JP2933941B2 - Control method, control device, culture method, and culture device for culture conditions of animal cells - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a control method and a control method of animal cell culture environment conditions, an animal cell culture method and a culture apparatus.

[Conventional technology]

Animal cells can produce proteins that cannot be produced by genetically modified bacteria, that is, proteins with added sugar chains, those with high molecular weight, and those with complex tertiary structures. Therefore, attention has been paid to the production of these proteins by culturing animal cells. In particular, establishment of a method for culturing animal cells on a large scale is urgently required.

When mass-culturing animal cells, it is difficult to apply a traditional microorganism culture method. The reason is that animal cells are weak against shearing force due to agitation because there is no cell wall, serum contained in the culture medium foams when aerated in the liquid, and the sterilization system is maintained for a long time because the generation time is several tens of hours And so on. In particular, since culturing is performed for a long period of time, a sensor for measuring the concentration of dissolved oxygen in a culture solution needs to be stably maintained for a long period of time. However, measurement may not be possible due to heat or pressure fluctuations during steam sterilization, or if used continuously for a long period of time, the internal liquid of the sensor may be depleted or a protein film may be formed on the liquid contact part of the sensor, making measurement impossible. If dissolved oxygen concentration cannot be measured due to sensor malfunction, culture cannot be maintained. That is, animal cells become toxic by oxygen when the oxygen supply is excessive, and the proliferation is reduced when the oxygen supply is insufficient.

Furthermore, an index indicating the optimal environmental condition of animal cells has not been known yet, and it has been difficult to quickly determine the cell growth state.

[Problems to be solved by the invention]

The prior art described above does not take into account the measures to be taken when the dissolved oxygen concentration becomes unmeasurable and diagnosing the proliferation activity of the cells, and it is difficult to continuously culture the cells for a long period of time. .

Therefore, an object of the present invention is to provide a method and an apparatus for stably culturing animal cells for a long period of time.

[Means for solving the problem]

The above object is achieved by calculating the ratio of the amount of lactic acid produced to the amount of glucose consumed in culturing animal cells, thereby diagnosing the growth state of the cells, and controlling the culture environment conditions.

That is, the present invention measures the concentration of glucose and the concentration of lactic acid in the culture solution of animal cells, and the ratio (L / G) of the amount of generated lactic acid to the amount of consumed glucose obtained from the measured values is within a predetermined range. If it deviates, a method for controlling the environmental conditions of animal cell culture characterized by controlling the concentration of dissolved oxygen and / or the concentration of glucose in the culture solution in accordance therewith, and the preferable range of the predetermined range is 0 to It is in the range of 0.6 (0 is not included). In addition, the control of the dissolved oxygen concentration and / or the glucose concentration in the culture solution in the case where the concentration deviates from the predetermined range is performed by increasing the dissolved oxygen concentration and / or decreasing the glucose concentration in the culture solution.

Further, the present invention provides a means for measuring glucose concentration and lactic acid concentration in a culture solution of animal cells, calculating a ratio between an amount of consumed glucose and an amount of generated lactic acid from the measured value, and inputting the calculated value in advance. Means for comparing with a predetermined range of values,
And a control device for controlling the environmental conditions for culturing animal cells, comprising means for controlling the concentration of dissolved oxygen and / or the concentration of glucose in the culture solution based on the comparison result.

Further, the present invention relates to a method for culturing an animal cell, the method for culturing an animal cell for controlling the above-mentioned culture environment conditions, and an apparatus for culturing an animal cell comprising the above-mentioned apparatus for controlling the culture environment conditions.

 Hereinafter, the present invention will be described in more detail.

Animal cells acquire energy in an aerobic environment by converting glucose into carbon dioxide and water through a tricarboxylic acid cycle. At this time, 38 mol of APT (adensinetriphosphate) can be produced from 1 mol of glucose, so that using a tricarboxylic acid cycle is a state suitable for growth. On the other hand, in an environment with insufficient oxygen supply, energy is obtained by converting glucose into lactic acid through a glycolysis pathway by the Pasteur effect. In this case, only 2 mol of APT can be produced from 1 mol of glucose, and using only the glycolytic pathway is in a state of poor glucose utilization efficiency. From this, it is considered that the supply state of oxygen and the growth activity can be estimated by using the ratio of the amount of lactic acid produced to the amount of glucose consumed as an index. Even when the oxygen supply condition is good, lactic acid is produced by the Crabtree effect [Crabtree: Biochemistry, 23 , 537-545 (1929)] when the glucose concentration in the culture solution is high. In this case as well, energy is obtained only through the glycolysis pathway without using the tricarboxylic acid cycle, and thus the cell growth activity is in a poor state.

The present inventors measured the concentration of glucose and the concentration of lactate in the culture solution, and used the ratio of the amount of generated lactic acid and the amount of consumed glucose (L / G) obtained from the measured values as an index, to determine the cell growth activity. The present inventors have found that culture environment conditions can be grasped and controlled by diagnosing, and the present invention has been completed.

The optimal conditions for the L / G ratio are in the range of 0 to 0.6 (excluding 0), and were obtained by culture experiments under the following various conditions.

Experimental Example 1 First, an air filter (manufactured by Germanic Science, 7.5 cm ² ) was attached to the upper lid of a tapping culture flask having a total capacity of 200 ml, so that an oxygen-containing gas flowed into the container. 80 ml of DM160-AU medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.) containing 10% newborn calf serum was charged into this container, and 1 × 1
0 ⁶ cells / ml in so as to seed cells JTS-1 strain (rat ascites hepatoma cells) were inoculated. Stirring speed 400rpm, temperature 37 ℃
And cultured in a CO ₂ incubator. Every two days, the whole medium was exchanged by centrifuging the culture solution (1200 rpm, 10 minutes). The initial glucose concentration of the culture at this time was 35
It was set to 00 ppm. 2 ml of culture solution before and after replacing the total volume of culture solution
Each sample was taken, the glucose concentration and the lactate concentration were measured with an enzyme sensor, and the cell concentration was counted with a hemocytometer.
The result is shown in FIG. The cells proliferated very well and reached a cell concentration of 1 × 10 ⁷ cells / ml on day 10.
The L / G at this time was initially high at 0.6, but gradually decreased and finally reached 0.3. From this, if L / G is 0.6 or less, the culture environment is under aerobic conditions, and the glucose supply is sufficient, and the cell growth activity is good, and environmental conditions suitable for growth are obtained. Can be determined.

[Experimental Example 2] Next, the concentration of glucose in the culture solution to be completely exchanged was adjusted to 10%.
The culture was carried out in a sealed state with the air filter removed and the air filter removed. The result is shown in FIG. The final cell concentration is 5.5 × 10 ⁶ cell / ml and L / G is 0.6
It exceeded the value. From this, it was found that under conditions where the glucose concentration was not excessive, when L / G exceeded 0.6, oxygen supply was insufficient and the cell growth ability was reduced.

[Experimental Example 3] Next, the concentration of glucose in the culture solution to be completely exchanged was adjusted to 10%.
The concentration was adjusted to 00 ppm, and an air filter was attached to the lid to perform culturing. FIG. 3 shows the results. Final cell concentration 7.5
× 10 ⁶ cells / ml. L / G decreases with time, 8
On the day, it became zero due to glucose depletion. Therefore, under the condition of glucose concentration of 1000 ppm, 1 × 10 ⁷ cells /
Glucose was found to be insufficient to obtain a high cell concentration of ml.

[Experimental Example 4] Next, a mouse-mouse hybridoma (parent strain P3-U)
Culture was performed using the 11D-11-1 strain described in 1). An air filter was attached to the tapping culture flask so that an oxygen-containing gas (variable in oxygen concentration) flowed into the container. 80 ml of serum-free medium E-RDF + RD
-1 (manufactured by Far Eastern Pharmaceutical Co., Ltd.) was charged, and seed cells (11D-11-1) were adhered to 1 × 10 ⁶ cells / ml. The culture was performed in a CO ₂ incubator at a stirring speed of 300 rpm and a temperature of 37 ° C. Every day, the medium was completely exchanged by centrifuging the culture solution (1200 rpm, 10 minutes). The initial glucose concentration of the culture solution was 3423 ppm. Before and after the exchange of the total amount of the culture solution, 2 ml of the culture solution was sampled, glucose and lactic acid concentrations were measured with an enzyme sensor, and the cell concentration was counted with a hemocytometer. The result is shown in FIG. Proliferation stopped at a cell concentration of 3 × 10 ⁶ cells / ml for 4 days after culture, and L / G was 0.
The value exceeded 6. However, when the oxygen concentration in the oxygen-containing gas was set to 60%, on the seventh day, the cell concentration was 5 × 10 5
^It became ⁶ cells / ml, and L / G also dropped to 0.4. From this, if L / G is 0.6 or less, the culture conditions are under aerobic conditions, and the glucose supply is sufficient, and cell growth activity is good and environmental conditions suitable for growth are obtained. Can be determined.

[Experimental example 5] Next, the cells were cultured under a constant condition of 20% oxygen concentration in the oxygen-containing gas. The results are shown in FIG. Cell concentration is 3
On the day, it reached 3 × 10 ⁶ cells / ml, but did not grow any more.
/ G exceeded 0.6 throughout the culture period. From this, under conditions where the glucose concentration is not excessive, L / G
If the value exceeds 0.6, oxygen supply is insufficient, and it can be seen that the proliferation ability of cells decreases.

From the results of each of the experimental examples described above, it was clarified that the cell growth state could be diagnosed by taking the ratio of the amount of consumed glucose to the amount of produced lactic acid. In particular, when L / G is in the range of 0 <L / G <0.6, the conditions of supply of glucose and oxygen are suitable because the cell growth is good.

Therefore, in the method for controlling the culture environment conditions of the present invention, when L / G deviates from a predetermined range, preferably from 0 to 0.6 (not including 0), the dissolved oxygen concentration in the culture solution and / or Control glucose concentration.

This control can be performed by increasing the dissolved oxygen concentration in the culture solution and / or decreasing the glucose concentration. For example, when L / G exceeds 0.6, first increase the oxygen supply amount and check whether L / G becomes 0.6 or less. If L / G still does not fall below 0.6, it can be controlled by reducing the glucose supply.

An example of a culture device provided with the control device of the present invention will be specifically described with reference to FIG. The culture tank 1 contains a culture solution 2 in which cells or cells immobilized on microcarriers or the like are suspended, and is suspended by rotating a stirring blade 4 using a motor 3. The oxygen supply method may be any of top surface ventilation, submerged ventilation, membrane ventilation, and the like. The oxygen-containing gas is supplied to the culture tank 1 through the conduit 13, and the supply amount is controlled by the flow control valve 6. In the glucose-containing liquid tank, a medium containing glucose or an aqueous glucose solution is stored. The glucose-containing solution is supplied to the culture tank 1 through the conduit 14.
And the supply amount is controlled by the flow rate control valve 7. The culture method includes a semi-batch culture and a continuous culture. For mass production, continuous culture is desirable. The culture supernatant is separated by the cell and culture solution separation means 10, and the culture supernatant is drawn out of the culture tank by using the culture supernatant sending pump 11 through the conduit 17. The concentration of lactic acid and glucose in the culture solution 2 may be measured using an enzyme sensor, liquid chromatography, or the like. The glucose concentration detecting means 8 and the lactic acid concentration detecting means 9 are installed and measured. These data are input to the arithmetic control means 12,
By outputting signals to the flow control valves 6 and 7, the supply of oxygen and the supply of substrate are controlled.

(Operation)

The present inventors experimentally demonstrated that the ratio (L / G) between the concentration of lactic acid produced and the amount of glucose consumed in the culture was correlated with the concentration of dissolved oxygen in the culture and the growth state of the cells. I found it. This L / G is within a predetermined range, especially 0 to 0.6.
(0 is not included), the cell growth is good. Therefore, by keeping L / G within the above range, the culture environment conditions for animal cells are well controlled.
And in this invention, L / G is maintained in the said range by controlling the dissolved oxygen concentration and / or glucose concentration in a culture solution.

〔Example〕

Hereinafter, examples of the present invention will be specifically described,
The invention is not limited in any way by this.

Example 1 An air filter (manufactured by Germanic Science, Inc., 7.5
cm ² ) to allow the oxygen-containing gas to flow into the container. 80 ml of DM16 containing 10% newborn calf serum
A 0-AU medium (manufactured by Far East Pharmaceutical Co., Ltd.) was prepared, and seed cells JTC-1 (rat ascites hepatoma cells) were inoculated at 1 × 10 ⁶ cells / ml. Agitation was performed at 400 rpm and the temperature was 37 ° C., and the cells were cultured in a CO ₂ incubator. Centrifuge the culture every day (1200rpm,
10 minutes), the culture supernatant was removed, and a fresh medium was added. Before and after the exchange of the total amount of the culture solution, the culture solution was sampled by 2 ml each, glucose and lactic acid concentrations were measured by an enzyme sensor, and the cell concentration was counted by a hemocytometer. Further, the glucose concentration in the medium to be exchanged was calculated and adjusted so that L / G was in the range of 0 <L / G <0.6. Here, it is assumed that the glucose consumption rate per JTC-1 strain cell is 1 × 10 ⁻⁷ mg / cell / day, and when L / G is 0.6 or more, this value is decreased, and when L / G becomes 0.4 or less. Thus, the supply amount was calculated by increasing this value for safety. The results of the culture are shown in FIG. The cell concentration reached 1 × 10 ⁷ cells / ml on day 5. L / G was high on the first day, but gradually declined to 0.4 on the third day.
Then, when the supply amount of glucose was increased, L / G increased. Except for the first day, L / G is in the range of 0 <L / G <0.6, cell proliferation is good, and 1 × 10 ⁷ cells / m
l high-density cultivation could be performed.

〔The invention's effect〕

ADVANTAGE OF THE INVENTION According to this invention, the culture environment conditions of animal cells can be controlled stably for a long period of time, and therefore, animal cells can be stably cultured for a long period of time.

[Brief description of the drawings]

FIGS. 1, 2, 3, 4 and 5 show the experimental results, FIG. 6 is a schematic diagram of the culture apparatus of the present invention, and FIG. 7 is a view showing the experimental results of one embodiment of the present invention. 1 ... culture tank, 2 ... culture solution, 3 ... motor, 4 ...
Stirring blade, 5 ... Glucose-containing liquid tank, 6,7 ... Flow control valve, 8 ... Glucose concentration detection means, 9 ... Lactate concentration detection means, 10 ... Separation means for cell and culture solution, 11 ... Culture supernatant feed pump, 12 …… Calculation control means, 13, 14, 15, 16, 17
……conduit.

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Norio Shimizu 1-280 Higashi-Koigakubo, Kokubunji-shi, Tokyo Inside the Basic Research Laboratory, Hitachi, Ltd. (56) References Biotechnology and Bioengineering, 27 (6), 837-841 (1985) (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 5/00-5/28 C12M 3/00-3/10 BIOSIS (DIALOG)

Claims (5)

(57) [Claims] The present invention relates to a method for measuring the concentration of glucose and the concentration of lactic acid in a culture solution of animal cells, wherein the ratio (L / G) of the amount of produced lactic acid to the amount of consumed glucose obtained from the measured values is from 0 to 0.6.
A method for controlling the environmental conditions for culturing animal cells, wherein the concentration of dissolved oxygen and / or the concentration of glucose in the culture solution is controlled accordingly when the value deviates from the range (however, 0 is not included). 2. The animal according to claim 1, wherein the concentration of dissolved oxygen and / or glucose in the culture is controlled by increasing the concentration of dissolved oxygen and / or decreasing the concentration of glucose in the culture. Control method of cell culture environment conditions. 3. A means for measuring a glucose concentration and a lactic acid concentration in a culture solution of animal cells, calculating a ratio (L / G) between an amount of lactic acid generated and an amount of consumed glucose from the measured values, and Means for comparing the value with a value in the range of 0 to 0.6 (but not including 0) which has been input in advance, and means for controlling the dissolved oxygen concentration and / or glucose concentration in the culture solution based on the comparison result. An apparatus for controlling environmental conditions for culturing animal cells, comprising: 4. A method for culturing animal cells, which comprises controlling the culture environment conditions according to claim 1 or 2. 5. An apparatus for culturing animal cells, comprising the apparatus for culturing environmental conditions according to claim 3, wherein the apparatus for culturing animal cells is provided.