JP2020124169A - Culture apparatus and culture method - Google Patents

Abstract

To provide a culture apparatus and a culture method, which are capable of performing a plurality of culture processes with different amounts of culture solutions and different culture techniques in a single culture tank in stages.SOLUTION: A culture apparatus 100 comprises a culture tank 1 for culturing cells in a culture solution, a stirrer 2 for stirring the culture solution, and a replenishing device (5, 7) for replenishing the culture tank 1 with a fresh medium. A lower space 110 in the culture tank 1 has a shape with a narrower inner width than an upper space 120, projecting downward. The culture method comprises steps of statically culturing cells in the culture solution in the lower space 110 in the culture tank 1, replenishing the culture tank 1 with the fresh medium after the static culture, and culturing the cells under stirring in the culture solution increased by supplementing the fresh medium.SELECTED DRAWING: Figure 1

Description

The present invention relates to a culture device and a culture method suitable for cell expansion culture.

Biopharmaceuticals including antibody drugs are produced by culturing cells and separating and purifying substances produced by the cells from contaminants. The cells producing the target substance are subjected to expansion culture until the number of cells suitable for producing the substance is reached, and then subjected to production culture using a large-scale culture solution. Generally, floating cell lines using CHO cells and the like are used for the production of antibody drugs and the like.

FIG. 17: is a figure explaining the flow of the conventional expansion culture.
As shown in FIG. 17, conventional general expansion culture is performed by stepwise increasing the volume of the culture solution from a small scale of several mL to a large scale of several thousand L. The cells proliferate in the culture medium to produce the target substance, and finally, the target substance is mass-produced in the culture medium expanded to several thousand L.

In ordinary batch culture, when cells grow and the cell density in the culture medium increases, the cells are likely to die due to nutrient depletion and accumulation of waste products secreted by the cells. Continuing batch culture balances the increase in cell number due to proliferation with the decrease in cell number due to death, which causes the cell density in the culture medium to reach a peak and allows the cells to grow to a number suitable for the production of the substance. Becomes difficult.

On the other hand, according to the expansion culture in which the culture scale is expanded stepwise, nutrients are supplied and secreted in order to replenish the culture medium in which the cells are cultured with fresh medium at each scale-up. Waste is diluted. In the expansion culture, since the culture scale is expanded in a state where the cells easily grow, the cell density in the culture solution is unlikely to reach the top, and a large number of cells suitable for the production of the substance can be obtained.

As shown in FIG. 17, a general substance production process includes a step A in which a culturing operation is manually performed in a safety cabinet, a step B in which a culturing operation is performed semi-automatically using a bioreactor, and a purpose for cells. And the stage C of the production culture for producing the substance. In general, scale-up is performed a plurality of times in each of the manual stage A and the semi-automated stage B.

The magnification for expanding the culture scale is generally a constant magnification through a plurality of scale-ups. The magnification for enlarging the culture scale is usually set to about 5 to 10 times. If the cell density is lowered too much by supplementing the fresh medium, the culture efficiency after scale-up will be deteriorated, so that a multiplication factor that maintains the cell density above a certain level is set.

As shown in FIG. 17, small-scale static culture using a culture container is performed at the beginning of the expansion culture in which the amount of the culture solution is about several mL to several hundred mL. As the culture container, in addition to the flask 53 shown in the figure, a dish, a bottle, a well plate, etc. are also used. The seed cells prepared in the vial 52 and the like are transferred and subcultured to culture vessels of various sizes and various shapes. When the amount of culture solution reaches several hundred mL, stirring culture or shaking culture using the spinner flask 54 may be performed.

Generally, the initial expansion culture using the flask 53 and the spinner flask 54 is performed in an incubator controlled to a constant culture temperature and a constant gas concentration. A culture container using a gas-exchangeable cap, a silicon stopper, etc. is allowed to stand still in an incubator in which the atmosphere inside the chamber is controlled to be constant to adjust the culture environment in the container. When expanding the culture scale, the culture medium is manually transferred in a safety cabinet.

In addition, as shown in FIG. 17, large-scale agitation culture using the bioreactor 55 is performed in the latter stage of the expansion culture when the volume of the culture solution reaches about several liters. Bioreactors 55 having various capacities are prepared, and the bioreactors 55 are connected to each other by a transportation system including steel pipes and tubes.

Generally, the latter-stage expansion culture using the bioreactor 55 is performed in a culture environment in which the culture temperature and the gas concentration are variably controlled. The culture fluid in the bioreactor 55 is maintained at a culture temperature of around 37° C. under forced ventilation, the dissolved oxygen concentration and the carbon dioxide concentration are controlled. When enlarging the culture scale, automatic transfer of the culture solution is performed aseptically by a transportation system including steel pipes and tubes.

Heretofore, with regard to expansion culture in which the culture scale is expanded stepwise, measures against problems associated with transfer of culture medium have been studied. At the time of expansion culture, it is necessary to manually transfer the culture solution, which causes a problem of work complexity. In addition, since shearing force, pressure, etc. are applied to the cells during the transfer of the culture solution, there is a problem that the cells are damaged. Further, since work is performed in a semi-open space such as a safety cabinet, there is a problem of contamination. In order to solve these problems, there has been proposed a technique that makes it unnecessary to transfer the culture solution.

Patent Document 1 describes a cell culture method in which the bottom surface of a container made of a flexible material is partially raised to be partitioned into a plurality of compartments, cells are cultured in each compartment, and then the compartments are dissolved. In Patent Document 1, the transition from the curing culture to the expansion culture is realized by eliminating the bulge generated in the container made of a flexible material.

JP, 2016-140295, A

During the expansion culture of cells, as described in Patent Document 1, a problem regarding the complexity of the work of transferring the culture solution, a problem regarding the handling of the culture solution and cells that do not damage the cells, and the transfer of the culture solution In addition to the problems related to contamination such as time, there are problems related to reproducibility of expansion culture and problems related to equipment used for expansion culture.

For example, in the initial expansion culture that requires manual work, the number of seeded cells and the number of transferred cells vary depending on the work due to differences in operator's procedures. Therefore, there is a problem that variations occur in the production amount of the target substance, the purity of the target substance, the quality of the product such as the non-denaturing degree of protein. Further, since manual work is required, incidental equipment such as a safety cabinet is required, which causes a problem that the culture facility for containment becomes large.

Further, in the latter-stage expansion culture using a bioreactor, an individual bioreactor is required for each culture scale. Therefore, there is a problem that a place for installing a large number of bioreactors is required. When connecting the bioreactors to each other with steel pipes, tubes, etc., a space for laying these pipes and piping work are required, and there is a risk that cells are damaged or cells are killed in the pipes.

Generally, when carrying out expansion culture with floating cells as the culture target and a large scale as the final target, static culture with less mechanical stress is performed at the beginning of expansion culture, and the latter stage of expansion culture where the culture scale has expanded to some extent. In addition, it is preferable to perform stirring culture which is advantageous for gas exchange efficiency and substance diffusion. Therefore, during the expansion culture, not only the transfer of the culture solution but also the switching of the culture method is desired. There is a demand for a technique capable of changing the amount of culture solution and the culture method while maintaining a high specific cell growth rate and viability and maintaining good culture efficiency.

Therefore, an object of the present invention is to provide a culturing apparatus and a culturing method capable of stepwise performing a plurality of culturing steps with different amounts of culturing liquid and culturing methods in one culturing tank.

As a result of diligent research, the present inventors have optimized the shape and structure of the inner space of the culture tank to perform a plurality of culture steps with different culture liquid amounts and different culture methods, and to increase the specific growth rate and viability of cells. The present invention has been completed by finding that it can be carried out stepwise in one culture tank without significantly reducing the temperature.

That is, the culture device according to the present invention to solve the above problems, a culture tank for culturing cells in a culture solution, a stirrer for stirring the culture solution, and a fresh medium in the culture tank. The lower space in the culture tank has a narrower inner width than the upper space and has a shape protruding downward.

Further, the culture method according to the present invention, statically culturing cells in a culture medium in the lower space of the culture tank, after the static culture, supplement the fresh medium in the culture tank, the fresh medium of The cells are cultivated with stirring in the culture medium increased by supplementation.

The culturing apparatus and culturing method according to the present invention can perform a plurality of culturing steps in which the amount of culturing liquid and the culturing method are different from each other in a single culturing tank.

It is a figure which shows typically an example of the culture apparatus which concerns on embodiment of this invention. It is a figure explaining the shape and structure of the internal space of the culture tank with which a culture device is equipped. It is a figure which shows the relationship between the liquid surface area-volume ratio of a culture solution, and the survival rate of a cell. It is a figure which shows the relationship between the liquid surface area-volume ratio of a culture solution, and the amount of cell growth. It is a figure which shows the example of a shape of the lower space of the culture tank with which a culture device is equipped. It is a figure which shows the example of a shape of the lower space of the culture tank with which a culture device is equipped. It is a figure which shows the example of a shape of the lower space of the culture tank with which a culture device is equipped. It is a figure which shows the example of a shape of the lower space of the culture tank with which a culture device is equipped. It is a figure which shows the example of a shape of the lower space of the culture tank with which a culture device is equipped. It is a figure which shows the example of a shape of the lower space of the culture tank with which a culture device is equipped. It is a figure which shows the example of a shape of the lower space of the culture tank with which a culture device is equipped. It is a figure which shows the constructional example of the culture tank with which a culture device is equipped. It is a figure which shows the constructional example of the culture tank with which a culture device is equipped. It is a flowchart which shows the culture method which concerns on embodiment of this invention. It is a figure which shows typically an example of the culture apparatus which concerns on embodiment of this invention. It is a figure explaining the shape and structure of the internal space of the culture tank with which a culture device is equipped. It is a figure explaining the flow of the conventional expansion culture. It is a figure which shows the result of having performed the expansion culture in a single culture tank.

<Culture device (batch culture type)>
First, a culture device according to an embodiment of the present invention will be described with reference to the drawings. In addition, in the following respective drawings, the same components are denoted by the same reference numerals, and a duplicate description will be omitted.

FIG. 1 is a diagram schematically showing an example of a culture device according to an embodiment of the present invention.
As shown in FIG. 1, a culture device 100 according to the present embodiment includes a culture tank 1, an agitator 2, an air diffuser 3, a temperature control device 4, a culture medium tank 5, a valve 6, and a supply pump 7. The analyzer 8, the controller 9, the recovery tank 10, the supply pipe 20, and the discharge pipe 21 are provided.

The culture device 100 is a device for expanding and culturing cells, and has a function of replenishing the culture tank 1 in the expansion culture with a fresh medium (culture solution) to expand the culture scale. In the culturing apparatus 100, the culture scale is expanded stepwise by supplementing the culture tank 1 with a fresh medium, and a multi-step culturing process is performed. The culturing apparatus 100 shown in FIG. 1 is configured such that each culturing process is performed in a batch culturing mode in which a medium is not supplied during culturing.

In the culture device 100, the culture system can be switched from static culture to stirring culture while the culture scale is being expanded stepwise. Therefore, batch culture type expansion culture with a large scale as the final target can be performed in a single culture tank 1. As the culture tank 1, a culture vessel having a proper shape and structure of the internal space is used in order to improve the efficiency of such expansion culture.

The cells to be cultured in the culture device 100 are not particularly limited, but cells acclimated to floating cells and cells having low adhesiveness are preferably used. The cells to be cultured include, for example, various monoclonal antibodies such as human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies, various physiologically active substances, and various other useful raw materials such as pharmaceutical raw materials, chemical raw materials, and food raw materials. Cells that produce the useful substances of are preferred.

Specific examples of the cells to be cultured include animal cells such as Chinese hamster ovary cells (CHO cells), baby hamster kidney cells (BHK cells), human fetal kidney cells (HEK cells), mouse myeloma cells, and other blood cells. Can be mentioned. However, the cells to be cultured are not limited to animal cells, and may be insect cells, plant cells, microalgae, cyanobacteria, bacteria, yeasts, fungi, algae, yeasts and the like.

The culture tank 1 is a tank for culturing cells in a culture solution, and is a closed reactor in which a space for containing the culture solution is sealed. The culture tank 1 can be formed of an appropriate material such as stainless steel, aluminum alloy, plastic, or glass. Alternatively, the culture tank 1 can be provided as a single-use type flexible culture bag. The flexible culture bag can be formed using a multilayer film made of a resin such as ethylene vinyl acetate, ethylene vinyl alcohol, low density polyethylene, or polyamide.

As shown in FIG. 1, the culture tank 1 includes a stirrer 2 for stirring the culture solution, and an air diffuser 3 for aerating the culture solution with air, oxygen, nitrogen, carbon dioxide, etc. Be prepared. As the air diffuser 3, various devices such as a ring sparger, a micro sparger, a sintered metal sparger, a membrane sparger, and an air diffuser can be used by connecting them to the gas supply device. In addition to the air diffuser 3 for aerating the culture solution, the culture tank 1 can also include an air diffuser (not shown) for aerating a predetermined gas in the gas phase portion.

Further, the culture tank 1 is provided with a temperature adjusting device 4 for adjusting the temperature in the tank around the culture tank 1. As the temperature adjusting device 4, for example, an appropriate device such as a water jacket, an air jacket, or an electric heater can be used.

The culture tank 1 can be equipped with a sensor (not shown) in order to measure the culture conditions in the tank and the culture state of cells online. As the sensor, for example, a temperature sensor such as a thermocouple, a dissolved oxygen sensor, a carbon dioxide sensor, a pH sensor, a cell counter using a capacitance meter or an absorptiometer, and a liquid level sensor can be provided. The data measured by the sensor can be output to the analyzer 8. Further, the culture tank 1 can be provided with a sampling port used for collecting the culture solution in order to measure the culture conditions in the tank and the culture state of the cells off-line.

As shown in FIG. 1, the culture tank 1 is provided with a replenishing device (5, 7) for replenishing the culture medium with fresh medium. A supply pipe 20 for supplying a fresh medium into the culture tank 1 is connected to the culture tank 1. At the other end of the supply pipe 20, a culture medium tank 5 which constitutes a replenishing device is connected. Further, the supply pipe 20 is provided with a supply pump 7 which constitutes a replenishing device.

The medium tank 5 aseptically stores the prepared sterilized fresh medium until the time of supplementing the fresh medium for expanding the culture scale. The culture device 100 may include a plurality of medium tanks 5. Each medium tank 5 is provided with a valve 6 that opens and closes the conduit of the supply pipe 20.

The supply pump 7 is provided to supply the fresh medium from the medium tank 5 to the culture tank 1. When enlarging the culture scale, a part of the plurality of valves 6 may be opened to replenish the culture tank 1 with fresh medium. By sequentially using only some of the medium tanks 5, it is possible to minimize the risk of contamination associated with supplementation of fresh medium.

The analyzer 8 is provided to analyze the state of cells being cultured in the culture tank 1 and the state of the culture solution in the culture tank 1. Items to be analyzed include the amount of state having a correlation with cell proliferation, the concentration of culture solution components, osmotic pressure, and the like. The growth state of cells can be determined by analyzing the amount of state having a correlation with the growth of cells and the concentration of the culture solution components. That is, it is possible to judge the progress of the culturing process, whether the cells have sufficiently grown in a certain culture scale.

State quantities that correlate with cell growth include cell number density, specific growth rate, survival rate, metabolic reaction rate of cells (metabolic production rate of target substance, metabolic consumption rate of nutrients, metabolic secretion rate of waste products, etc.) , Cell cumulative metabolism (cumulative metabolic production of target substance, cumulative metabolic consumption of nutrients, cumulative metabolic secretion of waste products, etc.), cell cycle (proportion of cells in G2 phase and M phase in cell population) Etc. Moreover, examples of the concentration of the culture solution components include concentrations of glucose, glutamine, lactic acid, ammonia, amino acids, vitamins, metabolites, growth factors, serum components and the like.

The cell number density, specific proliferation rate, and survival rate among the state quantities that correlate with cell proliferation can be determined by measuring the cell number. The number of cells can be measured by microscopic observation using a hemocytometer, dry weight method, turbidity method, capacitance method, NAD measurement for quantifying oxidized NAD ⁺ or reduced NADH, and various types of flow cytometry, etc. It can be measured using a measuring method. Moreover, the number of living cells and the number of dead cells can be counted by utilizing the discrimination by the trypan blue staining method. The specific growth rate can be determined by measuring the change over time in the number of viable cells.

Of the state quantities that correlate with cell growth, the metabolic reaction rate of cells (metabolic production rate of target substances, metabolic consumption rate of nutrients, metabolic secretion rate of waste products, etc.) and cumulative metabolic rate of cells (purpose The cumulative metabolic production amount of a substance, the cumulative metabolic consumption amount of nutrients, the cumulative metabolic secretion amount of waste products, etc.) can be obtained by the following method by conducting an actual culture test in advance.

The metabolic production rate of the target substance can be obtained by measuring the time-dependent change in the concentration of the target substance produced and converting it into the production amount per unit time per unit number of cells. When the target substance is a protein, for example, the concentration of the target substance can be quantified by an ELISA (Enzyme-Linked ImmunoSorbent Assay) method. Using an antibody that causes an antigen-antibody reaction with the protein to be measured, fluorescence analysis of the label, enzyme activity analysis, etc. 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 metabolic consumption rate of nutrients and the metabolic secretion rate of waste products can be determined by measuring the change over time in the concentration in the culture tank and converting it into the amount of change per unit time and the number of cells. The concentration of nutrients and the concentration of waste products can be measured by High Performance Liquid Chromatography (HPLC), Liquid Chromatography-Mass spectrometry (LC/MS), Liquid chromatography tandem mass spectrometry (LC/MS-MS). The cells can be quantified by subjecting the culture medium to gas chromatography mass spectrometry (GC/MS), gas chromatography tandem mass spectrometry (GC/MS-MS) and the like.

The cumulative metabolic production amount of the target substance, the cumulative metabolic consumption amount of nutrients, and the cumulative metabolic secretion amount of waste products can be obtained by a method of integrating the obtained metabolic rate or a method of actually measuring the cumulative amount. The metabolic reaction rate of cells (the metabolic production rate of a target substance, the metabolic consumption rate of nutrients, the metabolic secretion rate of waste products) can also be determined by conducting an actual culture test in advance and analyzing the intracellular metabolic flux.

Further, among the state quantities having a correlation with the proliferation of cells, the cell cycle can be obtained by performing flow cytometry using the discrimination by Propidium Iodide (PI) staining method.

While cells in G2 phase and M phase are about to start cell division, cells in G1 phase and G0 phase have time until cell division, and G0 phase has an indefinite length. A high proportion of cells in the G2 phase or M phase of the cell cycle is a state in which most of the cultured cells are about to start cell division, and it can be said that the cells are prone to proliferate. On the other hand, a state in which the proportion of cells in the G1 phase or G0 phase of the cell cycle is high is a state in which most of the cultured cells are in the interphase, and it can be said that the growth of the cells is suppressed.

By performing fluorescence detection by flow cytometry and calculating the integrated area of each curve on the frequency distribution, the number and ratio of cells in the G2 phase or the M phase can be known. When the number and ratio of such cells are used as an index for judging the progress of the culturing process, the culture scale can be expanded efficiently in time while giving priority to the state in which the cell population easily grows.

The control device 9 is provided to control the cell culture conditions and the operation of the culture device 100. The control device 9 operates, stops, outputs the rotation of the agitator 2, operates and stops the air diffuser 3, operates the ventilation amount, operates and stops the temperature control device 4, adjusts the temperature, opens and closes the valve 6, and supplies a pump. The operation, stop, discharge output, etc. of No. 7 are controlled by an appropriate control method such as PID control, proportional control, ON/OFF control and the like.

Data necessary for controlling the cell culture conditions can be preset and stored in the control device 9 in accordance with the type of cell, the purpose of expansion culture, and the like. Further, in order to control the operation of the culture device 100, a predetermined culture schedule can be set. The culture schedule should be set according to the target culture amount of cells and the target production amount of the target substance so that the size of the culture can be increased a specified number of times or the culture method can be switched at a specified time during the expansion culture. You can

The control device 9 controls each device to perform an operation of gradually increasing the amount of the culture solution in the culture tank 1 and switching the culture system from static culture to stirring culture during the process. The control device 9 receives the analysis data acquired by the analysis device 8, determines based on the analysis data whether or not the culture process of each culture scale is completed, and according to the determination result, the culture scale by supplementing the fresh medium. And the switching from static culture to agitation culture by activating the agitator 2.

The recovery tank 10 is provided to recover the cultured cells and the substances produced by the cells. The culture tank 1 is connected to a discharge pipe 21 for discharging cells cultured in the tank and substances produced by the cells. The recovery tank 10 is connected to the other end of the discharge pipe 21. The cells recovered in the recovery tank 10 can be used for larger-scale production culture. Alternatively, the target substance can be separated and purified from the contaminants by subjecting it to a separation and purification treatment together with the substance produced by the cells.

Here, the shape and structure of the internal space of the culture tank 1 will be specifically described.

FIG. 2 is a diagram for explaining the shape and structure of the internal space of the culture tank provided in the culture device.
As shown in FIG. 2, the culture tank 1 included in the culture apparatus 100 has an inner width that is not uniform in the height direction, and is relatively deep inside the tank and a lower space 110 having a relatively narrow inner width. And an upper space 120 having a wide inner width. In the culture tank 1 shown in FIG. 2, the lower space 110 is in the shape of a cone protruding downward from the culture tank 1. The upper space 120 has an inner width that is uniform in the height direction, but the lower space 110 has an inner width that is narrower than the lower end of the upper space 120.

The lower space 110 of the culture tank 1 is used for static culture. In the culturing step of statically culturing cells, the culture tank 1 is filled with a culture solution until the liquid surface reaches a predetermined height in the lower space 110. The height of the liquid surface in the lower space 110 can be rounded up step by step by supplementing with fresh medium according to a predetermined culture schedule, as indicated by lines I to IV in FIG.

The upper space 120 of the culture tank 1 is used for stirring culture. In the culture step of culturing cells with stirring, the culture solution is put into the culture tank 1 until the liquid surface reaches a predetermined height in the upper space 120. The height of the liquid surface in the upper space 120 can be rounded up step by step by supplementing with fresh medium according to a predetermined culture schedule, as indicated by lines V to VI in FIG.

As shown in FIG. 2, the culture tank 1 can be provided with a mechanical stirring type stirrer 2. The mechanical stirring type stirrer 2 includes a stirring blade 2a for stirring the culture solution, a rotating shaft 2b for rotating the stirring blade 2a, and a motor 2c for rotating the rotating shaft 2b. The stirring blade 2a may have any suitable shape such as a turbine blade, a paddle blade, a propeller blade, or a lattice blade. Further, the number of blade stages, blade diameter, blade height, etc. of the stirrer 2 can be set to appropriate conditions depending on the culture scale and the like as long as the stirrer 2a fits in the upper space 120.

<Culture experiment>
Here, the results of confirming the relationship between the shape and structure of the internal space of the culture tank 1 and the cell culture efficiency by a culture experiment are shown.

In culturing cells, temperature, dissolved oxygen concentration, other gas concentration, etc. are important as environmental factors. In static culture, gas exchange is carried out through the interface between the culture solution and the gas phase, so that the cell culture efficiency is affected by the area S of the interface between the culture solution and the gas phase and the volume V of the culture solution. It is thought to be. In order to examine the validity of such prediction and the appropriate range of the area S/volume V, the cells are statically cultured under various culture conditions, and the area S/volume V, the cell viability, and the cell proliferation amount are measured. I investigated the relationship with.

(experimental method)
As a test cell, a floating cell line CHO cell was used. As the medium, Dulbecco's Modified Eagle's Medium (DMEM medium) was used. As the culture vessel, two types of flasks (T75 flask, T225 flask) having different surface areas on the culture surface were used.

As shown in Table 1, a liquid medium was placed in each of the two types of flasks such that the liquid surface area S of the culture solution and the culture solution amount V were various conditions. In addition, the test cells were seeded in the medium at a cell density of 0.2×10 ⁶ cells/mL. Then, the gas composition of the gas phase part was 5% CO _{2 in the} atmosphere, the temperature of the culture solution was 37° C., and the dissolved oxygen concentration of the culture solution was adjusted to 40% of the saturated dissolved oxygen concentration, and the culture conditions were adjusted to 2 The culture was allowed to stand for a day.

After static culture, the number of live cells and dead cells was counted using a live-dead cell autoanalyzer Vi-Cell (manufactured by Beckman Coulter, Inc.), and the ratio of live cells in all cells (cell viability) and culture were performed. The total number of living cells (proliferation amount of cells) after that was determined.

Table 1 shows the type of the culture vessel used in the culture experiment, the liquid surface area S of the culture solution (area S of the interface between the culture solution and the gas phase), the culture solution volume V (volume V of the culture solution), the liquid surface area. -Volume ratio S/V (ratio S/V between the area S of the interface between the culture solution and the gas phase and the volume V of the culture solution) is shown.

(Experimental result 1)
FIG. 3 is a diagram showing the relationship between the liquid surface area-volume ratio of the culture solution and the cell viability.
In FIG. 3, the horizontal axis represents the liquid surface area-volume ratio S/V [cm ⁻¹ ] of the culture solution in the culture experiment, and the vertical axis represents the cell viability [%] confirmed after the culture experiment. .. The plot of ● is the result of the culture experiment using the T75 flask, and the plot of ▲ is the result of the culture experiment using the T225 flask.

As shown in FIG. 3, both in the case of using the T75 flask and the case of using the T225 flask, it was confirmed that the smaller the S/V, the higher the cell viability. Regardless of the type of culture vessel, the survival rate is remarkably high in the case of S/V≦8 [cm ⁻¹ ] and exceeds 90% in the case of S/V≦4 [cm ⁻¹ ] A high survival rate was obtained.

(Experimental result 2)
FIG. 4 is a diagram showing the relationship between the liquid surface area-volume ratio of the culture solution and the cell growth amount.
In FIG. 4, the horizontal axis represents the liquid surface area-volume ratio S/V [cm ⁻¹ ] of the culture solution in the culture experiment, and the vertical axis represents the relative value of the number of viable cells confirmed after the culture experiment. The plot of ● is the result of the culture experiment using the T75 flask, and the plot of ▲ is the result of the culture experiment using the T225 flask.

As shown in FIG. 4, both in the case of using the T75 flask and in the case of using the T225 flask, it was confirmed that the smaller the S/V, the larger the cell proliferation amount. Regardless of the type of culture vessel, when S/V≦8 [cm ⁻¹ ], the specific growth rate increases and the number of viable cells tends to increase, and when S/V≦4 [cm ⁻¹ ] , The maximum number of viable cells was obtained.

From these experimental results, it is understood that in static culture, the culture result is determined by S/V regardless of the size and shape of the culture container. S/V preferably satisfies S/V≦8 [cm ⁻¹ ] and S/V≦4 [from the viewpoint of maintaining high specific growth rate and viability of cells and achieving good culture efficiency. It is more preferable to satisfy [cm ⁻¹ ].

It is generally known that cell growth is inhibited by high concentration of carbon dioxide. When normal stationary culture is continued, the carbon dioxide concentration of the culture solution rises due to lysis from the gas phase and respiration of cells, and thus the growth of cells is more likely to be inhibited as the culture time elapses. On the other hand, when the S/V is small, it takes time to dissolve carbon dioxide from the gas phase part and to diffuse carbon dioxide throughout the culture solution. Therefore, the specific growth rate of cells is increased, and the number of viable cells obtained at the peak of the logarithmic phase is increased, so that the culture efficiency of static culture is improved.

Therefore, in the lower space 110 of the culture tank 1, the volume of the culture solution contained in the lower space 110 is V [cm ³ ] and the area of the interface between the culture solution contained in the lower space 110 and the gas phase is S [cm ² ]. Then, it is preferable that the liquid surface area-volume ratio S/V has a shape that satisfies the relationship of the following mathematical expression (I).
S/V≦8 [cm ⁻¹ ] (I)

When the lower space 110 of the culture tank 1 has a shape satisfying the relationship of the formula (I), the specific growth rate and survival rate of cells can be increased regardless of the size and shape of the culture tank 1. Therefore, the culture efficiency of static culture can be improved for any culture scale that is gradually increased in size. In addition, the survival rate of cells is increased, and impurities are less likely to be generated in the culture broth, so that the efficiency of separation and purification of substances produced by cells and the quality of products can be improved. From the viewpoint of further improving the culture efficiency, S/V≦4 [cm ⁻¹ ] is more preferable.

<Example of culture tank shape>
The lower space 110 of the culture tank 1 has an inner width narrower than that of the upper space 120 and can be provided in any shape as long as it has a shape protruding downward of the culture tank 1. The lower space 110 of the culture tank 1 preferably has a shape that satisfies the relationship of the mathematical formula (I), but may have a shape that satisfies the relationship of the mathematical formula (I) only in a state where a predetermined amount of the culture solution is placed. However, it may have a shape that satisfies the relationship of the formula (I) in all the states in which an arbitrary amount of the culture solution is added.

The lower space 110 of the culture tank 1 can be provided, for example, in such a shape that the inner width becomes narrower toward the lower side of the culture tank 1. As such a shape, for example, a stepped shape in which the inner width is gradually narrowed toward the lower side of the culture tank 1, or the inner width is continuously narrowed toward the lower side of the culture tank 1. A taper shape etc. are mentioned.

5, 6 and 7 are views showing examples of the shape of the lower space of the culture tank provided in the culture device.
As shown in FIGS. 5, 6 and 7, the lower space 110 of the culture tank 1 can be provided in a cone shape. FIG. 5 is a diagram illustrating an inverted conical cone shape, FIG. 6 is a diagram illustrating an inverted quadrangular pyramid cone shape, and FIG. 7 is an inverted triangular pyramid cone shape. It is a figure which illustrates. 5, FIG. 6 and FIG. 7, reference numeral 50 indicates the culture solution contained in the lower space 110, reference symbol S indicates the area of the interface between the culture solution 50 contained in the lower space 110 and the gas phase, and the reference numeral h indicates the height of the culture solution 50 placed in the lower space 110.

When the lower space 110 is provided in the shape of a cone, the volume V of the culture medium 50 contained in the lower space 110 is V=S·h/3, and the S/V of the culture medium 50 is 3/h. It is preferable that S/V≦8 [cm ⁻¹ ] is satisfied, and it is more preferable that S/V≦4 [cm ⁻¹ ] is satisfied. Therefore, when the lower space 110 is provided in a cone shape under this condition. It can be said that the height h of the culture medium 50 placed in the lower space 110 is preferably larger than 3/8 cm, and more preferably larger than 3/4 cm.

When the lower space 110 is provided in the shape of a cone, the lower space 110 of the culture tank 1 has a cross-sectional area s [cm ² ] at an arbitrary height and a lower area than the height, similarly to the culture solution 50. It can be said that the volume v [cm ³ ] preferably satisfies s/v≦8 [cm ⁻¹ ] and more preferably s/v≦4 [cm ⁻¹ ]. Therefore, when the lower space 110 is provided in a cone shape under this condition, it can be said that the total height of the lower space 110 is preferably larger than 3/8 cm, and more preferably larger than 3/4 cm.

FIG. 8 is a diagram showing an example of the shape of the lower space of the culture tank provided in the culture device.
As shown in FIG. 8, the lower space 110 of the culture tank 1 can be provided in a columnar shape. FIG. 8: is a figure which illustrates the rectangular parallelepiped columnar shape. In FIG. 8, reference numeral 50 represents the culture solution contained in the lower space 110, reference symbol S represents the area of the interface between the culture solution 50 contained in the lower space 110 and the gas phase, and reference symbol h represents the lower space 110. The height of the culture solution 50 placed in the table is shown.

When the lower space 110 is provided in a columnar shape, the volume V of the culture medium 50 contained in the lower space 110 is V=S·h, and the S/V of the culture medium 50 is 1/h. Since it is preferable that S/V≦8 [cm ⁻¹ ] is satisfied and it is more preferable that S/V≦4 [cm ⁻¹ ] is satisfied, the lower space 110 is provided in a columnar shape under this condition. It can be said that the height h of the culture solution 50 placed in the lower space 110 is preferably larger than 1/8 cm, and more preferably larger than 1/4 cm.

When the lower space 110 is provided in a columnar shape, the lower space 110 of the culture tank 1 has a cross-sectional area s [cm ² ] at an arbitrary height and lower than the height in the same manner as the culture medium 50. It can be said that the volume v [cm ³ ] preferably satisfies s/v≦8 [cm ⁻¹ ] and more preferably s/v≦4 [cm ⁻¹ ]. Therefore, when the lower space 110 is provided in the shape of a column under this condition, the total height of the lower space 110 is preferably larger than 1/8 cm, and more preferably larger than 1/4 cm.

FIG. 9 is a diagram showing an example of the shape of the lower space of the culture tank provided in the culture device.
As shown in FIG. 9, the lower space 110 of the culture tank 1 can be provided in a partially spherical shape. FIG. 9: is a figure which illustrates the spherical shape. In FIG. 9, reference numeral 50 indicates the culture solution 50 placed in the lower space 110, reference numeral S indicates the area of the interface between the culture solution 50 placed in the lower space 110 and the gas phase, and reference numeral r indicates the sphere. The radius indicates the radius, the symbol h indicates the height of the culture medium 50 contained in the lower space 110, and the symbol c indicates the radius of the interface between the culture medium 50 contained in the lower space 110 and the gas phase.

When the lower space 110 is provided in a spherical shape, the volume V of the culture solution 50 placed in the lower space 110 is V=π·h(3c ² +h ² )/6, and the culture solution 50 placed in the lower space 110 is 50. The area S of the interface between the and the gas phase is S=π·h(2r−h). It is preferable that S/V≦8 [cm ⁻¹ ] is satisfied, and it is more preferable that s/v≦4 [cm ⁻¹ ] is satisfied. Therefore, when the lower space 110 is provided in a spherical shape under this condition. The height h [cm] of the culture solution 50 placed in the lower space 110 is V [cm ³ ] the volume of the culture solution 50 placed in the lower space 110, and the volume of the culture solution 50 placed in the lower space 110 and the gas phase Where S[cm ² ] is the area of the interface, r[cm] is the radius of the spherical shape, and c[cm] is the radius of the interface between the culture solution 50 and gas phase contained in the lower space 110. In addition to the formula (I), it is preferable to satisfy the following formulas (II) and (III).
V=π·h(3c ² +h ² )/6 (II)
S=π·h(2r-h) (III)

In addition, when the lower space 110 of the culture tank 1 is provided in a pyramidal shape, in addition to the illustrated shape example, it may be another polygonal pyramid such as an inverted pentagonal pyramid, an inverted hexagonal pyramid, or an inverted elliptic cone. It can also be provided. Further, they can be provided in a polygonal pyramid shape with rounded corners or a polygonal pyramid shape with slightly curved sides, which is substantially equivalent to these shapes. Regarding these shapes, the height of the culture solution and the total height of the lower space 110 are preferably larger than 3/8 cm, and more preferably larger than 3/4 cm.

When the lower space 110 of the culture tank 1 is provided in a columnar shape, in addition to the illustrated shape example, other polygonal columnar shapes such as a cubic shape, a triangular columnar shape, a pentagonal columnar shape, and a hexagonal columnar shape, a columnar shape, an elliptic columnar shape. Etc. can also be provided. Further, it may be provided in a polygonal column with rounded corners or a polygonal column with slightly curved sides, which is substantially equivalent to these shapes. Regarding these shapes, the height of the culture solution and the total height of the lower space 110 are preferably larger than 1/8 cm, and more preferably larger than 1/4 cm.

10 and 11 are diagrams showing examples of the shape of the lower space of the culture tank provided in the culture device.
As shown in FIGS. 10 and 11, the lower space 110 of the culture tank 1 may be provided in other discontinuous shapes in addition to the illustrated pyramidal shape, columnar shape, and spherical shape. FIG. 8: is a figure which illustrates the discontinuous shape of a reverse truncated cone shape which notched a part of reverse cone. FIG. 8: is a figure which illustrates the multistage convex discontinuous shape which combined the rectangular parallelepiped from which size differs mutually.

In addition, the lower space 110 of the culture tank 1 has a spherical truncated cone shape in which a part of a sphere is cut out by two parallel surfaces, or an inverted truncated cone shape in which a part of an inverted polygonal cone, an inverted elliptical cone, etc. is cut out. It can also be provided in a discontinuous shape. Further, the lower space 110 of the culture tank 1 is a multi-step convex discontinuous shape that is a combination of polygonal columns, cylinders, elliptic columns, inverted polygonal truncated cones, inverted truncated cones, inverted elliptical truncated cones, spheres, etc. of different sizes. Can also be provided.

<Structure example of culture tank>
The culture tank 1 is of an upper mechanical agitation type in which the agitator 2 is arranged on the upper side in FIG. The mechanical stirrer 2 of the upper mechanical stirring type is installed such that all the stirring blades 2 a are located in the upper space 120 and the lowermost end of the stirring blade 2 a is located above the lower space 110. The rotary shaft 2b supports the stirring blade 2a from above the culture tank 1. The rotating shaft 2b is parallel to the central axis of the culture tank 1, hangs from the center of the top of the culture tank 1 to the center of the upper space 120, and fixes the stirring blade 2a to the lower part of the center of the upper space 120.

According to such an upper mechanical agitation type structure, it becomes difficult for cells to adhere and aggregate to the agitating blade 2a and the rotating shaft 2b during static culture using the lower space 110. Therefore, it is possible to prevent a strong mechanical stress from being applied to the cells at the time of the transition to the stirring culture, and an inaccurate counting of the cell number.

However, the culture tank 1 may be applied with other stirring methods instead of the upper mechanical stirring method as shown in FIG. For example, in addition to a mechanical stirring type equipped with a rotary shaft and a motor, a magnetic stirring type in which a stirring blade is rotated by magnetic force may be used. Further, a shaking stirring type without an agitating blade or an air flow stirring type may be used.

FIG. 12 is a diagram showing a structural example of a culture tank provided in the culture device.
As shown in FIG. 12, the culture tank 1 may be a lower mechanical stirring type in which the stirrer 2 is arranged on the lower side. The lower mechanical stirring type stirrer 2 is installed such that all the stirring blades 2a are located in the upper space 120, and the lowermost end of the stirring blades 2a is located above the lower space 110. On the other hand, the rotating shaft 2b supports the stirring blade 2a from below the culture tank 1 which is eccentric from the central axis of the culture tank 1. The rotating shaft 2b is inclined with respect to the central axis of the culture tank 1, extends through the lower space 110 from the outside separated from the center of the bottom of the culture tank 1 to the central side of the upper space 120, and the stirring blade 2a. Is fixed to the lower part near the center of the upper space 120.

According to such a lower mechanical stirring type structure, even if the motor 2c is not fixed to the upper portion, it becomes difficult for cells to adhere and aggregate to the stirring blade 2a and the rotating shaft 2b during stationary culture using the lower space 110. .. Therefore, it is possible to prevent a strong mechanical stress from being applied to the cells at the time of the transition to the stirring culture, and an inaccurate counting of the cell number.

FIG. 13 is a diagram showing a structural example of a culture tank provided in the culture device.
The culture tank 1 may be of a shaking and stirring type without a stirring blade. A shaking and stirring type stirrer 2 shown in FIG. 13 includes a motor 2c for rotating a rotating shaft 2b, a shaking table 2d for supporting the culture tank 1, and a hub (rotating member) for driving the shaking motion of the shaking table 2d by the rotating motion. 2e, an eccentric pin (conversion member) 2f that converts the rotational movement of the hub 2e into a shaking movement, and a belt 2g (transmission member) that transmits the rotational movement of the rotating shaft 2b to the hub 2e. The hub 2e is located below the shaking table 2d and is rotatably supported by bearings (not shown). The shaking table 2d is supported in a shakeable state by a hub 2e and an eccentric pin 2f, and a support member (not shown) configured similarly to the hub 2e and the eccentric pin 2f.

The center axis of the culture tank 1 supported by the shaking table 2d is eccentric from the center axis of the hub (rotating member) 2e. The eccentric pin 2f is eccentrically provided on the upper surface of the hub 2e from the central axis of the hub 2e, and the tip of the eccentric pin 2f is rotatably connected to the shaking table 2d. A rotary shaft 2b connected to the hub 2e and the motor 2c is provided with a pulley, and a belt 2g is stretched around the pulley. When the motor 2c operates, the hub 2e rotates by the rotation of the rotating shaft 2b, and the eccentric pin 2f moves around the central axis of the hub 2e. The shaking table 2d can guide the trajectory by a guide (not shown) so that the eccentric pin 2f can perform a swinging shaking motion and a reciprocating shaking motion.

In such a shake stirring type structure, the center axis of the culture tank 1 and the rotation axis of the shaking table 2d do not coincide with each other, and the position and the direction are displaced, so that the culture tank 1 is eccentric, and Are mixed. Since it is not necessary to install a stirring blade or a rotary shaft in the culture tank 1, cells do not adhere or aggregate to the stirring blade or the rotary shaft. Therefore, it is possible to prevent a strong mechanical stress from being applied to the cells at the time of the transition to the stirring culture, and an inaccurate counting of the cell number. Further, when a single-use type culture bag is used as the culture tank 1, it is easy to attach/detach the agitator 2 and hermetically seal the culture tank 1.

The shaking and stirring type stirrer 2 shown in FIG. 13 is configured such that the culture tank 1 is shaken and stirred by using the hub 2e, the eccentric pin 2f, and the like, but the stirrer 2 is the shaking motion of the shaking table 2d. Other mechanisms such as a slider/crank mechanism and a cam mechanism, or a combination thereof may be used as a mechanism for converting the rotational movement of the rotating member that drives the shaft into a shaking movement. The shaking and stirring type stirrer 2 may be provided with a device for performing other shaking motions such as horizontal reciprocating motion, vertical reciprocating motion, two-dimensional or three-dimensional orbital motion, and seesaw type tilting motion. Good.

<Culturing method (batch culture method)>
Next, a culturing method according to an embodiment of the present invention will be described with reference to the drawings together with a process when the culturing apparatus 100 is used.

FIG. 14 is a flowchart showing the culture method according to the embodiment of the present invention.
FIG. 14 illustrates a flow of expanding and culturing cells that produce a target substance using the culturing apparatus 100. In the culturing apparatus 100, the amount of the culture solution in the culture tank 1 is expanded stepwise by supplementing the fresh medium, and a plurality of culturing steps having different culture solution amounts are performed stepwise. During the process, the culture system is switched from static culture to stirring culture, and static culture and stirring culture in a culture medium increased by supplementation with fresh medium are performed stepwise in a single culture tank 1.

First, the culture conditions necessary for culturing cells are set in the culture device 100 (step S10). In the culturing apparatus 100, the expansion culturing in which the culturing scale is expanded stepwise is carried out in accordance with a predetermined culturing schedule so that the size is increased a specified number of times and the culturing method is switched at a specified time. ..

The items of the culture conditions to be set include a culture schedule, for example, the target passage number of cells for static culture and agitation culture (the target value of the number of size-ups), and the culture of the culture tank 1 for each stepwise culture process. The liquid volume (culture scale) can be mentioned. Further, the culture temperature, the gas composition of the gas phase portion in the stationary culture, the rotation speed of the stirrer 2 in the stirring culture, the dissolved oxygen concentration, the pH and the like can be mentioned. In order to determine the progress of each culturing process, the target state amount of cells (target value such as cell number density) for each culturing process, the target culturing time for each culturing process, or the culture solution for each culturing process The target concentration of the component can be set.

Then, the cells to be expanded and cultured are seeded in the medium (step S20). Fresh medium is placed in the culture tank 1 until the liquid surface reaches a predetermined height in the lower space 110. Then, the air diffuser 3, the temperature controller 4, etc. are controlled so that the set culture conditions are achieved, and the cell suspension diluted to an appropriate cell number density is aseptically charged. It is preferable that the culture tank 1 is provided with an input port for seeding cells at a predetermined height in the lower space 110.

Then, the cells are statically cultured in the culture medium in the lower space 110 of the culture tank 1 (step S30). The static culture is performed while the agitator 2 is stopped and the culture scale is maintained such that the liquid surface has a predetermined height in the lower space 110.

During static culture, the gas concentration/gas composition of the gas phase portion in the culture tank 1 is controlled to be constant by aeration of air, oxygen, carbon dioxide, nitrogen or the like. Further, the temperature of the culture solution is controlled to be constant. According to such constant control, the cells are less likely to be disturbed, and the cells can be stably grown to a predetermined cell density. It is preferable that the gas concentration and the gas composition of the gas phase portion are the same as the atmosphere in the normally used incubator.

Then, during static culture, an index value indicating the growth state of cells is acquired (step S40). As an index value indicating the growth state of cells, a state quantity having a correlation with the growth of cells, for example, cell number density, specific growth rate, survival rate, metabolic reaction rate of cells, cumulative metabolic rate of cells, cell cycle, etc. , Can be obtained by actual measurement by the analyzer 8. The actual measurement may be performed online using a sensor, or may be performed offline by sampling the culture solution.

Alternatively, the culture time for each culture process may be acquired by a timer as an index value indicating the growth state of cells. Further, the concentration of the culture solution component for each culture step may be acquired by actual measurement by the analyzer 8. When the relationship with the growth state of cells has been confirmed in advance, not only the state quantity of cells, but also the lapse of the culture time after the start of the culture process on a certain culture scale, the change in the concentration of the culture solution components, Can be used as an indicator of the growth state of.

When the index value indicating the growth state of the cells is acquired during static culture, it is determined whether or not the statically cultured cells have reached the target growth state (step S50). The control device 9 receives the analysis data acquired by the analysis device 8, and presets the index value acquired by actual measurement for the state quantity of the cell, the culture time, or the concentration of the culture solution component, and is preset. Compare with the target value. The progress of the culturing process can be determined based on whether or not the index value acquired by actual measurement exceeds the target value.

If the result of determination is that the target growth state has not been reached (step S50; No), static culture of the same culture scale is continued in the lower space 110 of the culture tank 1 (step S30).

On the other hand, as a result of the determination, when the target proliferation state has been reached (step S50; Yes), it is determined whether or not the switching condition necessary for switching the culture method is satisfied (step S60). The controller 9 compares the actual passage number (number of times of size-up) in static culture with the target passage number defined as the culture schedule. The culture system can be switched depending on whether or not the parameter representing the actual passage number has reached the target passage number.

For example, when the height of the liquid surface in the lower space 110 is rounded up as indicated by lines I to IV in FIG. 2, the number of passages during static culture is four times. When the relationship with the passage number is confirmed in advance, the state amount of cells, or the concentration of the culture solution component, the index value obtained by actual measurement, and the preset target value in static culture , May be compared. It is also possible to determine the progress of subculture based on whether or not the index value acquired by actual measurement exceeds the target value in static culture, and switch the culture method.

If the result of determination is that the target passage number has not been reached (step S60; No), the supply pump 7 is activated, and the culture tank 1 is replenished with a predetermined amount of fresh medium from the medium tank 5 and the culture scale of the next stage is reached. The amount of culture solution is expanded to (step S70). At this time, the control device 9 adds a parameter representing the actual passage number. Then, the stationary culture of the expanded culture scale is continued in the lower space 110 of the culture tank 1 (step S30).

On the other hand, when the target passage number is reached as a result of the determination (step S60; Yes), the culture system is switched from static culture to stirring culture, the stirrer 2 is operated, and the lower space 110 and the upper space 120 of the culture tank 1 are activated. In step S80, the cells are cultivated with stirring in the culture medium increased by supplementing the fresh medium. The stirring culture is performed while stirring the culture solution by the stirring blade 2a located in the upper space 120 in the culture tank 1 and maintaining the culture scale such that the liquid surface has a predetermined height in the upper space 120.

During stirring culture, the culture state is monitored by a temperature sensor, a dissolved oxygen sensor, a carbon dioxide sensor, a pH sensor, or the like. The culture temperature is controlled within a predetermined range by the temperature controller 4. The dissolved oxygen concentration and carbon dioxide concentration of the culture solution are controlled within a predetermined range by aeration of air, oxygen, carbon dioxide, nitrogen, etc. by the air diffuser 2. Further, the pH of the culture solution is controlled by addition of an alkaline solution or aeration of carbon dioxide.

Subsequently, an index value indicating the growth state of the cells is acquired during the stirring culture (step S90). As the index value indicating the growth state of the cells, the state quantity of the cells, the culture time, or the concentration of the culture solution component can be acquired as in the case of the process in the static culture (see step S40).

When the index value indicating the growth state of the cells is obtained during the stirring culture, it is determined whether or not the cells in the stirring culture have reached the target growth state (step S100). The control device 9 receives the analysis data acquired by the analysis device 8, and presets the index value acquired by actual measurement for the state quantity of the cell, the culture time, or the concentration of the culture solution component, and is preset. The target value for each culture process is compared. The progress of the culturing process can be determined based on whether or not the index value obtained by actual measurement exceeds the target value for each culturing process.

If the result of determination is that the target growth state has not been reached (step S100; No), stirring culture at the same culture scale is continued in the lower space 110 and the upper space 120 of the culture tank 1 (step S80).

On the other hand, as a result of the determination, when the target proliferation state has been reached (step S100; Yes), it is determined whether or not the end condition necessary for ending the culture is satisfied (step S110). The controller 9 compares the actual passage number (number of times of size-up) in the stirring culture with the target passage number defined as the culture schedule. The continuation/termination of the expansion culture is determined depending on whether or not the parameter representing the actual passage number has reached the target passage number.

For example, when the height of the liquid surface in the upper space 120 is rounded up as shown by the lines V to VI in FIG. 2, the passage number during stirring culture is two times. If the relationship with the number of passages is confirmed in advance, the state amount of the cells, or, for the concentration of the culture solution components, the index value obtained by actual measurement, and the target value in the preset stirring culture, May be compared. It is also possible to judge the progress of the passage and determine whether to continue or end the expansion culture based on whether or not the index value obtained by the actual measurement exceeds the target value in the stirring culture.

As a result of the determination, if the target passage number has not been reached (step S110; No), the supply pump 7 is operated, and a predetermined amount of fresh medium is replenished from the medium tank 5 into the culture tank 1, and the next-stage culture scale is supplied. The amount of culture solution is expanded to (step S120). At this time, the control device 9 adds a parameter representing the actual passage number. Then, the stirring culture of the expanded culture scale is continued in the lower space 110 and the upper space 120 of the culture tank 1 (step S80).

On the other hand, if the result of determination is that the target passage number has been reached (step S110; Yes), the stirrer 2 is stopped and expansion culture is completed (step S130). After that, the process moves to a production culture process for mass-producing the target substance or a separation and purification process for collecting and purifying the substance produced by the cells.

According to the culture device 100 and the culture method described above, since the fresh medium can be replenished in the culture tank having a predetermined shape and structure, the culture solution volume is increased stepwise, and the culture method is static culture in the middle thereof. The expansion culture for switching from the culture culture to the stirring culture can be performed using the single culture tank 1. In general culture vessels and bioreactors, the bottom is often flat and the shape/structure is not optimized. Therefore, the lower space 110 of the culture tank 1 has a shape that satisfies the relationship of the formula (I), If the structure of an appropriate stirring system is used, the specific growth rate and survival rate of the cells become high. Therefore, it is possible to carry out a plurality of culturing steps in which the amount of culturing solution and the culturing method are different from each other stepwise in one culturing tank while maintaining good culturing efficiency.

In particular, according to the culture apparatus 100 and the culture method described above, since it is possible to eliminate the need for transferring the culture solution, there is a problem regarding the complexity of the transfer operation of the culture solution and a problem regarding the handling of the culture solution and the cells. Therefore, it is possible to solve the problem of contamination when transferring the culture solution. According to the culturing apparatus 100, it is possible to automate all the culturing steps in a completely closed system, and it becomes difficult for manual work to intervene. Therefore, the reproducibility of culturing and the production amount/quality of a target substance are improved. be able to. Further, since it is not necessary to prepare an individual bioreactor for each culture scale, it is possible to downsize the culture device and the culture facility. Further, since the batch culture type expansion culture is performed, the culture environment can be easily stabilized as compared with the perfusion culture type.

<Culture device (perfusion culture type)>
Next, another embodiment of the present invention having a different culture method will be described with reference to the drawings.

FIG. 15: is a figure which shows typically an example of the culture apparatus which concerns on embodiment of this invention.
As shown in FIG. 15, the culture device 200 according to the present embodiment includes a culture tank 1, a stirrer 2, an air diffuser 3, a temperature control device 4, a culture medium tank 5, a valve 6, and a supply pump 7. , Analysis device 8, control device 9, recovery tank 10, cell separation device 11, extraction pump 12, suction pump 13, supply pipe 20, extraction pipe 22, return pipe 23, and collection pipe 24 is provided.

The culturing device 200 is a device for expanding and culturing cells, like the above-mentioned culturing device 100, and has a function of replenishing the culture tank 1 during expansion culture with fresh medium (culture solution) to expand the culture scale. Have In the culturing apparatus 200, the culture scale is stepwise expanded by supplementing the culture tank 1 with a fresh medium, and a multi-step culturing process is performed. The culture apparatus 200 shown in FIG. 15 performs each culture step in the form of perfusion culture (continuous culture) in which a medium is continuously supplied during the culture and the same amount of the medium is continuously discharged. Has been done.

In the culture device 200, the culture system can be switched from static culture to agitation culture while the culture scale is being expanded stepwise. Therefore, perfusion culture type expansion culture with a final target of large scale can be performed in a single culture tank 1. The configuration of the culturing device 200 and the main operating method are substantially the same as those of the culturing device 100 described above, except for the cell separation device 11 and the corresponding pumps and pipes.

As shown in FIG. 15, a pull-out tube 22 for pulling out the culture solution and the cultured cells from the culture tank 1 is connected to the culture tank 1 of the culture device 200. The other end of the drawing tube 22 is connected to a cell separation device 11 that separates the culture solution drawn from the culture tank 1 from the cells. The drawing tube 22 is provided with a drawing pump 12 for drawing the culture solution and the cultured cells from the culture tank 1 to the cell separation device 11.

A return pipe 23 for returning the cells separated from the culture solution into the culture tank 1 is connected to the cell separation device 11. The other end of the return pipe 23 is connected to the culture tank 1. In addition, the cell separation device 11 is connected to a collection tube 24 for collecting the culture solution in which the cells have been separated. The recovery tank 10 is connected to the other end of the recovery pipe 24. The collection tube 24 is provided with a suction pump 13 that sucks the culture solution from the cell separation device 11.

The cell separation device 11 is, for example, a Tangential Flow Filtration (TFF) method of flowing a liquid in a direction orthogonal to the separation membrane, a liquid is caused to flow in a direction orthogonal to the separation membrane, and suction and release to and from the separation membrane are performed. Alternate Tangential Flow Filtration (ATF) method, gravity sedimentation separation method, and sonic separation method in which differences in size and density are separated by ultrasonic vibration can be used. it can.

FIG. 16 is a diagram for explaining the shape and structure of the internal space of the culture tank provided in the culture device.
As shown in FIG. 16, the culture tank 1 included in the culture apparatus 200 has an inner width that is not uniform in the height direction, and is relatively deep in the tank with a lower space 110 having a relatively narrow inner width. And an upper space 120 having a wide inner width. The drawing tube 22 is connected to the bottom of the lower space 110 that projects downward in the culture tank 1.

<Culturing method (perfusion culture method)>
Next, a culturing method according to an embodiment of the present invention will be described with reference to the drawings together with a process when the culturing apparatus 200 is used.

In the culturing device 200, as in the culturing device 100, the amount of the culture solution in the culture tank 1 is increased stepwise by supplementing the fresh medium, and a plurality of culturing steps having different culture solution amounts are performed stepwise. During the process, the culture system is switched from static culture to stirring culture, and static culture and stirring culture in a culture medium increased by supplementation with fresh medium are performed stepwise in a single culture tank 1. Perfusion culture type expansion culture using the culture device 200 can be performed in the flow shown in FIG.

In the culture device 200, the perfusion culture may be performed only in the static culture process (see step S30 in FIG. 14) or in the agitated culture process (see step S80 in FIG. 14) only. However, both of these may be performed. Further, among the culturing process of static culturing and the culturing process of stirring culturing, only the culturing process of a predetermined culturing scale may be performed. The culturing step without perfusion culturing can be operated by a batch culturing method.

In the culturing device 200, the culturing conditions necessary for culturing cells can be set similarly to the culturing device 100 described above. The items of the culture conditions to be set include, in addition to the items of the culture device 100, a culture schedule that defines a culture process for performing perfusion culture, and a perfusion flow rate for each culture process.

During the perfusion culture, the extraction pump 12 is operated to extract the culture solution and the cultured cells from the culture tank 1. Further, the supply pump 7 is operated to replenish the culture solution from the medium tank 5 into the culture tank 1. Then, the cell separation device 11 separates the extracted culture fluid and cells from each other. Then, the separated cells are returned to the culture tank, and the suction pump 13 is operated to collect the culture solution containing the substance produced by the cells in the recovery tank 10.

When performing perfusion culture in the culture process of static culture (see step S30 in FIG. 14), while maintaining the stirrer 2, while maintaining the culture scale such that the liquid surface has a predetermined height in the lower space 110. Perfusion culture is performed under culture conditions that are constantly controlled. When the culture process of static culture is perfusion culture, nutrient depletion and accumulation of waste products secreted by cells are less likely to occur. Therefore, it is possible to grow to a higher cell density per a predetermined culture scale. Since the final number of cells increases, the culture tank 1 itself can be downsized.

On the other hand, when performing perfusion culture in the culture process of stirring culture (see step S80 in FIG. 14), while stirring the culture solution, while maintaining the culture scale at which the liquid surface has a predetermined height in the upper space 120. Perform perfusion culture under variably controlled culture conditions. When the culture process of agitation culture is perfusion culture, not only can the cells be grown to a higher cell density per predetermined culture scale, but also the substances secreted by the cells into the culture medium can be continuously recovered. Since it is possible to continuously produce the target substance in a state where the cell growth is hardly inhibited, it is possible to realize high productivity.

According to the culturing apparatus 200 and the culturing method described above, a fresh medium can be replenished in the culturing tank having a predetermined shape and structure. Therefore, as in the batch culturing type culturing apparatus 100 and the culturing method described above, the amount of the culture solution can be increased. Can be increased stepwise, and in the middle of this, expansion culture in which the culture system is switched from static culture to stirring culture can be performed using a single culture tank 1. When the lower space 110 of the culture tank 1 has a shape satisfying the relationship of the mathematical formula (I) and has a structure of an appropriate stirring system, the specific growth rate of cells and the survival rate become high. Therefore, it is possible to carry out a plurality of culturing steps in which the amount of culturing solution and the culturing method are different from each other stepwise in one culturing tank while maintaining good culturing efficiency.

In particular, according to the culture device 200 and the culture method described above, since perfusion culture type expansion culture is performed, it is possible to grow to a higher cell density as compared with the batch culture type. , Can be more easily miniaturized. Even when cells secrete a large amount of a substance that inhibits growth, a large number of cells and a large amount of a target substance can be obtained by setting a culture schedule in which the number of size ups is reduced.

Although the present invention has been described above, the present invention is not limited to the above-described embodiments and modifications, and various modifications can be made without departing from the spirit of the present invention. For example, the present invention is not necessarily limited to those having all the configurations of the above-described embodiments and modifications. A part of the configuration of a certain embodiment or a modified example is replaced with another configuration, a part of the configuration of a certain embodiment or a modified example is added to another form, a configuration of a certain embodiment or the modified example Can be omitted.

For example, the number of culture medium tanks 5, the arrangement of other devices, the connection of pipes, and the like in the culture devices 100 and 200 are not limited. The culture tank 1 can have an appropriate form shown in the shape example and the structure example, and not only the stirrer 2 but also the arrangement of the air diffuser 3 and the connection position of the pipes are similar to those of the stirrer 2. Can be optimized.

In addition to the air diffuser 3, the culture devices 100 and 200 may include an air diffuser (not shown) for passing a predetermined gas through the gas phase. In such a case, the air diffuser 3 that diffuses the culture medium may be opened in either the lower space 110 or the upper space 120.

Further, in the culture devices 100 and 200, the culture tank 1 and the replenishing device (5, 7) may be connected in advance, or may be connected during culture. When a single-use type culture bag is used as the culture tank 1, the agitator 2 may be fixed in advance so as to have a predetermined arrangement, or may be fixed at a predetermined position during culture.

Further, the culture devices 100 and 200 are provided with a culture medium tank 5 and a supply pump 7 as a replenishing device. However, gravity or pressure difference may be used instead of the supply pump 7 to supply the fresh medium. In such a case, the replenishing device may include only the medium tank 5 that replenishes the fresh medium by utilizing gravity or a pressure difference. As the culture medium tank 5, various containers, a flexible bag, or the like may be provided.

Further, in the culturing method using the culturing devices 100 and 200 (see FIG. 14), the production culturing is performed after the expansion culturing in the culturing tank 1 is completed. However, the production culture step may be performed using the culture tank 1, or may be performed by transferring the culture tank 1 or the recovery tank 10 to another culture tank.

Further, the culturing method using the culturing devices 100 and 200 (see FIG. 14) may have a culturing schedule in which the culturing step of static culturing and the culturing step of stirring culturing are each one step culturing step. In the above-mentioned culture method, the operation of the feed pump 7 and the stirrer 2 may be changed to appropriate conditions, or fed-batch expansion culture may be performed within a range where the culture scale does not change significantly. Good.

Hereinafter, the present invention will be specifically described with reference to Examples, but the technical scope of the present invention is not limited thereto.

During the stepwise expansion of the culture scale, a single culture vessel is used for expansion culture that switches the culture method from static culture to agitation culture, in the case of batch culture type and perfusion culture type. The results of culturing were compared.

(experimental method)
As a test cell, a floating cell line CHO cell was used. As the medium, a liquid medium of DMEM medium was used. Frozen CHO cells were thawed in a water bath at 37° C. and fresh medium was added to prepare a cell suspension having a cell density of 0.2×10 ⁶ cells/mL. 5 mL of this cell suspension was placed in a culture tank, the gas composition of the gas phase was adjusted to 5% CO _{2 in the} atmosphere, and the temperature of the culture solution was adjusted to 37° C., and expansion culture was started.

The culture scale of expansion culture, the number of times of size increase, the timing of switching the stirring method, and the like were the same as those in the conventional example shown in FIG. Table 2 shows an outline of the culture schedule for expansion culture.

As shown in Table 2, static culture was performed from the culture step I to the culture step III. From the culture step IV to the culture step IX, stirring culture was performed. The expansion of the culture scale was performed by supplementing a fresh medium at 37° C. so that the amount of the culture solution would be the set value, when the preset culture time had elapsed. After the completion of the culture step IX, the process proceeded to a separation and purification step in which the substance produced by the cells was collected and purified.

The number of viable cells and the cell survival rate were determined at the end of the culture process of each culture scale. Then, the culture results were compared between the case where the whole culture process was a batch culture system and the case where the whole culture process was a perfusion culture system.

FIG. 18: is a figure which shows the result of performing expansion culture in a single culture tank.
In FIG. 18, the left axis is the number density of live cells confirmed in the culture tank [×10 ⁵ cells/mL], and the right axis is the cell survival rate [%] confirmed in the culture tank, the horizontal axis. Indicates the culture time [day]. Plots of ▲ are results of number density of live cells in batch culture, plots of △ are results of viability in batch culture, plots of ● are results of number density of live cells in perfusion culture, The plot is the result of survival rate in perfusion culture.

As shown in FIG. 18, it was confirmed that a high survival rate and a certain number of viable cells could be obtained by both the batch culture method and the perfusion culture method. When the results of the batch culture method and the results of the perfusion culture method were compared, it was confirmed that in the case of the perfusion culture method, the maximum value of the number density of viable cells was increased by 5 times or more.

This result means that, when performing expansion culture as shown in FIG. 17, it is possible to scale up from 75 L to 2000 L in one step, and a 400 L scale culture tank can be omitted. It can be said that a single tank which is smaller than that in the conventional method can be used as the culture tank. Further, in the case of the perfusion culture method, no decrease in the survival rate was observed during the passage, and it can be said that a more excellent culture efficiency can be realized as compared with the batch culture method.

100 Cultivator 200 Cultivator 1 Cultivation tank 2 Stirrer 2a Stirring blade 2b Rotating shaft 2c Motor 2d Shaking table 2e Hub (rotating member)
2f Eccentric pin (conversion member)
2g Belt 3 Air diffuser 4 Temperature control device 5 Medium tank 6 Valve 7 Supply pump 8 Analysis device 9 Control device 10 Recovery tank 11 Cell separation device 12 Extraction pump 13 Suction pump 20 Supply pipe 21 Discharge pipe 22 Extraction pipe 23 Return pipe 24 Recovery tube 50 Culture solution 52 Vial 53 Flask 54 Spinner flask 55 Bioreactor 110 Lower space 120 Upper space

Claims (14)

A culture tank for culturing cells in a culture medium,
A stirrer for stirring the culture solution,
A replenishing device for replenishing a fresh medium in the culture tank,
The lower space in the culture tank is a culture device having an inner width narrower than that of the upper space and protruding downward. The culture device according to claim 1, wherein
In the lower space in the culture tank, the volume of the culture solution contained in the lower space is V [cm ³ ] and the area of the interface between the culture solution contained in the lower space and the gas phase is S [cm ² ]. Then, the following formula (I);
S/V≦8 [cm ⁻¹ ] (I)
A culture device having a shape that satisfies the relationship of. The culture device according to claim 2, wherein
The lower space in the culture tank has a cone shape,
A culture device in which the height of the culture solution contained in the lower space is larger than 3/8 cm. The culture device according to claim 2, wherein
The lower space in the culture tank has a columnar shape,
A culture apparatus in which the height of the culture solution contained in the lower space is larger than 1/8 cm. The culture device according to claim 2, wherein
The lower space in the culture tank has a spherical shape,
The height h [cm] of the culture solution contained in the lower space is V [cm ³ ] the volume of the culture solution contained in the lower space, and the culture solution contained in the lower space and the gas phase are When the area of the interface is S [cm ² ], the radius of the spherical shape is r [cm], and the radius of the interface between the culture solution and the gas phase in the lower space is c [cm], Formulas (I), (II) and (III);
S/V≦8 [cm ⁻¹ ] (I)
V=π·h(3c ² +h ² )/6 (II)
S=π·h(2r-h) (III)
Culture device that satisfies the relationship of. The culture device according to claim 1, wherein
The stirrer has a stirring blade and a rotating shaft for rotating the stirring blade,
The stirring blade is located in the upper space in the culture tank,
The culture device in which the rotating shaft supports the stirring blade from above the culture tank. The culture device according to claim 1, wherein
The stirrer has a stirring blade and a rotating shaft for rotating the stirring blade,
The stirring blade is located in the upper space in the culture tank,
The rotating shaft supports the stirring blade from below the culture tank, which is eccentric from the central axis of the culture tank. The culture device according to claim 1, wherein
The agitator has a shaking table that supports the culture tank, a rotating member that drives the shaking motion of the shaking table, and a conversion member that converts the rotational motion of the rotating member into the shaking motion,
A culture device in which a central axis of the culture tank supported by the shaking table is eccentric from a central axis of the rotating member. The culture device according to claim 1, wherein
An extraction tube for extracting the culture solution and the cells from the culture tank,
A cell separation device for separating the culture solution and the cells withdrawn from the culture tank,
And a return pipe for returning the cells separated from the culture solution into the culture tank. Statically culturing the cells in the culture medium in the lower space of the culture tank,
After the static culture, supplement the fresh medium in the culture tank,
A culturing method in which the cells are cultivated with stirring in a culture medium increased by supplementing the fresh medium. The culture method according to claim 10, wherein
In the static culture, a culture method in which the gas concentration in the gas phase part in the culture tank is controlled to be constant. The culture method according to claim 10, wherein
In the stirring culture, a culture method in which the culture solution is stirred by a stirring blade located in an upper space in the culture tank. The culture method according to claim 10, wherein
In at least one of the static culture and the agitated culture, a culture method in which the amount of culture liquid in the culture tank is expanded by supplementing with fresh medium, and cultures having different culture liquid amounts are performed. The culture method according to claim 10, wherein
In at least one of the static culture and the agitation culture, together with drawing the culture solution and the cells from the culture tank, supplement the culture solution in the culture tank, and the culture solution and the cells withdrawn And a method of performing perfusion culture in which the separated cells are returned to the culture tank.

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