JP2020005511A - Culture method and culture apparatus - Google Patents

Abstract

To provide a culture method and a culture apparatus each of which allows for efficient production of a substance by means of continuous culture of a cell.SOLUTION: A culture method for continuously culturing a cell is provided that comprises: measuring a state quantity having correlation with cell proliferation during cell culture; controlling the supply flow rate of culture medium supplied to a culture tank to a first flow rate value when a measured value of the state quantity is a preset value set beforehand or more; and controlling the supply flow rate of culture medium to a second flow rate value smaller than the first flow rate value when a measured value is less than a predetermined value. A culture apparatus 100 comprises: a culture tank 1 for culturing a cell; a supply pump P1 which supplies culture medium to the culture tank 1; a temperature regulator 4 which is capable of cooling culture medium supplied to the culture tank 1; an analyzer 8 which measures the state quantity having correlation with cell proliferation during cell culture; and a control device 9 which controls the supply flow rate of culture medium on the basis of the measured value of the state quantity.SELECTED DRAWING: Figure 12

Description

  The present invention relates to a culture method and a culture apparatus for continuously culturing cells that produce a target substance.

  Biopharmaceuticals, including antibody drugs, are produced by culturing cells and purifying substances produced by the cells. Methods for culturing cells include batch culture in which the medium is not supplied during the culture, fed-batch culture in which the medium is supplied during the culture but is not discharged until the end of the culture (semi-batch culture), or continuous culture during the culture. And a continuous culture (perfusion culture) in which the same amount of medium is continuously discharged.

  According to batch culture, the quality of the product tends to vary from culture to culture, but the risk of contamination can be dispersed and reduced. On the other hand, according to fed-batch culture, the concentration of the product can be increased, and purification costs and medium costs can be reduced when performing large-scale culture. In the field of industrial substance production, for this reason, fed-batch culture has mainly been used.

  However, fed-batch cultivation requires a large cultivation facility, and in recent years, a change to continuous culturing has been widely studied. According to the continuous culture, not only the size of the culture equipment can be reduced, but also the culture environment can be easily maintained constant, so that the quality and production amount of the product can be stabilized. Regarding continuous culture, a technique for controlling cell growth during culture has been proposed to improve the production amount of a target substance.

  US Pat. No. 5,059,097 discloses establishing a mammalian cell culture in a serum-free culture medium, inducing cell growth arrest by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5 mM or less, and -A method comprising maintaining mammalian cells in a growth arrested state by perfusion with a serum-free perfusion medium having an asparagine concentration (see claim 1). Recombinant protein production is said to be increased compared to cultures where the cells are not subjected to cell growth arrest induced by L-asparagine (see claim 51).

JP 2014-520534 A

  In the production of substances by cells, it is desired to grow the cells to a high cell number density in order to obtain a large amount of the target substance. However, in continuous culture, cell growth and production of the target substance proceed at the same time, so that during the production of the target substance, the cells die not a little, and a large amount of impurities are generated by cell decomposition. When the impurity concentration in the culture solution increases, the number of purification steps and the purification cost increase, and the quality of the product is impaired.

  In continuous culture, the passage of cells proceeds during production of the target substance, and the accumulation of mutations may alter the structure and metabolic pathway / efficiency of the target protein, thereby impairing the quality of the product. Can be a problem. In addition, in continuous culture, it is necessary to supply more medium than the cells need to supply and discharge the medium continuously, and to purify low-concentration target substances from the discharged culture solution. There is also a need for improvements in operating and refining costs.

  Therefore, an object of the present invention is to provide a culture method and a culture device that can efficiently perform substance production by continuous cell culture.

  In order to solve the above problems, a culture method according to the present invention is a culture method for continuously culturing cells, wherein a state quantity having a correlation with the growth of the cells is measured during cell culture, and the state quantity is measured. When the value is outside the range of the preset set value and indicates that the growth rate of the cells is high, the supply flow rate of the medium supplied to the culture tank is controlled to the first flow rate value, and the measured value is When the growth rate of the cells is low within the range of the set value, the supply flow rate of the medium is controlled to a second flow rate value smaller than the first flow rate value.

  Further, the culture apparatus according to the present invention is a culture apparatus for continuously supplying a medium to a culture tank, and continuously culturing cells while continuously discharging the medium, and a culture tank for culturing cells. And a supply pump for supplying a culture medium to the culture tank, a temperature control device capable of cooling the culture medium supplied to the culture tank, and measuring a state quantity having a correlation with the growth of the cell during the culture of the cell. An analysis device, and a control device that controls a supply flow rate of the culture medium based on the measured value of the state quantity is provided.

  The culture method and the culture apparatus according to the present invention can efficiently perform substance production by continuous culture of cells.

It is a figure which shows the growth curve of a cell, and the quantitative change of the product which a cell produces. It is a figure which shows the relationship between the glucose concentration, the production rate of IgG, the specific growth rate, and the survival rate. It is a figure which shows the relationship between glutamine density | concentration and IgG production rate, specific growth rate, and survival rate. It is a figure which shows the relationship between the concentration of lactic acid, the production rate of IgG, the specific growth rate, and the survival rate. It is a figure which shows the relationship between the density | concentration of ammonia, the production rate of IgG, specific growth rate, and the survival rate. It is a figure which shows the relationship between the density | concentration of glucose and the density | concentration of glutamine, and a specific growth rate. It is a figure which shows the relationship between the density | concentration of glucose and the density | concentration of lactic acid, and a specific growth rate. It is a figure which shows the relationship between the density | concentration of glucose and the density | concentration of ammonia, and a specific growth rate. It is a figure which shows the relationship between the density | concentration of glutamine and the density | concentration of lactic acid, and a specific growth rate. It is a figure which shows the relationship between the density | concentration of glutamine and the density | concentration of ammonia, and a specific growth rate. It is a figure which shows the relationship between the density | concentration of lactic acid and the density | concentration of ammonia, and a specific growth rate. It is a figure which shows an example of a structure of a culture apparatus typically. It is a flowchart which shows the process at the time of the continuous culture in a culture apparatus. It is a figure which shows the analysis method of intracellular metabolic flux. It is a figure showing the concept of a cell cycle. FIG. 4 is a diagram illustrating the relationship between the fluorescence intensity obtained by nuclear staining and the number of cells.

  Hereinafter, a culture method and a culture apparatus according to an embodiment of the present invention will be described with reference to the drawings.

  The culture method according to the present embodiment relates to a method for continuously culturing cells that produce a target substance. The continuous culture is a culture method in which a medium is continuously supplied to a culture tank and the supplied medium is continuously discharged from the culture tank. In continuous culture, the supply flow rate of the liquid culture medium supplied to the culture tank and the discharge flow rate of the liquid culture medium discharged from the culture tank are equal to each other. The term “continuous” as used herein refers to continuous control of medium supply and discharge, and medium supply and discharge may be intermittent.

  Examples of cells to be cultured include animal cells such as Chinese hamster ovary cells (CHO cells), baby hamster kidney cells, and mouse myeloma cells. The cells to be cultured may be plant cells, microalgae, cyanobacteria, insect cells, bacteria, yeasts, fungi, algae, yeasts, and the like.

  As the target substance, any substance such as various physiologically active substances, pharmaceutical raw materials, chemical raw materials, food raw materials, and functional substances having other usefulness can be produced. In the culture method according to the present embodiment, a particularly preferable purpose of the culture is production of antibodies using floating cells. As the antibody, for example, any of a monoclonal antibody, a polyclonal antibody, a humanized antibody, a human antibody, and other antibodies may be produced.

FIG. 1 is a diagram showing a growth curve of a cell and a quantitative change in a product produced by the cell.
As shown in FIG. 1, the cells proliferate remarkably in the logarithmic growth phase, and after a lapse of the culture time, the cells enter a stationary phase, where growth and death are balanced, and thereafter the number of deaths increases. On the other hand, the amount of product produced by the cells increases with the passage of the culture time, and continues to increase even when the cells enter the stationary phase. The product amount P is represented by the following formula (I), where V is the production rate of a substance by cells and N is the number of cells.
P = ∫V × Ndt (I)

  In order to increase the final production amount of the target substance, it is effective to produce the substance in a culture system in which both the production rate V and the number of cells N are large, as represented by the formula (I). In addition, in view of shortening the time required for substance production, as shown in FIG. 1, it can be said that it is appropriate to grow the cells to a high cell number density in the early stage of culture in which the cell number density tends to increase. Cell proliferation is accompanied by an increase in dead cells and impurities, waste of nutrients and substrates, accumulation of mutations due to subculture, and so on. It may be appropriate to produce the substance.

  Therefore, in the culture method according to the present embodiment, control for switching the culture environment is performed so that the cells have a growth rate suitable for the time period during continuous culture. By controlling the switching of the culture environment, the first stage of the culture, in which the cell number density tends to increase, is defined as the culture period for increasing the cell number density, and the culture environment in which the cells can easily proliferate is increased to increase the cell growth rate. On the other hand, the latter stage of the culture, in which the cell number density is increased, is set as the production stage for producing the target substance, and the culture environment in which the cell proliferation is suppressed is reduced to reduce the cell growth rate (see FIG. 1).

  The control for switching the culture environment is performed based on a state quantity having a correlation with the cell growth. The state quantity used as an index includes, for example, cell number density, cell specific growth rate, cell viability, cell metabolic reaction rate (metabolic production rate of target substance, metabolic consumption rate of nutrients, metabolic secretion rate of waste products) Etc.), cumulative metabolic rate of cells (cumulative metabolic production of target substance, cumulative metabolic consumption of nutrients, cumulative metabolic secretion of waste products, etc.), cell cycle (of cells in G2 phase and M phase in cell population) Ratio) and the like which are correlated with the doubling time of the cell population.

  Control for switching the culture environment measures a state quantity having a correlation with cell growth during cell culture, and when the measured value of the state quantity falls within a range of a preset set value. To do. As the measurement value, the value of the state quantity directly measured in the culture system can be used. Alternatively, using a simulation based on a previous culture test, an estimated value estimated from the value of the state quantity directly measured in the culture system can be used.

  The set value to be compared with the measured value is a state quantity having a correlation with the cell growth, indicating that the cell growth rate is low, and setting a range of the value of the state quantity corresponding to a state suitable for producing a substance. , Can be set in advance based on the results of preliminary tests and the like. As the set value, it is preferable to set the range of the value of the state quantity corresponding to the later stage of the logarithmic growth phase, the transition time from the logarithmic growth phase to the stationary phase, the initial phase of the stationary phase, and the like.

  When such a set value range is set, the period in which the measured value measured in the culture system is out of the set value range overlaps the logarithmic growth phase of the cells, and the culture environment in which the cells can easily grow should be established. It becomes equivalent to the culture period. On the other hand, the period when the measured value measured in the culture system is within the range of the set value overlaps with the stationary period of the cell, and corresponds to the production period in which the culture environment in which cell growth is suppressed should be set. .

  For example, when the state quantity having a positive correlation with the cell growth is used as an index, the measured value of the state quantity having a positive correlation with the cell growth remains large, and the set value and the measurement value set for the positive correlation remain. When the difference from the threshold value exceeds the threshold value (out of the range of the set value and indicates that the growth rate of the cells is high), the cells are growing, and thus the culture conditions in the culture period are set. . On the other hand, when the measured value of the state quantity having a positive correlation with the cell growth decreases and the difference between the set value set for the positive correlation and the measured value becomes equal to or smaller than the threshold (within the range of the set value) (When it indicates that the growth rate of the cells is low), the cells have begun to enter the stationary phase and are switched to production phase conditions.

  Alternatively, when the state quantity having a negative correlation with the cell growth is used as an index, the measured value of the state quantity having a negative correlation with the cell growth remains small, and the set value and the measurement value set for the negative correlation are used. When the difference from the threshold value exceeds the threshold value (out of the range of the set value and indicates that the growth rate of the cells is high), the cells are growing, and thus the culture conditions in the culture period are set. . On the other hand, when the measured value of the state quantity having a negative correlation with the cell growth becomes large and the difference between the set value set for the negative correlation and the measured value becomes equal to or less than the threshold value (within the range of the set value) (When it indicates that the growth rate of the cells is low), since the growth of the cells has begun to stagnate, the conditions are switched to the production phase.

  As a control method for switching the culture environment, a method for controlling the supply flow rate of the culture medium supplied to the culture tank can be used. When the measured value of the state quantity having a correlation with the cell growth is outside the range of the preset set value and indicates that the cell growth rate is high, the supply flow rate of the medium is suitable for the cell growth. The medium flow rate is controlled to a high flow rate value (first flow rate value), and when the measured value of the state quantity is within the range of the preset set value and indicates that the cell growth rate is low, the supply flow rate of the culture medium is reduced. It can be controlled to a low flow value (second flow value) smaller than the high flow value (first flow value). The high flow rate value is the minimum flow rate that allows the growth of the same cell number density as in the production phase.

  When the control for reducing the supply flow rate of the medium is performed in such a manner during the continuous culture, the concentration of the medium component supplied to the culture tank decreases, and the concentration of the metabolite that inhibits the growth in the culture tank increases. Therefore, the culture environment in the culture tank is switched to a culture environment in which cell growth is suppressed.

  It is preferable to control the supply flow rate of the culture medium so that the concentration of the predetermined component in the culture tank changes beyond a threshold value (boundary value). Components that change the concentration include, for example, carbon sources such as glucose, glutamine, amino acids such as glutamic acid, ammonium salts, nitrogen sources such as ammonia, vitamins, inorganic salts, other nutrients such as serum components, and cells. And metabolites that inhibit cell growth, such as lactic acid and ammonia, which are metabolically produced. In the control of the supply flow rate of the culture medium, in particular, at least one of the concentration of glucose in the culture tank, the concentration of glutamine in the culture tank, the concentration of lactic acid in the culture tank, and the concentration of ammonia in the culture tank is adjusted. Is preferred.

  Here, the results of a culture experiment showing the relationship between the concentration of each component and the growth rate of cells and the production rate of a target substance for glucose, glutamine, lactic acid, and ammonia are shown.

  As test cells, Chinese Hamster Ovary cells (CHO cells) into which the gene of immunoglobulin G (Immunoglobulin G: IgG) was introduced were used. As a medium, a liquid medium obtained by adding 10% of fetal bovine serum (FBS) to Dulbecco's Modified Eagle's Medium (DMEM medium) was used.

  First, test cells were seeded in a medium (DMEM + 10% FBS) so as to have a cell density of 0.2 cells / mL, and cultured for 3 days in continuous culture. Then, the medium was replaced with a medium in which any of glucose, glutamine, lactic acid and ammonia was changed to various concentrations, and the cells were further cultured for one day. Thereafter, the increase in the amount of IgG produced (production rate), the increase in the amount of cells (specific growth rate), and the ratio of viable cells to all the cells (survival rate) in each series after culturing were determined.

FIG. 2 is a graph showing the relationship between the concentration of glucose and the production rate, specific growth rate, and survival rate of IgG.
FIG. 2 shows the results when the concentration of glucose in the medium was changed to 0, 0.2, 0.4, 1.5, 4, and 6 g / L. As shown in the figure, the lower the glucose concentration, the lower the specific growth rate, but the higher the production rate.

As shown by the broken line in FIG. 2, considering the specific growth rate = 0.025 h ⁻¹ , it can be said that it is preferable to adjust the glucose concentration to 0.4 g / L or more in the culture period. On the other hand, it can be said that it is preferable to adjust the glucose concentration to less than 0.4 g / L during the production period.

FIG. 3 is a graph showing the relationship between the concentration of glutamine and the production rate, specific growth rate, and survival rate of IgG.
FIG. 3 shows the results obtained by changing the glutamine concentration in the medium to 0, 0.2, 0.4, 1.5, 4, and 6 mM. As shown, the lower the glutamine concentration, the lower the specific growth rate and production rate.

As shown by the broken line in FIG. 3, in view of the specific growth rate = 0.025 h- ¹ , it can be said that it is preferable to adjust the concentration of glutamine to 1.5 mM or more in the culture period. On the other hand, it can be said that it is preferable to adjust the concentration of glutamine to less than 1.5 mM during the production period. From the viewpoint of maintaining a somewhat high production rate, it can be said that the concentration in the production period is more preferably 0.4 mM or more and less than 1.5 mM.

FIG. 4 is a diagram showing the relationship between the concentration of lactic acid and the production rate, specific growth rate, and survival rate of IgG.
FIG. 4 shows the results when the concentration of lactic acid in the medium was changed to 0, 0.2, 0.4, 1.5, and 3.5 g / L, respectively. As shown in the figure, the higher the concentration of lactic acid, the lower the production rate, specific growth rate, and survival rate.

As shown by the broken line in FIG. 4, it can be said that it is preferable to adjust the concentration of lactic acid to 1.0 g / L or less during the culture period in consideration of the specific growth rate = 0.025 h ^-1 . On the other hand, it can be said that it is preferable to adjust the concentration of lactic acid to a concentration exceeding 1.0 g / L during the production period. From the viewpoint of maintaining a high survival rate of about 90% or more, it can be said that the concentration during the production period is more than 1.0 g / L and 5.0 g / L or less.

FIG. 5 is a graph showing the relationship between the concentration of ammonia and the production rate, specific growth rate, and survival rate of IgG.
FIG. 5 shows the results when the concentration of ammonia in the medium was changed to 0, 2, 6, 10, 20, and 30 mM, respectively. As shown in the figure, the higher the concentration of ammonia, the lower the production rate, specific growth rate, and survival rate.

As shown by the broken line in FIG. 5, in view of the specific growth rate = 0.025 h ⁻¹ , it can be said that it is preferable to adjust the concentration of ammonia to 2.0 mM or less in the culture period. On the other hand, it can be said that it is preferable to adjust the concentration of ammonia to a concentration exceeding 2.0 mM in the production period. From the viewpoint of maintaining a high survival rate of about 90% or more, it can be said that the concentration in the production phase is more than 2.0 mM and 20 mM or less.

FIG. 6 is a graph showing the relationship between the concentration of glucose / glutamine and the specific growth rate.
FIG. 6 shows the results when the concentration of glucose in the medium was changed to 0 to 2.5 g / L and the concentration of glutamine was changed to 0 to 2.5 mM. When the two concentrations were adjusted, the specific growth rate increased as the glucose concentration and the glutamine concentration increased, and the specific growth rate decreased as the glucose concentration and the glutamine concentration decreased. Compared with FIGS. 2 and 3 in which the concentration of one type of component is adjusted, the effect is shown in the direction in which the concentration of each component is added, and the concentration of glucose that should be lowered to reduce the specific growth rate is one type. Was higher than when the component was adjusted.

As shown by the thick line in FIG. 6, in consideration of the specific growth rate = 0.025 h ⁻¹ , in the culture period, the glucose concentration was adjusted to 1.5 g / L or more and the glutamine concentration was adjusted to 1.5 mM or more. It can be said to be preferable. On the other hand, it can be said that it is preferable to adjust the glucose concentration to less than 1.5 g / L and the glutamine concentration to less than 1.5 mM during the production period.

FIG. 7 is a diagram showing the relationship between the concentration of glucose / the concentration of lactic acid and the specific growth rate.
FIG. 7 shows the results when the concentration of glucose in the medium was changed to 0 to 3.0 g / L and the concentration of lactic acid was changed to 0 to 2.5 g / L. Compared to FIGS. 2 and 4 in which the concentration of one kind of component is adjusted, the effects of the concentration of each kind of component are shown to be opposite to each other. The concentration of lactic acid, which should be higher than in the case of adjusting the component and should be increased to reduce the specific growth rate, was higher than in the case of adjusting one type of component.

As shown by the thick line in FIG. 7, considering the specific growth rate = 0.025 h ⁻¹ , in the culture period, the glucose concentration was 1.5 g / L or more and the lactic acid concentration was 2.0 g / L or less. It can be said that it is preferable to adjust to. On the other hand, it can be said that it is preferable to adjust the glucose concentration to less than 1.5 g / L and the lactic acid concentration to more than 2.0 g / L during the production period.

FIG. 8 is a diagram showing the relationship between the concentration of glucose / the concentration of ammonia and the specific growth rate.
FIG. 8 shows the results when the concentration of glucose in the medium was changed to 0 to 5.0 g / L and the concentration of ammonia was changed to 0 to 2.5 mM. Compared with FIGS. 2 and 5 in which the concentration of one kind of component is adjusted, the effect of each component is opposite to each other, and the concentration of glucose that should be lowered in order to decrease the specific growth rate is one kind. It was higher than when adjusting the components.

As shown by the thick line in FIG. 8, considering the specific growth rate = 0.025 h ⁻¹ , in the culture period, the glucose concentration was adjusted to 1.5 g / L or more and the ammonia concentration was adjusted to 2.0 mM or less. It can be said to be preferable. On the other hand, in the production period, it can be said that it is preferable to adjust the glucose concentration to be less than 1.5 g / L and the ammonia concentration to be more than 2.0 mM.

FIG. 9 is a diagram showing the relationship between the concentration of glutamine / the concentration of lactic acid and the specific growth rate.
FIG. 9 shows the results of changing the concentration of glutamine in the medium to 0 to 3.0 mM and the concentration of lactic acid to 0 to 2.5 g / L. Compared with FIGS. 3 and 4 in which the concentration of one kind of component is adjusted, the effects of the concentrations of each kind of component are opposite to each other, and the concentration of glutamine to be lowered in order to reduce the specific growth rate is one kind of glutamine. The concentration of lactic acid, which was lower than in the case of adjusting the component and which should be increased to reduce the specific growth rate, was higher than in the case of adjusting one type of component.

As shown by the bold line in FIG. 9, in consideration of the specific growth rate = 0.025 h ⁻¹ , during the culture period, the glutamine concentration was adjusted to 1.0 mM or more and the lactic acid concentration was adjusted to 2.0 g / L or less. It can be said to be preferable. On the other hand, in the production period, it can be said that it is preferable to adjust the glutamine concentration to less than 1.0 mM and the lactic acid concentration to more than 2.0 g / L.

FIG. 10 is a graph showing the relationship between the glutamine concentration / ammonia concentration and the specific growth rate.
FIG. 10 shows the results obtained by changing the concentration of glutamine in the medium to 0 to 5.0 mM and the concentration of ammonia to 0 to 2.5 mM. Compared to FIGS. 3 and 5 in which the concentration of one kind of component was adjusted, the effects of the concentrations of each component were opposite to each other, and the concentration of glutamine, which should be lowered in order to reduce the specific growth rate, was one kind. The concentration of ammonia, which should be higher to lower the specific growth rate than in the case of adjusting one component, was lower than that in the case of adjusting one type of component.

As shown by the thick line in FIG. 10, considering the specific growth rate = 0.025 h ⁻¹ , in the growth phase, the glutamine concentration should be adjusted to 1.0 mM or more and the ammonia concentration to 1.5 mM or less. Is preferred. On the other hand, in the production period, it can be said that it is preferable to adjust the glutamine concentration to less than 1.0 mM and the ammonia concentration to more than 1.5 mM.

FIG. 11 is a diagram showing the relationship between the concentration of lactic acid / the concentration of ammonia and the specific growth rate.
FIG. 11 shows the results obtained by changing the concentration of lactic acid in the medium to 0 to 5.0 g / L and the concentration of ammonia to 0 to 2.5 mM. Compared to FIGS. 4 and 5 in which the concentration of one kind of component is adjusted, the effect is shown in the direction in which the concentration of each component is added, and the concentration of lactic acid that should be increased to reduce the specific growth rate is one kind. Was higher than when the component was adjusted.

As shown by the thick line in FIG. 11, considering the specific growth rate = 0.025 h ⁻¹ , the lactic acid concentration was adjusted to 2.0 g / L or less and the ammonia concentration was adjusted to 2.0 mM or less in the growth phase. It can be said to be preferable. On the other hand, in the production period, it can be said that it is preferable to adjust the concentration of lactic acid to a concentration exceeding 2.0 g / L and the concentration of ammonia to a concentration exceeding 1.5 mM.

  Therefore, in controlling the supply flow rate of the culture medium, when adjusting the concentration of one component contained in the culture solution, it can be said that the concentration of the component is preferably set to the condition shown in Table 1 below. When adjusting the concentrations of a plurality of components contained in the culture solution, it can be said that the concentrations of the components are preferably set to the conditions shown in Table 2 below. Since these components are components common to various cells, they are highly reliable and effective indicators.

  As shown in Table 1, when the concentration of one component is adjusted to exceed a threshold value (boundary value), it is necessary to cause a large change in concentration. However, since the growth rate of cells can be easily reduced, culture It is possible to carry out efficient continuous culture by clearly separating the production phase from the production phase.

  Further, as shown in Table 2, if the concentrations of a plurality of components that promote proliferation are adjusted to exceed a threshold value (boundary value), it is not necessary to cause a large change in the concentration of each component. Can be reduced. On the other hand, when only the concentration of the metabolite that inhibits growth is adjusted to exceed the threshold value (boundary value), the concentration of other medium components and the like is kept relatively high, and the effect on cells is further reduced. In some cases, stagnation of metabolic production of target substances, modification of proteins, and the like can be suppressed.

  As a control method for switching the culture environment, a combination of control of the supply flow rate of the culture medium and control of the temperature of the culture medium can be used. When the measured value of the state quantity having a correlation with the cell growth is out of the range of the preset set value and indicates that the cell growth rate is high, the supply flow rate of the medium is set to a high flow rate suitable for growth. Value (first flow rate value), the temperature of the culture medium is controlled to a high temperature range (first temperature range) suitable for growth, and the measured value of the state quantity is set in a preset value range. And when the cell growth rate is low, the supply flow rate of the culture medium is controlled to a low flow rate value (second flow rate value) smaller than the high flow rate value (first flow rate value) suitable for growth, The temperature of the culture medium can be controlled to a low temperature range (second temperature range) lower than a high temperature range (first temperature range) suitable for growth.

  During the continuous culture, when the control for decreasing the supply flow rate of the culture medium and the control for decreasing the temperature of the culture medium are performed as described above, the concentration of the culture medium component supplied to the culture tank decreases, and the metabolic production that inhibits the growth is reduced. The concentration of the product in the culture tank increases, and the temperature of the culture solution in the culture tank also decreases. Therefore, the culture environment in the culture tank can be reliably switched to a culture environment in which cell growth is suppressed.

  The temperature of the medium is, specifically, from a high temperature range of 35 ° C to 39 ° C, more preferably 36 ° C to 38 ° C, and a low temperature of 29 ° C to 35 ° C, more preferably 31 ° C to 33 ° C. Switching to a temperature range is preferred.

  Next, the configuration of a culture apparatus that can be used in the above-described culture method and the operation method thereof will be described.

FIG. 12 is a diagram schematically illustrating an example of the configuration of a culture device.
As shown in FIG. 12, the culture apparatus 100 according to the present embodiment includes a culture tank 1, a stirrer 2, an air diffuser 3, a temperature control device 4, a medium container 5, a cell separation device 6, a collection tank. 7, an analyzer 8, a controller 9, a supply pump P1, a discharge pump P2, and a suction pump P3.

  The culture tank 1 is a closed container for culturing cells in a liquid medium continuously supplied and discharged. The culture tank 1 is provided with a stirrer 2 for stirring the culture solution and an air diffuser 3 for aerating oxygen, air, carbon dioxide gas, and the like to the culture solution. Further, the culture tank 1 is provided with a temperature control device 4 for adjusting the temperature in the tank. The culture tank 1 is provided with sensors (not shown) for measuring temperature, pH, dissolved oxygen concentration, carbon dioxide concentration and the like.

  The temperature control device 4 is composed of, for example, a water jacket type heat exchanger or the like, and is capable of cooling the culture medium supplied to the culture tank. The temperature of the culture medium is switched from a high temperature range to a low temperature range during continuous culture by the temperature control device 4 that can cool the culture medium. The temperature control device 4 is preferably a device that performs both heating and cooling, from the viewpoint of maintaining the culture environment in the culture tank 1 at an optimum temperature.

  As shown in FIG. 12, the culture tank 1 is connected to a culture vessel 5 via a pipe. As the medium container 5, a plurality of containers can be used for continuous culture. A valve V is provided at the outlet of each medium container 5. When any of the valves V is opened during the continuous culture, the culture medium prepared in the culture medium container 5 is continuously supplied to the culture tank 1 at a predetermined flow rate by the supply pump P1.

  The culture tank 1 is connected to a cell separation device 6 via a pipe. At the time of continuous culture, the culture solution in the culture tank 1 is withdrawn by the discharge pump P2 at a flow rate equal to the supply flow rate, and sent to the cell separation device 6. The cell separation device 6 is a device that separates cells from a culture solution, and is configured by, for example, a device based on various principles such as filtration separation, gravity sedimentation separation, centrifugation, ultrasonic coagulation separation, and membrane separation.

  The cells separated by the cell separation device 6 are returned to the culture tank 1 and continuous culture is continued. On the other hand, the culture solution from which the cells have been separated by the cell separation device 6 is sent to the collection tank 7 by the suction pump P3. Since the target substance produced by the cells is collected in the collection tank 7 together with the medium components, waste products, and the like, the collected culture solution is subjected to a subsequent purification treatment or the like.

  The analyzer 8 is a device that analyzes the state of the cultured cells and the state of the culture solution. Items to be analyzed include a state quantity, a concentration of a medium component, an osmotic pressure, and the like that have a correlation with cell growth. The state quantity includes cell number density, specific growth rate, viability, metabolic reaction rate of cells, accumulated metabolic rate of cells, cell cycle, and the like. Examples of the concentration of the medium component include concentrations of a target substance, glucose, glutamine, lactic acid, ammonia, amino acids, vitamins, metabolites, growth factors, serum components, and the like.

  In addition, the analyzer 8 has a function of estimating an estimated value from a value directly measured in the culture system using a simulation based on a preliminary culture test for a state quantity correlated with cell growth. Can be The measurement result and the estimation result of the state quantity are output to the control device 9.

  The control device 9 controls the supply flow rate of the supply pump P1 that supplies the culture medium to the culture tank 1 and the cooling temperature of the culture medium by the temperature control device 4 based on the measured value of the state quantity that has a correlation with the cell growth. . Further, the control device 9 controls a culture environment factor of the culture tank 1 and a rotation speed of the stirrer 2. The culture environment factors include culture temperature, pH, dissolved oxygen concentration, carbon dioxide concentration and the like.

FIG. 13 is a flowchart showing a process during continuous culture in the culture device.
FIG. 13 illustrates a flow of a process of performing a single control for switching the culture environment during the continuous culture in the culture device 100.

  First, culture conditions necessary for culturing cells are set in the culture device 100 (step S1). Items to be set include the amount of culture solution in the culture tank 1, the culture temperature, the pH, the dissolved oxygen concentration, the carbon dioxide concentration, the rotation speed of the stirrer 2, the supply flow rate of the culture medium, and the like. Regarding the supply flow rate of the medium, individual control target values are set for each of the culture period and the production period. In addition, a set value as a start condition of control for switching the culture environment and an end condition for ending the continuous culture are set. When each item is input to the controller 9, the stirrer 2, the air diffuser 3, the temperature controller 4, and the like are controlled, and the culture environment in the culture tank 1 is adjusted to a culture environment suitable for the culture period.

  Subsequently, cells producing the target substance are seeded on a medium (step S2). At a stage where the culture environment in the culture tank 1 is adjusted to a culture environment suitable for the growth phase, or at a stage immediately before that, cells are aseptically introduced into the culture tank 1 to start culture. Cells may be introduced in an appropriate number and state of cells, but it is necessary to pre-culture in a culture environment equivalent to that in the growth phase, and to introduce a cell suspension adjusted to 5 to 10 times the cell number density in the case of seeding. Is preferred.

  Subsequently, the cells are cultured and grown in the culture tank 1 (step S3). The culture temperature during the growth phase is preferably controlled at 37 ° C. ± 2 ° C. from the viewpoint of maintaining a high growth rate. In the growth phase, the cells are grown by continuous culture, but batch culture may be performed temporarily as a step prior to continuous culture.

  Subsequently, the culture solution is analyzed during the continuous culture (culture during the culture period) (step S4). The analyzer 8 measures a state quantity having a correlation with cell proliferation, and acquires a measured value by direct measurement or estimation. The analysis of the culture solution may be performed continuously or intermittently during continuous culture. Further, a culture temperature, a pH, a dissolved oxygen concentration, a carbon dioxide concentration, and the like are monitored by a sensor (not shown).

  Subsequently, it is determined whether or not the culture environment in the culture tank 1 matches the culture conditions of the culture period set in advance (step S5). It is determined whether or not the culture environment factors such as the culture temperature, pH, dissolved oxygen concentration, and carbon dioxide concentration obtained by the analysis of the culture solution do not deviate from the range of the culture conditions in the growth phase set in step S1. Is done.

  As a result of the determination, if the culture environment in the culture tank 1 does not conform to the culture conditions in the culture period (Step S5; No), the culture environment is controlled to conform to the culture conditions (Step S6). The control target values of the stirrer 2, the diffuser 3, the temperature control device 4, etc. are updated so that the analyzed items do not deviate from the range of the culture conditions in the growth phase set in step S1, and the PID control and the proportional control are performed. The culture environment is adjusted by ON / OFF control. Thereafter, the process is returned, and the culture in the culture period is continued under appropriate culture conditions (step S3).

  On the other hand, as a result of the determination, if the culture environment in the culture tank 1 matches the culture condition in the culture period (Step S5; Yes), the process proceeds, and it is determined whether the state of the cell matches the switching condition. A determination is made (step S7). It is determined whether or not the measured value of the state quantity having a correlation with the cell growth obtained by the analysis of the culture solution is within the range of the set value of the control for switching the culture environment set in step S1.

  As a result of the determination, if the state of the cells does not conform to the switching condition (Step S7; No), the process is returned to the original state because the switching from the growth phase to the production phase is not appropriate. Is continued (step S3).

  On the other hand, as a result of the determination, if the state of the cell conforms to the switching condition (Step S7; Yes), since the switching from the growth phase to the production phase is appropriate, the processing is advanced and control for switching the culture environment is performed. (Step S8). The switching of the culture environment is performed by controlling the supply flow rate of the culture medium supplied to the culture tank, or by controlling the supply flow rate of the culture medium and controlling the temperature of the culture medium.

  Subsequently, the cells are cultured in the culture tank 1 to produce the target substance (Step S9). When the temperature of the medium is switched, the culture temperature in the production phase is preferably controlled to 32 ° C. ± 3 ° C. from the viewpoint of more surely suppressing cell growth.

  Subsequently, the culture solution is analyzed during the continuous culture (culture during the production phase) (step S10). The analyzer 8 measures a state quantity having a correlation with cell proliferation, and acquires a measured value by direct measurement or estimation. The analysis of the culture solution may be performed continuously or intermittently during continuous culture. Further, a culture temperature, a pH, a dissolved oxygen concentration, a carbon dioxide concentration, and the like are monitored by a sensor (not shown).

  Subsequently, it is determined whether or not the culture environment in the culture tank 1 conforms to the culture conditions in the production period that are set in advance (Step S11). It is determined whether or not the culture environment factors such as the culture temperature, pH, dissolved oxygen concentration, and carbon dioxide concentration obtained by the analysis of the culture solution do not deviate from the range of the culture conditions in the production period set in step S1. Is done.

  As a result of the determination, if the culture environment in the culture tank 1 does not conform to the culture conditions in the production phase (Step S11; No), the culture environment is controlled to conform to the culture conditions (Step S12). The control target values of the stirrer 2, the diffuser 3, the temperature control device 4, etc. are updated so that the analyzed items do not deviate from the range of the culture conditions in the growth phase set in step S1, and the PID control and the proportional control are performed. The culture environment is adjusted by ON / OFF control. Thereafter, the process is returned, and the culture in the production phase is continued under appropriate culture conditions (step S9).

  On the other hand, as a result of the determination, if the culture environment in the culture tank 1 conforms to the culture conditions in the production phase (Step S11; Yes), the process proceeds, and it is determined whether the state of the cells conforms to the termination condition. A determination is made (step S13). It is determined whether or not the measured value of the state quantity having a correlation with the cell growth obtained by the analysis of the culture solution satisfies the termination condition for terminating the continuous culture set in step S1.

  The termination condition is a state quantity that has a correlation with cell growth, and the range of the value of the state quantity corresponding to a state in which cell death is remarkable and is not suitable for producing a substance is determined by a preliminary test result or the like. It can be set in advance based on this. When the measured value is lower than such an end condition value, it is preferable to end the continuous culture, because the death of the cells causes contamination and the like to proceed.

  As a result of the determination, if the state of the cell does not conform to the termination condition (Step S13; No), the process is returned to the end of the continuous culture because the termination of the continuous culture is not appropriate, and the culture in the production phase is continued under the culture conditions in the production phase. (Step S9).

  On the other hand, as a result of the determination, if the state of the cell conforms to the termination condition (step S13; Yes), the process is advanced, the culture is stopped, and the production of the target substance is terminated because the end of the continuous culture is appropriate. I do.

  In the above process, the control for switching the culture environment is performed once during the continuous culture, but may be performed a plurality of times. When the culture environment is switched a plurality of times, different values can be set as index values (measured values) of the control for switching the culture environment. When the culture environment is switched a plurality of times, at least one of the culture period and the production period is divided into a plurality of stages. Steps S3 to S7 and steps S9 to S13 (see FIG. 13) can be repeated according to the number of times of switching during any of the culture period and the production period.

  Hereinafter, a specific method of analyzing a state quantity having a correlation with cell proliferation will be described.

<Cell number density, specific growth rate, viability>
The cell number density, the specific growth rate, and the survival rate among the indexes indicating the state of the cells can be obtained by the following method.

The survival rate can be determined as a ratio of living cells to all cells. The number of cells can be measured by various methods such as microscopic observation using a hemocytometer, dry weight method, turbidity method, capacitance method, NAD measurement for quantifying oxidized NAD ⁺ and reduced NADH, and flow cytometry. It can be measured using a measuring method. In addition, the number of living cells can be measured by utilizing the determination by trypan blue staining.

The specific growth rate can be determined by measuring the change over time in the number of living cells. The cell growth rate v is represented by the following formula (II), where X is the number of living cells, μ is the specific growth rate, and t is the time.
v = dX / dt = μX (II)

  Since the cell growth rate v has a linear relationship between the number of viable cells and time as represented by the formula (II), it can be determined from measurements of two or more different culture times. Since the number of measurements is small and the error may not be negligible, a large number of measurements may be performed during the logarithmic growth phase, and the measurement results shown on the logarithmic graph may be linearly approximated by the least square method.

<Metabolic reaction rate / cumulative metabolic rate>
Among the indices indicating the state of the cell, metabolic reaction rates (metabolic production rate of target substance, metabolic consumption rate of nutrients, metabolic secretion rate of waste products, etc.) and cumulative metabolic rate (cumulative metabolic production of target substance, nutrient The cumulative metabolic consumption, the cumulative metabolic secretion of waste products, etc.) can be determined by the following method.

  The metabolic production rate of the target substance can be determined by measuring the time-dependent change in the concentration of the target substance to be produced and converting it into the amount of production per unit time per unit number of cells. For example, when the target substance is a protein, the concentration of the target substance can be determined by an ELISA (Enzyme-Linked ImmunoSorbent Assay) method. Using an antibody that produces an antigen-antibody reaction with the protein to be measured, label fluorescence analysis, enzyme activity analysis, and the like are performed. As the ELISA method, any of a direct method, an indirect method, a sandwich method, a competitive method and the like may be used.

  The rate of metabolic consumption of nutrients and the rate of metabolic secretion of waste products can be determined by measuring the change over time in the concentration in the culture tank and converting the change per unit time per unit number of cells. The concentration of nutrients and the concentration of wastes can be measured by High Performance Liquid Chromatography (HPLC), Liquid Chromatography-Mass spectrometry (LC / MS), and Liquid Chromatography Tandem Mass Spectrometry (LC / MS-MS). Gas chromatography mass spectrometry (GC / MS), gas chromatography tandem mass spectrometry (GC / MS-MS), or the like can be used to quantify the cells by removing the cells.

  The cumulative metabolic production of the target substance, the cumulative metabolic consumption of nutrients, and the cumulative metabolic secretion of waste products can be obtained by a method of integrating the obtained metabolic rate over time or a method of actually measuring the cumulative amount.

FIG. 14 is a diagram showing a method of analyzing intracellular metabolic flux.
As shown in FIG. 14, the metabolic reaction rate (the rate of metabolic production of the target substance, the rate of metabolic consumption of nutrients, and the rate of metabolic secretion of waste products) can also be determined by analyzing the intracellular metabolic flux. By utilizing the results of the analysis, it is possible to estimate the metabolic production rate of the target substance, the metabolic consumption rate of nutrients, the metabolic secretion rate of waste products, and the like in calculation.

  A metabolic reaction model used for analyzing intracellular metabolic flux can be constructed by performing a simulation based on an initial model created based on a known metabolic pathway, and correcting the result with the result of a culture experiment. Known metabolic pathways include, for example, glycolysis, gluconeogenesis, citrate cycle, glyoxylate cycle, oxidative phosphorylation, pentose phosphate cycle, reductive pentose phosphate cycle, urea cycle, β oxidation, amino acid biosynthesis, nucleotide Various metabolic pathways such as metabolism, glycogen synthesis, lipid biosynthesis, fatty acid biosynthesis, cholesterol biosynthesis, purine synthesis, pyrimidine synthesis, and shikimate pathway can be used.

  In culture experiments, cells of interest are cultured using a substrate labeled with a radioactive isotope of carbon. Then, isotope ratios of metabolites / intermediate metabolites individually generated through each metabolic pathway are measured by GC / MS. For example, the cultured cells are collected from a culture tank, and after fixing the cells, various metabolites and intermediate metabolites necessary for analysis are extracted. Then, for derivatization for GC / MS, after adding 2% methylhydroxylamine hydrochloride and reacting at 55 ° C. for 2 hours, N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide (MTBSTFA) After adding 45 μL of a 1% tert-butyldimethylchlorosilane (t-BDMCS) solution and reacting at 37 ° C. for 1 hour, analysis is performed by GC / MS.

  The analysis conditions of GC / MS-MS are, for example, using DB-35 ms (manufactured by GL Sciences, length 30 m) as a column, and increasing the temperature: 3.5 ° C./min from 100 ° C. to 300 ° C. Inlet temperature: 270 ° C., carrier gas: helium, carrier gas flow rate: 1 mL / min.

  On the other hand, in the simulation, first, a metabolic reaction model is assumed for a metabolic reaction chained and branched in a cell, and initial values of metabolic reaction rates (R1 to R8 in FIG. 14) of each metabolic reaction are given as random numbers. Then, in the steady state, the ratio of the isotope contained in each metabolite / intermediate metabolite is calculated, and this calculated value is compared with the measurement result of the culture experiment. If there is a statistically significant difference between the calculated value and the measurement result, the metabolic reaction rate value is corrected by a numerical solution based on the QP (Quadratic Programming) subproblem method so that the mutual mean square error is minimized. . Then, recalculation is performed under the corrected condition, and the calculation and correction are repeated until there is no significant difference between the calculated value and the measurement result.

  Since a state where there is no significant difference is a metabolic reaction model that is appropriate for calculation, the calculated metabolic reaction rate value or the like can be used as an estimated value. The use of such an analysis eliminates the need for frequent analysis and quantification during continuous culture, and eliminates the time-consuming restriction of performing analysis and quantification.

<Cell cycle>
Among the indices indicating the state of the cell, the cell cycle can be determined by performing flow cytometry using discrimination by propidium iodide (PI) staining.

FIG. 15 is a diagram showing the concept of the cell cycle.
As shown in FIG. 15, the cell cycle affects cell division / proliferation and intracellular metabolism. The cell cycle of eukaryotic cells consists of mitosis (M phase) and other interphases (G1, S, G2). The cell cycle is about 16 to 24 hours, the M phase is about 1 hour, the S phase is about 6 to 8 hours, the G2 phase is about 2 to 6 hours, and the time ratio between the M phase, G1, S phase and G2 phase Is approximately 1: 5: 7: 3. In the G1 phase, there are cells that interrupt the cell cycle due to various factors and enter the G0 phase and become dormant.

  Cells in the G2 and M phases are about to initiate cell division, while cells in the G1 and G0 phases have time to divide, and the G0 phase has an indefinite length. The culture period in which the culture environment in which the cells can easily proliferate should be a state in which the proportion of cells in the G2 or M phase of the cell cycle is high, that is, a state in which many of the cultured cells are about to start cell division. It can be said that it is preferable to match them. On the other hand, the production period, which should be a culture environment in which cell growth is suppressed, is a state in which the proportion of cells in the cell cycle in the G1 or G0 phase is high, that is, a state in which most of the cultured cells are in the interphase. It can be said that it is preferable to match with.

FIG. 16 is a diagram illustrating the relationship between the fluorescence intensity obtained by nuclear staining and the number of cells.
In FIG. 16, the vertical axis indicates the number of cells, and the horizontal axis indicates the fluorescence intensity of cells stained for nuclei by the PI staining method. When cells are cultured, and a cell population having a mixed cell cycle is subjected to nuclear staining and fluorescence detection by flow cytometry, specific profiles (C11, C12, C13) as shown in FIG. 16 are obtained.

  A curve C11 shows an example of a profile obtained from cells in the G1 or G0 phase. Since the replication of chromosomal DNA has not started in cells in the G1 or G0 phase, the detected fluorescence intensity is weak.

  A curve C12 shows an example of a profile obtained from cells in the S phase. Since cells in the S phase have begun replication of chromosomal DNA and the amount of DNA is in an increasing stage, the detected fluorescence intensity is not uniform and spreads over a wide range.

  On the other hand, a curve C13 shows an example of a profile obtained from cells in the G2 or M phase. In the cells in the G2 phase and the M phase, since sufficient DNA for cell division is synthesized, the detected fluorescence intensity is high.

  The profile shown in FIG. 16 is obtained as a frequency distribution of the number of cells. Therefore, when fluorescence is detected by flow cytometry and the integrated area of each curve on the frequency distribution is calculated, the number and ratio of cells in the G2 and M phases can be known. Such a cell cycle (the proportion of cells in the G2 phase and the M phase in the cell population) is correlated with cell growth, but does not have to wait for cell growth for analysis and is completed in about one hour. Therefore, the culture environment can be switched at an appropriate time.

  According to the above culturing method and culturing apparatus, the state quantity having a correlation with the cell growth is measured during the culturing of the cell, and control for switching the culture environment is performed based on the state quantity, so that the cell is suitable for the time. Continuous culture can be performed efficiently at the growth rate. Since the cell density can be efficiently increased in the growth phase in the early stage of the culture, the target substance can be produced in a large amount by sufficiently grown cells. On the other hand, during the late production phase, the target substance can be produced by suppressing cell growth, thus suppressing the increase of dead cells and impurities, waste of nutrient sources and substrates, accumulation of mutations due to passage, etc. As a result, the target substance can be highly purified and the operating cost and the purification cost can be reduced.

  Although the embodiment of the present invention has been described above, the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the spirit of the present invention. For example, a part of the configuration of an embodiment can be replaced with another configuration, or a part of the configuration of an embodiment can be omitted.

REFERENCE SIGNS LIST 100 culture device 1 culture tank 2 stirrer 3 diffuser 4 temperature control device 5 medium container 6 cell separation device 7 recovery tank 8 analysis device 9 control device P1 supply pump P2 discharge pump P3 suction pump V valve

Claims (10)

A method for continuously culturing cells, comprising:
Measuring the state quantity having a correlation with the proliferation of the cells during the culture of the cells,
When the measured value of the state quantity is outside the range of the preset set value and indicates that the growth rate of the cells is high, the supply flow rate of the medium supplied to the culture tank is set to the first flow rate value. Control and
A culture method for controlling the supply flow rate of the medium to a second flow rate value smaller than the first flow rate value when the measured value is within the range of the set value and indicates that the growth rate of the cells is low; . When the measured value is out of the range of the set value, while controlling the supply flow rate of the culture medium to the first flow rate value, and controlling the temperature of the culture medium to a first temperature range,
When the measured value is within the range of the set value, the supply flow rate of the culture medium is controlled to the second flow rate value, and the temperature of the culture medium is set to a second temperature range lower than the first temperature range. The culture method according to claim 1, which is controlled.   The said state quantity is a cell number density, a specific growth rate, a cell viability, a metabolic reaction rate, a cumulative metabolic rate, or a ratio of cells in the G2 phase and the M phase in the cell population. 3. The culture method according to 2.   The concentration of at least one of a concentration of glucose, a concentration of glutamine, a concentration of lactic acid, and a concentration of ammonia in the culture tank is controlled by controlling a supply flow rate of the culture medium. Culture method.   The concentration of glucose in the culture tank is 0.4 g / L or more when the measured value is out of the range of the set value, and the concentration is 0.4 g / L when the measured value is in the range of the set value. The concentration is adjusted to less than 4 g / L, or the concentration of glutamine in the culture tank is 1.5 mM or more when the measured value is out of the range of the set value, and the measured value is The culture method according to claim 1 or 2, wherein the concentration is adjusted to less than 1.5 mM when the concentration is within the range of the set value.   The concentration of lactic acid in the culture tank is 1.0 g / L or less when the measured value is out of the range of the set value, and 1. when the measured value is in the range of the set value. The concentration is adjusted to a concentration exceeding 0 g / L, or the concentration of ammonia in the culture tank is 2.0 mM or less when the measured value is out of the range of the set value. The culture method according to claim 1 or 2, wherein the concentration is adjusted to exceed 2.0 mM when the concentration is within the range of the set value.   The concentration of glucose in the culture tank is 1.5 g / L or more when the measured value is out of the range of the set value, and 1. when the measured value is in the range of the set value. When the concentration is adjusted to less than 5 g / L and the concentration of glutamine in the culture tank is outside the range of the set value, the concentration is 1.5 mM or more, and the measured value is the set value. Is adjusted to a concentration of less than 1.5 mM when the concentration is within the range of, or 2.0 g / l when the measured value is out of the range of the set value. L or less, when the measured value is within the range of the set value, the concentration is adjusted to more than 2.0 g / L, or the concentration of ammonia in the culture tank, Set value The culture according to claim 1 or 2, wherein the concentration is adjusted to 2.0 mM or less when out of the range, and to a concentration exceeding 2.0 mM when the measured value is within the set value range. Method.   The concentration of glutamine in the culture tank is 1.0 mM or more when the measured value is outside the range of the set value, and less than 1.0 mM when the measured value is within the range of the set value. And the concentration of lactic acid in the culture tank is 2.0 g / L or less when the measured value is out of the range of the set value, and the measured value is in the range of the set value. Is adjusted to a concentration exceeding 2.0 g / L when the concentration is within the range, or the concentration of ammonia in the culture tank is 1.5 mM or less when the measured value is outside the range of the set value. The culture method according to claim 1 or 2, wherein the concentration is adjusted to a concentration exceeding 1.5 mM when the measured value is within the range of the set value.   1. The concentration of lactic acid in the culture tank is 2.0 g / L or less when the measured value is out of the range of the set value, and 2. when the measured value is within the range of the set value. When the concentration is adjusted to more than 0 g / L, and the concentration of ammonia in the culture tank is outside the range of the set value, the concentration is 2.0 mM or less, and the measured value is the set value. The culture method according to claim 1 or 2, wherein the concentration is adjusted to more than 2.0 mM when the concentration is within the range of (3). A culture apparatus that continuously supplies a medium to a culture tank, and continuously cultures cells while continuously discharging the medium,
A culture tank for culturing cells,
A supply pump for supplying a culture medium to the culture tank,
A temperature control device capable of cooling the medium supplied to the culture tank,
An analyzer for measuring a state quantity having a correlation with the growth of the cell during the culture of the cell,
A controller that controls a supply flow rate of the culture medium based on the measured value of the state quantity.

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