JPS6296083A - Culture of antimal cell - Google Patents

Abstract

PURPOSE:To keep the pH of a culture liquid at a prescribed level with simple procedure and to enable the mass culture of animal cell in high efficiency, by using a culture liquid containing NaHCO3 at a concentration above a specific level and blasting CO2 gas into the medium to control the pH at a prescribed level. CONSTITUTION:An animal cell is cultured in a culture liquid containing >3.0g/L of NaHCO3 while blasting CO2 gas in the liquid to control the pH of the liquid to 7.0-7.5. The pH of the culture liquid can be controlled to the above level suitable for the culture of animal cell simply by blasting CO2 gas into the liquid. An interferon having high titer can be produced by applying the above process to the cell for producing interferon.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a method for culturing animal cells.

[Conventional technology]

Animal cell culture is used as a means for artificially producing useful substances that cells produce in vivo, such as vaccines, urokinase, and interferon.

As an animal cell culture method, in the case of tether-dependent cells, a culture method using a Lou bottle or a roller bottle is known, but it is considered to be quite difficult to culture a large amount of tether-dependent cells using this method. There is.

In other words, in this method, the cells only grow in a single layer on the bottom of the roux bottle or the inner surface of the roller bottle, making it extremely difficult to culture in large quantities.

This is because it is necessary to handle a large number of roux bottles or roller bottles, making handling extremely complicated, and it is considered almost impossible to control culture conditions such as pH and dissolved oxygen concentration in the medium at a constant level.

On the other hand, cells that can be cultured in suspension, such as established cell lines, are often cultured in tanks.

However, recently a culture method called microcarrier culture method suitable for mass culture of tether-dependent cells has been developed. This culture method involves inoculating seed cells into a medium suspended in microcarriers with positively charged chemical residues, collagen-coated microcarriers, or other microcarriers capable of adhering cells. :4. This is a method of culturing in a turbid state. The inoculated cells attach to the microcarrier surface and proliferate there.

Suspension culture using a tank as a culture method for animal cells is easy to handle, and it is also easy to control the culture conditions to the optimum value, so it is a culture method suitable for scaling up to industrial production scale. It can be said.

[Problem that the invention seeks to solve]

In animal cell culture, one of the important culture conditions to be controlled in any of the above methods is the pH of the culture solution.

The pH of the culture solution suitable for animal cell culture is approximately 7.0.
~7.5, and many cells are damaged at pH 7.8 or higher or pH 6.8 or lower.

A commonly used method for controlling the pH of a culture solution is to adjust the pH by blowing in carbon dioxide gas using sodium bicarbonate as a buffer. However, when performing large-scale culture using this method, the pH of the culture solution decreases as the number of cells increases in the latter half of the culture, and finally reaches a pH of 7.0.
There was a problem in that it was impossible to maintain the value within the range of ~7.5. If it is not possible to control the pH by just blowing in carbon dioxide gas, an alkali such as a caustic soda aqueous solution may also be added in order to prevent the pH from dropping and keep the pH constant. However, there are problems in that the device is complicated, the sterilization operation is complicated, and the control method is complicated.

The purpose of the present invention is to solve this problem, and to maintain the pH of the culture solution at a constant pH of 7.0 to 7.5, which is suitable for animal cell culture, by simply blowing carbon dioxide into the animal cell culture method. Our goal is to provide the following.

In particular, it provides a method suitable for culturing animal cells for the purpose of interferon production.

The technique for producing interferon in cultured cells is called the superinduction method, in which cells are stimulated with an interferon inducer and then treated with metabolic inhibitors such as cycloheximide and actinomycin D to induce interferon production. There are known methods for further enhancing the

The present inventors have focused on the above-mentioned microcarrier culture method and superinduction method using a medium buffered with sodium bicarbonate as a method for producing large quantities of interferon derived from cultured cells such as diploid cells, and The dissolved oxygen concentration and temperature in the medium during culture and interferon production process are controlled to optimal values, and carbon dioxide gas is mixed into the gas vented to the gas phase or liquid phase to lower the pH to 7.
．． Attempts have been made to produce interferon under constant control within the range of 0 to 7.5. However, when using a culture tank of IOL size or larger and using a culture solution in which sodium bicarbonate is used as a buffer and the pH is adjusted by blowing carbon dioxide gas, it is necessary to use a specified amount of sodium bicarbonate (Eagle MEM medium 1.25 to 2 .2g/I5, Dulbecco's modified Eagle medium 1.0-1.8g/L, 199 medium 1.3
~2.2g/L, Ham F12 medium 1.2-2.0g/L
, RPM11640 medium 1.0-3.0g/L, Fisher medium 1.2-2. With the addition of Og/L (m-) from Nisosui Pharmaceutical's catalogue, as the cells proliferate, the pH of the culture solution decreases, and eventually the pH decreases even if carbon dioxide gas is no longer blown into the animal cell culture. suitable p
H7.0-7.5 could not be maintained, and therefore a high unit of interferon could not be produced, and there was a limit to the titer of interferon produced.

In addition, in order to prevent a drop in pH and keep the pH constant, a method of adding an alkali such as aqueous caustic soda solution is also used, but as mentioned above, the equipment is complicated and the sterilization operation is complicated, and the control There was a problem that the method was complicated.

The purpose of the present invention is to solve this problem, to keep the pH of the culture solution constant in the pH range of 7.0 to 7.5, which is suitable for animal cell culture, by simply blowing carbon dioxide gas, and to produce the desired product. The object of the present invention is to provide an animal cell culture method that is highly effective in stably producing a certain interferon in higher units.

[Means for solving problems]

The above object of the present invention is achieved by adding more than 3.0 g/L of sodium bicarbonate to the culture solution and blowing carbon dioxide gas into it. When producing interferon using the microcarrier culture method, the pH is maintained at 7.0 to 7 throughout the cell culture process using microcarriers and the production process.
．． This is achieved by adding more than 3.0 g/L of sodium bicarbonate to the culture solution and blowing carbon dioxide gas into the culture solution to maintain a constant value within the range of 5.5 g/L.

Typically, an amount of sodium bicarbonate added of 3.0 g/L to 6.0 g/L will accomplish the purpose.

The amount of carbon dioxide gas blown into the culture varies depending on the content of sodium bicarbonate, but usually gas with a carbon dioxide concentration of 20% or less is blown into the culture to control the pH within a certain range.

Next, a method for culturing tether-dependent cells and producing interferon, which is a specific example of the present invention, will be described.

Microcarriers (in a medium) are suspended, seed cells are inoculated into the suspension, and the medium is cultured with gentle stirring while controlling the medium to an appropriate dissolved oxygen concentration and temperature.

Microcarriers include polysaccharides such as dextran, dextrin, starch, cellulose, and substituent derivatives thereof, or certain synthetic polymers such as polyvinyl alcohol, hydroxy-substituted acrylates or methacrylates, polystyrene, etc. Microparticles to which an appropriate amount of amine residues are bonded are used.

The microcarrier concentration varies depending on the microcarrier material and the type of cells to be cultured, but the microcarrier surface area per ml of medium is approximately 10CI+! -100
CII! Preferably, the concentration range is as follows.

The medium composition, serum concentration, etc. are appropriately selected depending on the type of cells to be cultured, cell concentration, etc. During the culture, the culture medium is changed as appropriate, and the culture is continued for several to 10 days until the surface of the microcarrier is almost completely covered with cells.

Before the proliferated cells are treated with an interferon-inducing agent, it is preferred that a low unit of interferon and a negatively charged water-soluble polymer be added and incubated for several hours to one day.

Techniques for treating cultured cells with an interferon inducer to produce interferon include a method in which cells are stimulated to produce interferon using an interferon inducer such as natural or synthetic RNA, and an interferon inducer called the calcium method is used. After stimulating the cells with an interferon inducer such as double-stranded RNA called the super induction method, which is a method in which an appropriate amount of calcium is added to the culture medium at or about 10 hours beforehand to further enhance interferon production, the cells are stimulated with an interferon inducer such as double-stranded RNA, and then transferred to cyclo. A method for further enhancing interferon production by treating cells with metabolic inhibitors such as Ximide and actinomycin D (US Pat. No. 3,773°924);
A method called the UV method is known in which cells are irradiated with ultraviolet rays for several hours before and after stimulation of cells using an interferon-inducing agent to further enhance interferon production.

In the above embodiments, the present invention provides that during the interferon induction treatment and also during the culture of the cells, sodium bicarbonate is added in an amount exceeding 3.0 g/L, typically 3.0 g/L.
The essence is to use a culture solution to which an amount of ~6.0 g/L has been added and to control the pH in the culture solution to a constant value within the range of 7.0 to 7.5 by blowing carbon dioxide gas.

The culture solution used in the present invention includes Eagle MEM medium,
Dulbecco's MEM medium and the like are preferably used.

The present invention will be specifically explained below with reference to Examples.

(Examples 1-2 and Comparative Examples 1-2) A 150L stainless steel culture tank with a stirrer was charged with human incubation in Eagle MEM 100L containing 5 v/v% newborn calf serum and 3.3 g/L of cross-linked dextran microcarriers having diethylaminoethyl groups. Approximately 1.2X1 diploid cells
Inoculate at a rate of 0' cells/ml and inoculate with gentle stirring.
°C, the dissolved oxygen concentration is 10% of the saturated solubility in air
Cultivation was performed four times for 6 days at 50%.

The amount of sodium bicarbonate added to the medium was 5.0 g/L for Examples 1 and 2, and 2.0 g/L for Comparative Examples 1 and 2.
It is 2g/L. The flow rate of carbon dioxide gas was regulated so that the pH during the culture was maintained at a value of 7.0 to 7.5.

Along the way, we conducted Doonjikota on the 1st, 3rd, and 5th days.

Next, the medium was replaced with Eagle's MEM medium containing 100 international units of interferon/ml of carboxyl methyl cellulose, and incubated at 37° C. for about 20 hours at a dissolved oxygen concentration of 10% to 50% of the saturated solubility in air.

Then 10 μg/m 1 cycloheximide and 5
Add 0 μg/ml poly(I):poly(C), incubate at 37°C for 4 hours, and further add 4 μg/ml.
Add ml actinomycin D and incubate for 1 hour at 37°C. After that, the medium was exchanged twice with Eagle MEM medium supplemented with 0.5 g/L methylcellulose, and
It was incubated at 7°C for about 2 days.

Table 1 shows changes in cell number, pH, and carbon dioxide flow rate during culture. When cultured in MEM medium supplemented with 2.2 g/L of sodium bicarbonate, the pH decreases from around the fourth day of culture, even though the flow rate of carbon dioxide gas is zero, and finally drops to below 7.0. On the other hand, when cultured in MEM medium supplemented with 5.0 g/L of sodium bicarbonate, it was possible to maintain pH 7.0 to 7.5, which is suitable for cell culture, until the end of culture.

The amount of interferon finally produced in the medium used was measured by the CPE inhibition method using FL cells and V. stomatitis virus, and converted to international units.

From this result, the pH of the culture solution was kept at 7.0 to 7.0 until the end of the culture.
It can be seen that in the example where the pH was controlled to be constant within the range of 7.5, a higher unit of interferon production was obtained compared to the comparative example where the pH decreased to 7.0 or less in the late stage of culture.

〔Effect of the invention〕

In the method of the present invention, the pH of the culture solution can be kept constant in the pH range of 7.0 to 7.5, which is suitable for animal cell culture, by simply blowing carbon dioxide gas during mass culture of animal cells. In particular, in the case of interferon-producing cells, high titer interferon can be produced by using the method of the present invention.

Claims (1)

[Claims] A method for culturing animal cells, comprising using a culture solution containing more than 3.0 g/L of sodium bicarbonate, and controlling the pH of the culture solution to a constant value by blowing carbon dioxide gas.