (4H) strain is S
The ES-MaBr-4 strain was designated as No. No. 8 medium, and the Spodoptera frugiperda-derived cell (93H) strain was NIAS-MaBr-93 strain. It is known as an insect cell line obtained by acclimation and subculture in each of 8 mediums (Japanese Patent Laid-Open No. 63-148983), and these can also be cultured by this method.

The virus that can be used in the present invention can be a virus that can be propagated in cell culture, and in particular, any virus that infects insect cells, such as nuclear polyhedrosis virus, granule disease virus, cytoplasmic polyhedrosis. Insect viruses that can be biologically eradicated by infecting crop-feeding insects such as viruses are mentioned.

The liquid medium that can be used in this method is MM medium containing fetal bovine serum [Contrib. Boyel Jhompson Inst
Vol. 22, pp. 435-460 (1964)] and No. 8 media and the like can be mentioned, but any liquid medium can be used depending on the cells to be used as long as it is a liquid medium in which the above-mentioned biological cells can grow.

In carrying out the method of the present invention, after injecting the medium into the culture tank, a sufficient amount of nitrogen gas is blown into the space above the culture solution to expel air. Then, pure oxygen gas is supplied through an oxygen gas supply pipe provided at the top of the culture tank to replace nitrogen gas. As the pure oxygen gas, use a commercially available culture-grade one, and use one that has been sterilized with a sterile filter.

The nozzle outlet of the oxygen gas supply pipe is blown directly into the air layer portion in the culture tank, and the position, the shape of the outlet, and the number of outlets are limited as long as the surface of the culture solution is not ruffled. It is not something that will be done. The cells may be inoculated with the virus at the time when the medium reaches the value of the dissolved oxygen amount described below, or the cells inoculated with the virus may reach the value of the dissolved oxygen amount described below in the medium. You may plant at the time.

The supply amount of oxygen gas varies depending on the volume of the gas phase portion of the culture tank, the volume of the medium contained in the culture tank, and the oxygen consumption of virus-infected cells during the culture, but the culture tank has a capacity of 5 liters. Then, from the extent that the oxygen gas flow meter does not move, 1
Keep it in the range of about liter / h. The dissolved oxygen content of the medium is 20% or less of the saturated dissolved oxygen content during the initial period of virus culture from the time of inoculation of the virus into the host cells in the medium to the start of the logarithmic growth phase of the virus, preferably 1 or less.
Maintain a value of 0-15%. At the start of the logarithmic growth phase of the virus, where the growth rate of the virus is obviously improved and the virus amount is rapidly increased, and thereafter, the dissolved oxygen amount is in the range of 50% or more of the saturated dissolved oxygen amount, preferably in the range of 60 to 80%. Maintain at.

The culture tank is provided with an exhaust pipe for discharging gas from the space above the culture liquid. A condenser is installed in this exhaust pipe to prevent the decrease of the amount of the culture medium liquid due to the evaporation of the culture liquid. It is preferable that the water vapor is condensed and returned to the culture tank. In addition, general equipment necessary for culturing cells in a culture tank, such as a temperature sensor, a pH sensor, and a pH adjusting device, is appropriately installed.

The present invention will be further described below with reference to test examples and examples.

Test Example 1 No. 3 having the composition shown in Table 1 below was blown with oxygen gas alone into the gas phase portion in the culture tank, but without aeration in the culture solution. Cultured cells of Spodoptera frugiperda (4H strain) were cultured in 8 medium without virus inoculation for 10 days (240 hours). The dissolved oxygen content in the culture solution was 15% of the saturated dissolved oxygen content until the second day of culture (up to 48 hours of culture time).
Was maintained at a constant value of 60% from the 3rd day of culture (72 hours of culture time). The oxygen consumption (oxygen demand, μ1 / hour) of the cultured cells in this culturing process was measured using a recipe meter (a part of the culture solution together with the cells was sampled and measured over time). The results are shown in Table 2. The cells on the 1st day of cell inoculation (culture time 24 hours) in the early stage of culture are about 1/2 of the oxygen consumption of the cells on the 3rd day of culture (culture time 72 hours) already in the logarithmic growth phase. It was found that it only needed a large amount.

[0031]

[Table 1] No. 8 Composition of medium Sodium chloride 7.0 g / 1 Potassium chloride 0.2 g / 1 Magnesium chloride hexahydrate 0.1 g / 1 Calcium chloride dihydrate 0.2 g / 1 D-glucose 4.0 g / 1 lactalbumin Hydrolyzate (Nutritional Biocheimicals Corp) 6.5 g / 1 Yeast rate (powdered yeast) (Difco) 5.0 g / 1 pH 6.5 ± 0.2

[Table 2] Test Example 2 Culture solution (cell concentration: 62.0 × 1) obtained from Test Example 1 on the 10th day of culture, which was the culture cell of Spodoptera litura (4H strain)
0 ⁵ cells / ml), the cells were separated from the culture medium and a new No. 8 suspended in medium (cell inoculation). Immediately after that, N containing cultured cells derived from Spodoptera frugiperda (4H strain)
o. A suspension of Spodoptera frugiperda nuclear polyhedrosis virus was added to 8 medium, and the virus was inoculated into cultured cells.

As in Test Example 1, the virus-infected cells were continuously cultured while maintaining the dissolved oxygen content in the culture solution at a value of 15% of the saturated dissolved oxygen content.

In the same manner as in Test Example 1, the oxygen consumption (oxygen demand, μl / hour) of virus-infected cultured cells was measured with a recipe meter. The results are shown in Table 3.

[0034]

[Table 3] Regarding the results in Table 3, on day 0 of culture of virus-inoculated cells (immediately after virus inoculation), oxygen consumption of cells (μl
/ Hr / 1 × 10 ⁵ cells) was 0.6 and decreased to 0.4 on the first day of virus culture, which is due to the pain of planting cells in a new medium and the effect of virus inoculation. Is recognized. On the 3rd day of culture of virus-inoculated cells (corresponding to the logarithmic growth phase of virus), the oxygen consumption of cells increased to 1.5, about 2.5 times as much as 0.6 on the 0th day of culture. Is recognized.

Furthermore, the value of 1.5 of the oxygen consumption on the 3rd day of culture in Table 3 was used as the 3rd day of culture of virus-uninfected cells shown in Table 2 of Test Example 1 (logarithmic growth phase of cells). It is recognized that the oxygen consumption value in () is increased by about 50% or more as compared with 0.9.

Example 1 A medium (No. 8 medium) having the composition shown in Table 1 was prepared,
After sterilization, 3.5 liters were dispensed to a small glass culture jar (capacity of 5 liters, New Brunswick). Nitrogen gas was sufficiently blown into the gas phase in the space above the jar to expel air, and then oxygen gas was blown in to replace nitrogen gas. The amount of oxygen gas supplied was adjusted, and the dissolved oxygen content of the medium was adjusted and set to a value of 15% of the saturated dissolved oxygen content while being measured by a dissolved oxygen meter. Then, this medium was inoculated with Spodoptera frugiperda-derived cells (4H strain) cultured in advance in a spinner bottle at an inoculation rate of 2 × 10 ⁵ cells / ml. Culturing was carried out for 6 days starting at 27 ° C. and stirring speed 60 rpm.

6 days after the start of cell culture, the cell concentration was 5 × 1.
0 was ⁵ / ml. Then, to this cell culture medium, 100 ml of a virus suspension obtained by dissolving polyhedra (10 ⁹ ) of Spodoptera frugiperda nuclear polyhedrosis virus in a 0.1 molar sodium carbonate aqueous solution was added and inoculated. After virus inoculation, the cells were cultured at a stirring speed of 60 rpm and a temperature of 27 ° C. for 12 days. While measuring the amount of dissolved oxygen in the medium for 3 days after starting the culture of virus-inoculated cells, blow oxygen gas into the air layer of the jar so that the oxygen concentration is 15% of the saturated amount of dissolved oxygen. , 4 days after the virus inoculation, the supply was made to be 60%. After 12 days of culture of virus-infected cells, the rate of cells producing virus polyhedra was 20%, and the amount of virus polyhedra produced was 1.2 × 10 ¹⁰ cells / l. The results are shown in Table 4 below.

Control Example 1 In accordance with Example 1 except that the virus-infected cells were cultured for 12 days while keeping the dissolved oxygen content of the medium constant at the value of 15% of the saturated dissolved oxygen content from the beginning of virus inoculation. Culture was performed. The results are shown in Table 4.

After 12 days of culturing the virus-infected cells, the rate of cells producing polyhedra was 2%, and the amount of polyhedra produced was 6.5 × 10 ⁸ cells / l.

Control Example 2 According to Example 1, except that the virus-infected cells were cultured for 12 days while keeping the dissolved oxygen amount of the medium constant at the value of 60% of the saturated dissolved oxygen amount from the beginning of virus inoculation. Culture was performed. The results are shown in Table 4.

After 3 days of culture of virus-infected cells, the cell concentration dropped to 1 × 10 ⁵ cells / ml or less, and after 12 days of culture of virus-infected cells, the rate of cells producing polyhedra was 1.
% Or less, and it was difficult to collect polyhedra.

Control Example 3 In accordance with Example 1 except that a gas obtained by mixing oxygen gas, nitrogen gas and carbon dioxide gas in air was used instead of pure oxygen gas and was blown into the medium at an aeration rate of 1 liter / min. Culture was performed. However, for 3 days after virus inoculation, the amount of dissolved oxygen in the medium was maintained at 15% of the saturated amount of dissolved oxygen by aeration of a mixed gas, and then the culture was increased to 60%. However, the amount of dissolved oxygen in the medium could not be maintained at 60% of the saturated amount of dissolved oxygen by aeration of the mixed gas, and the amount of dissolved oxygen gradually decreased with culturing, and became lower than the measurement limit value in the middle of the culture. It was The results are shown in Table 4.

The rate of cells producing virus polyhedra was 3%, and the amount of polyhedra produced was only 8.4 × 10 ⁸ cells / l.

[0044]

[Table 4] Example 2 A medium (No. 8 medium) prepared according to the composition shown in Table 1 was prepared, and after sterilization, 3.6 l was dispensed into a small glass jar (manufactured by Iwaya Co., Ltd. with a flat capacity of 5 l) to remove nitrogen. 5 liters of gas
Blown in. Four days after the virus inoculation obtained according to Example 1, the cell culture of the Spodoptera frugiperda nuclear polyhedrosis virus-infected cell was centrifuged (300 rpm) and 400 ml of the supernatant of the medium in which the cells had been removed was added to inoculate.

The dissolved oxygen content of the medium was adjusted to the saturated dissolved oxygen content of 10
%, And then the cells of Spodoptera frugiperda (4H strain) cultured in a spinner bottle in this medium in advance had an initial concentration of 5 × 1.
0 were inoculated ^five in addition to be / ml. Using a marine type impeller, stirring speed 120 rpm and temperature 2
The culture was performed at 7 ° C for 14 days. 3 days after the start of culturing, while measuring the dissolved oxygen content of the medium with a dissolved oxygen meter, blow oxygen gas into the gas portion of the jar at an aeration rate of 1 l / h so as to be 10% of the saturated dissolved oxygen content, and thereafter to 80%. Was supplied. After 14 days of culturing, the rate of cells producing polyhedra was 18%, and the production amount of polyhedra was 1 × 10 ^10.
The number was 1 / l.

As a control example, oxygen was supplied in the same manner as in Example 2 except that the dissolved oxygen content of the medium after the start of the culture was kept constant at 10% of the saturated dissolved oxygen content. The rate of cells produced was 1% or less, and it was difficult to collect polyhedra.

As another control example, oxygen was supplied in the same manner as in Example 2 except that the amount of dissolved oxygen in the medium after the start of culture was kept constant at 80% of the saturated amount of dissolved oxygen. The cell concentration dropped to 1 × 10 ⁵ cells / ml or less. After 12 days of culturing, the rate of cells producing polyhedra was 1% or less, and it was difficult to collect the polyhedra.

As yet another comparative example, the procedure of Example 2 was repeated except that air was used instead of oxygen gas, and air was blown at a rate of 1.2 l / min to measure the amount of dissolved oxygen in the medium. At the beginning of the culturing, the culture was cultivated so as to be 10% of the saturated dissolved oxygen amount and 80% of the saturated dissolved oxygen amount after the middle culturing period, but in the middle culturing period, the dissolved oxygen amount of the medium was 80% of the saturated dissolved oxygen amount.
The percentage of cells producing polyhedra could not be maintained at 1% or less. Polyhedron production is 1 × 10 ⁸ pieces / l
It was below.

[0049]

INDUSTRIAL APPLICABILITY As described above, the present invention reduces the amount of dissolved oxygen in the medium at the time of virus inoculation and at the beginning of virus infection, and supplies a large amount of oxygen when the virus is in the logarithmic growth phase. Can propagate and propagate a large amount of virus. Further, by using pure oxygen gas instead of a mixed gas of oxygen, nitrogen and carbon dioxide gas, the required amount can be promptly supplied when a large amount of oxygen is required. Furthermore, by the method of culturing while blowing oxygen gas into the gas portion in the culture tank, the harmful effect of oxygen gas on the cells is extremely small, and the culture solution can be stirred without causing foaming and oxygen can be rapidly supplied. it can. As described above, according to the present invention, virus infection and growth in cultured cells can be efficiently performed, and a large amount of virus can be produced.

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Claims (1)

[Claims] 1. Pure oxygen gas in a space above the liquid surface of a culture solution of virus-infected cells in a culture tank containing a liquid medium containing host cells infected with the virus after virus inoculation. By supplying by blowing alone from the oxygen gas supply pipe, the oxygen gas of the atmosphere or the main component of pure oxygen gas and a small amount of water vapor spontaneously evaporating from the culture solution, a small amount of carbon dioxide gas and a small amount of other species gas The atmosphere is formed to cover the liquid surface of the culture solution with the atmosphere, and at the same time, the liquid surface of the culture solution is prevented from wavy due to the disturbing action of the supply flow of pure oxygen gas. The above atmosphere is maintained from the oxygen gas supply pipe to the above atmosphere by maintaining the above atmosphere on the liquid surface of the culture solution and further adjusting the supply amount of pure oxygen gas and / or intermittently supplying pure oxygen gas. Supply of pure oxygen gas By controlling and controlling the supply of this pure oxygen gas, the dissolved oxygen amount in the liquid phase of the above-mentioned culture medium, that is, the medium, is changed from the time of inoculation of the virus to the host cells to the start of the logarithmic growth phase of the virus. In the initial stage of virus culture, the value is not more than 20% of the saturated dissolved oxygen content of the medium, but is maintained at a value almost or almost constant to meet the oxygen demand of the host cells, and during the subsequent logarithmic growth phase of the virus and After that, the dissolved oxygen content of the medium was adjusted to 50% of the saturated dissolved oxygen content.
A liquid culture method for virus-infected cells, which is maintained at a constant value or a substantially constant value at a level of not less than 100%, but lower than 100%.