JP7355498B2 - Culture method - Google Patents

Description

The present invention relates to a culture method using a culture device suitable for expanding cell culture.

Biopharmaceuticals, including antibody drugs, are manufactured by culturing cells and separating and purifying substances produced by the cells from impurities. Cells that produce a target substance are expanded and cultured until a cell number suitable for production of the substance is reached, and then subjected to production culture using a large-scale culture medium. Generally, floating cell systems using CHO cells and the like are used for the production of antibody drugs and the like.

FIG. 17 is a diagram illustrating the flow of conventional expansion culture.
As shown in FIG. 17, conventional expansion culture is performed by increasing the volume of the culture solution in stages from a small scale of several mL to a large scale of several thousand liters. The cells produce the target substance while proliferating in the culture solution, and eventually mass-produce the target substance in the culture solution expanded to several thousand liters.

In normal batch culture, when cells proliferate and the cell density in the culture medium increases, the cells tend to die due to nutrient depletion and accumulation of waste products secreted by the cells. If batch culture is continued, the increase in the number of cells due to proliferation is balanced by the decrease in the number of cells due to death, so the cell density in the culture solution reaches a plateau, making it difficult to grow the number of cells to a level suitable for producing the substance. becomes difficult.

On the other hand, according to expansion culture, which expands the culture scale step by step, the culture medium in which the cells are cultured is replenished with fresh medium each time the cells are scaled up, which replenishes nutrients and increases secretion. Waste products are diluted. In expansion culture, the culture scale is expanded in a state where cells are easy to proliferate, so the cell density in the culture solution is unlikely to reach a plateau, and a large amount of cells suitable for producing substances can be obtained.

As shown in Figure 17, a typical substance production process consists of stage A in which culture operations are performed manually in a safety cabinet, stage B in which culture operations are performed semi-automatically using a bioreactor, and It consists of a production culture step C to produce a substance. Generally, scale-up is performed multiple times in each of the manual stage A and the semi-automated stage B.

The magnification for expanding the culture scale is generally kept constant throughout multiple scale-ups. The magnification for expanding the culture scale is usually set to about 5 to 10 times. If the cell density decreases too much due to replenishment of fresh medium, the culture efficiency after scale-up will deteriorate, so a magnification that maintains a certain level of cell density is set.

As shown in FIG. 17, in the early stage of expansion culture when the amount of culture solution is about several mL to several hundred mL, small-scale stationary culture using a culture container is performed. In addition to the flask 53 shown in the figure, dishes, bottles, well plates, etc. are also used as culture containers. The seed cells prepared in the vial 52 or the like are transferred to culture vessels of various sizes and shapes and subcultured. When the amount of culture solution reaches several hundred mL, stirring culture or shaking culture using a spinner flask 54 may be performed.

Generally, initial expansion culture using the flask 53 or spinner flask 54 is performed in an incubator controlled at a constant culture temperature and constant gas concentration. A culture container using a gas-exchangeable cap, silicone stopper, etc. is placed in an incubator in which the internal atmosphere is controlled to be constant, and the culture environment inside the container is adjusted. When expanding the culture scale, the culture solution is manually transferred in a safety cabinet.

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

Generally, the latter stage expansion culture using the bioreactor 55 is performed in a culture environment where the culture temperature and gas concentration are variably controlled. The culture solution in the bioreactor 55 is maintained at a culture temperature of around 37° C. under forced aeration, the dissolved oxygen concentration and carbon dioxide concentration are controlled. When expanding the culture scale, automatic transfer of the culture solution is performed aseptically using a transport system consisting of steel pipes, tubes, etc.

Conventionally, with regard to expansion culture in which the culture scale is expanded step by step, countermeasures have been considered for problems associated with transferring the culture solution. When expanding culture, it is necessary to manually transfer the culture solution, which poses a problem of complicated work. Furthermore, since shearing force, pressure, etc. are applied to the cells during the transfer of the culture solution, there is a problem in that the cells are damaged. Furthermore, 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, techniques have been proposed that make 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 partition it into a plurality of compartments, cells are cultured in each compartment, and then the compartments are dissolved. In Patent Document 1, the transition from curing culture to expansion culture is realized by eliminating the protuberance caused in a container made of a flexible material.

Japanese Patent Application Publication No. 2016-140295

When expanding cell culture, there are issues related to the complexity of transferring the culture solution, issues related to the handling of the culture solution and cells in a way that does not damage the cells, and transfer of the culture solution, as described in Patent Document 1. In addition to issues related to contamination such as time, there are also issues related to the reproducibility of expanded culture and issues with the equipment used for expanded culture.

For example, in initial expansion culture that requires manual labor, the number of cells to be seeded and the number of cells to be transferred will vary depending on the operator's technique. Therefore, a problem arises in that variations occur in the quality of the product, including the production amount of the target substance, the purity of the target substance, and the degree of non-denaturation of the protein. Furthermore, since manual labor is required, additional equipment such as a safety cabinet is required, and there is also the problem that the culture facility for containment becomes large.

In addition, in late-stage expansion culture using bioreactors, separate bioreactors are required for each culture scale. Therefore, there is a problem in that a large number of locations for installing bioreactors are required. When bioreactors are connected to each other with steel pipes, tubes, etc., space for laying these and piping work are required, and there is a risk that cells will be damaged or killed during the piping.

Generally, when expanding culture with the ultimate goal of culturing floating cells on a large scale, static culture with less mechanical stress is performed at the beginning of expansion culture, and at the later stage of expansion culture when the culture scale has expanded to a certain extent. In addition, it is preferable to perform agitation culture, which is advantageous for gas exchange efficiency and substance diffusion. Therefore, during expansion culture, it is desirable not only to transfer the culture solution but also to switch the culture method. There is a need for a technology that can change the amount of culture solution and culture method while maintaining a high specific growth rate and survival rate of cells and maintaining good culture efficiency.

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a culture method in which a plurality of culture steps using different amounts of culture solution and culture methods can be performed in a stepwise manner in a single culture tank.

As a result of intensive research, the present inventors have found that by optimizing the shape and structure of the internal space of the culture tank, multiple culture processes with different culture solution volumes and culture methods can be performed with different cell specific growth rates and survival rates. The present inventors have discovered that the process can be carried out in stages in one culture tank without significantly reducing the yield, and have completed the present invention.

That is, in order to solve the above-mentioned problems, the culture method according to the present invention is a culture method using a culture device for expansion culture that performs agitation culture after static culture, and the culture device is a culture method that uses a culture tank for culturing cells in the culture tank, a stirrer for stirring the culture solution in the culture tank, and a replenishing device for replenishing the culture tank with fresh culture medium, and a lower space in the culture tank. has a downwardly protruding shape with an inner width narrower than the upper space, and the lower space in the culture tank has a volume of V [cm ³ ] of the culture solution put in the lower space, and When the area of the interface between the culture solution and the gas phase is S [cm ² ], the following formula (I); S/V≦8 [cm ⁻¹ ] ... (I) is satisfied. The agitator has a stirring blade and a rotating shaft for rotating the stirring blade, and the stirring blade is located only in an upper space within the culture tank, and in the culture method, Cells are statically cultured in a culture solution in the lower space of the culture tank, and after the static culture, a fresh medium is replenished into the culture tank, and the cells are cultured in the culture solution whose volume has been increased by replenishing the fresh medium. Culture the cells with agitation .

In the culture method according to the present invention, a plurality of culture steps using different amounts of culture solution and culture methods can be performed in a stepwise manner in one culture tank.

FIG. 1 is a diagram schematically showing an example of a culture device according to an embodiment of the present 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. FIG. 2 is a diagram showing the relationship between the surface area-volume ratio of a culture solution and the survival rate of cells. FIG. 3 is a diagram showing the relationship between the surface area-volume ratio of a culture solution and the amount of cell proliferation. It is a figure which shows the example of a shape of the lower space of the culture tank with which a culture apparatus 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 apparatus 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 apparatus 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 apparatus 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 apparatus 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 apparatus 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 apparatus is equipped. It is a figure showing an example of structure of a culture tank with which a culture device is equipped. It is a figure showing an example of structure of a culture tank with which a culture device is equipped. 1 is a flowchart showing a culture method according to an embodiment of the present invention. FIG. 1 is a diagram schematically showing an example of a culture device according to an embodiment of the present 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 conventional expansion culture. FIG. 3 is a diagram showing the results of expanded 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. Note that common components in the following figures are denoted by the same reference numerals, and redundant explanation 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, the culture apparatus 100 according to the present embodiment includes a culture tank 1, an agitator 2, an aeration device 3, a temperature control device 4, a culture medium tank 5, a valve 6, and a supply pump 7. , an analyzer 8 , a controller 9 , a recovery tank 10 , a supply pipe 20 , and a discharge pipe 21 .

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

In the culture apparatus 100, the culture method can be switched from static culture to agitation culture while expanding the culture scale step by step. Therefore, batch culture expansion culture with the ultimate goal of large-scale cultivation can be performed in the single culture tank 1. The culture tank 1 is one in which the shape and structure of the internal space are optimized in order to improve the efficiency of such expanded culture.

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

Specific examples of cells to be cultured include animal cells such as Chinese hamster ovary cells (CHO cells), baby hamster kidney cells (BHK cells), human embryonic 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, yeast, fungi, algae, yeast, etc.

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

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

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

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. Examples of the sensor include 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 absorption photometer, a liquid level sensor, and the like. Data measured by the sensor can be output to the analyzer 8. Furthermore, the culture tank 1 can be equipped with a sampling port used to collect a culture solution in order to measure the culture conditions in the tank and the culture state of cells off-line.

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

The medium tank 5 aseptically stores the prepared sterilized fresh medium until it is replenished with fresh medium to expand the culture scale. The culture device 100 can include a plurality of culture medium tanks 5. Each culture medium tank 5 is equipped with a valve 6 that opens and closes the supply pipe 20 .

The supply pump 7 is provided to supply fresh culture medium from the culture medium tank 5 to the culture tank 1 . When expanding the culture scale, only some of the plurality of valves 6 can be opened to replenish the culture tank 1 with fresh culture medium. By sequentially using only some of the culture medium tanks 5, the risk of contamination associated with replenishment of fresh culture medium can be minimized.

The analyzer 8 is provided to analyze the state of cells cultured in the culture tank 1 and the state of the culture solution in the culture tank 1. Items to be analyzed include state quantities that correlate with cell proliferation, concentration of culture solution components, osmotic pressure, and the like. The growth state of cells can be determined by analyzing the state quantities that correlate with cell growth and the concentrations of culture solution components. That is, it is possible to determine whether the cells have sufficiently proliferated on a certain culture scale and the progress of the culture process.

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

Among state quantities that correlate with cell proliferation, cell number density, specific growth rate, and survival rate can be determined by measuring cell number. Cell counts can be determined using various methods such as microscopic observation using a hemocytometer, dry weight method, turbidity method, capacitance method, NAD measurement to quantify oxidized NAD ⁺ and reduced NADH, and flow cytometry. It can be measured using a measuring method. Further, the number of living cells and the number of dead cells can be counted using discrimination by trypan blue staining. The specific growth rate can be determined by measuring changes in the number of living cells over time.

In addition, among the state quantities that correlate with cell proliferation, the metabolic reaction rate of the cell (metabolic production rate of target substances, metabolic consumption rate of nutrients, metabolic secretion rate of waste products, etc.) and the cumulative metabolic amount of cells (target Cumulative metabolic production of substances, cumulative metabolic consumption of nutrients, cumulative metabolic secretion of waste products, etc.) can be determined by the following method by conducting an actual culture test in advance.

The metabolic production rate of the target substance can be determined by measuring changes over time in the concentration of the produced target substance and converting it into the production amount per unit time and 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. Fluorescence analysis of labels, enzyme activity analysis, etc. are performed using antibodies that cause antigen-antibody reactions with the protein to be measured. As the ELISA method, any of the direct method, indirect method, sandwich method, competitive method, etc. 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 in concentration in the culture tank over time and converting it into the amount of change per unit time and per unit number of cells. The concentration of nutrients and waste products can be measured using 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), etc. can be used to quantify the culture solution from which cells have been removed.

The cumulative metabolic production of the target substance, the cumulative metabolic consumption of nutrients, and the cumulative metabolic secretion of waste products can be determined by integrating the determined metabolic rate over time or by actually measuring the cumulative amount. Cell metabolic reaction rates (metabolic production rate of target substances, metabolic consumption rate of nutrients, metabolic secretion rate of waste products) can also be determined by conducting actual culture tests in advance and analyzing intracellular metabolic fluxes.

Furthermore, among the state quantities that correlate with cell proliferation, the cell cycle can be determined by flow cytometry using propidium iodide (PI) staining.

Cells in the G2 phase and M phase are about to start cell division, while cells in the G1 phase and G0 phase have some time before cell division, and the G0 phase has an indefinite length. A state in which the proportion of cells in the G2 or M phase of the cell cycle is high is a state in which most of the cultured cells are about to start cell division, and can be said to be a state in which the cells are likely 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 cell proliferation can be said to be suppressed.

By detecting fluorescence using flow cytometry and calculating the integral area of each curve on the frequency distribution, it is possible to know the number and proportion of cells in the G2 phase or M phase. If the number and proportion of such cells are used as an index for determining the progress of the culture process, it is possible to expand the culture scale in a time-efficient manner while prioritizing a state in which the cell population can easily proliferate.

The control device 9 is provided to control cell culture conditions and operation of the culture device 100. The control device 9 controls the operation, stop, and rotation output of the agitator 2, the operation, stop, and ventilation amount of the aeration device 3, the operation, stop, and regulated temperature of the temperature control device 4, the opening and closing of the valve 6, and the supply pump. The operation, stop, discharge output, etc. of 7 are controlled by an appropriate control method such as PID control, proportional control, on/off control, etc.

Data necessary for controlling cell culture conditions can be set and stored in the control device 9 in advance according to the type of cells, the purpose of expansion culture, and the like. Furthermore, a predetermined culture schedule can be set to control the operation of the culture apparatus 100. The culture schedule should be determined according to the target culture volume of cells, target production volume of the target substance, etc. so that the size can be increased a specified number of times during expansion culture, and the culture method can be switched at specified times. I can do it.

The control device 9 controls each device to increase the amount of culture solution in the culture tank 1 in stages and to switch the culture method from static culture to agitation culture in the middle. The control device 9 receives the analysis data acquired by the analyzer 8, determines whether the culture process of each culture scale is completed based on this analysis data, and, according to the determination result, restarts the culture scale by replenishing fresh medium. and switching from static culture to agitation culture by starting the agitator 2.

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

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

FIG. 2 is a diagram illustrating the shape and structure of the internal space of the culture tank provided in the culture apparatus.
As shown in FIG. 2, the culture tank 1 included in the culture apparatus 100 has an internal width that is not uniform in the height direction, and a lower space 110 having a relatively narrow inner width and a lower space 110 that has a relatively narrow inner width. The upper space 120 has a wide inner width. In the culture tank 1 shown in FIG. 2, the lower space 110 has a conical shape that projects downward from the culture tank 1. The upper space 120 has a uniform inner width in the height direction, but the lower space 110 has an inner width 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 culture process of statically culturing cells, a culture solution is poured into the culture tank 1 until the liquid level reaches a predetermined height in the lower space 110. The height of the liquid level in the lower space 110 can be rounded up in stages by replenishing fresh medium according to a predetermined culture schedule, as shown by lines I to IV in FIG.

The upper space 120 of the culture tank 1 is used for stirring culture. In the culture process of agitating cells, a culture solution is poured into the culture tank 1 until the liquid level reaches a predetermined height in the upper space 120. The height of the liquid level in the upper space 120 can be rounded up in stages by replenishing fresh medium according to a predetermined culture schedule, as shown by lines V to VI in FIG.

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

<Culture experiment>
Here, we will show the results of a culture experiment that confirmed the relationship between the shape and structure of the internal space of the culture tank 1 and the cell culture efficiency.

In cell culture, temperature, dissolved oxygen concentration, and other gas concentrations are important environmental factors. In static culture, gas exchange occurs through the interface between the culture solution and the gas phase, so 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 that In order to examine the validity of such predictions and the appropriate range of area S and volume V, cells were cultured statically under various culture conditions and the area S, volume V, cell survival rate, and cell proliferation amount were determined. We investigated the relationship between

(experimental method)
As the test cells, CHO cells, which are a floating cell system, were used. Further, as a medium, Dulbecco's Modified Eagle's Medium (DMEM medium) was used. Two types of flasks (T75 flask and T225 flask) with different culture surface areas were used as culture vessels.

As shown in Table 1, a liquid medium was placed in each of the two types of flasks so that the surface area S of the culture solution and the amount V of the culture solution were under various conditions. Further, test cells were seeded into the medium at a cell density of 0.2×10 ⁶ cells/mL. Then, the culture conditions were adjusted such that the gas composition of the gas phase 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 40% of the saturated dissolved oxygen concentration. It was statically cultured for several days.

After static culture, the number of live and dead cells was counted using a live/dead cell autoanalyzer Vi-Cell (manufactured by Beckman Coulter), and the percentage of live cells in all cells (cell viability) and the culture The total number of viable cells (amount of cell proliferation) was determined.

Table 1 shows the type of culture container 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), and the liquid surface area. - Indicates the volume ratio S/V (ratio S/V of the area S of the interface between the culture solution and the gas phase and the volume V of the culture solution).

(Experiment result 1)
FIG. 3 is a diagram showing the relationship between the liquid surface area-volume ratio of the culture solution and the cell survival rate.
In FIG. 3, the horizontal axis shows the liquid surface area-volume ratio S/V [cm ^-1 ] of the culture solution in the culture experiment, and the vertical axis shows the cell survival rate [%] confirmed after the culture experiment. . The ● plots are the results of culture experiments using T75 flasks, and the ▲ plots are the results of culture experiments using T225 flasks.

As shown in FIG. 3, it was confirmed that the smaller the S/V, the higher the cell survival rate, both when using a T75 flask and when using a T225 flask. Regardless of the type of culture vessel, when S/V≦8 [cm ^-1 ], the survival rate becomes significantly high, and when S/V≦4 [cm ^-1 ], it reaches well over 90%. A high survival rate was obtained.

(Experiment result 2)
FIG. 4 is a diagram showing the relationship between the surface area-volume ratio of the culture solution and the amount of cell proliferation.
In FIG. 4, the horizontal axis shows the liquid surface area-volume ratio S/V [cm ⁻¹ ] of the culture solution in the culture experiment, and the vertical axis shows the relative value of the number of living cells confirmed after the culture experiment. The ● plots are the results of culture experiments using T75 flasks, and the ▲ plots are the results of culture experiments using T225 flasks.

As shown in FIG. 4, it was confirmed that the smaller the S/V, the greater the amount of cell proliferation in both cases when a T75 flask was used and when a T225 flask was used. Regardless of the type of culture container, when S/V≦8 [cm ^-1 ], the specific growth rate becomes high and the number of viable cells increases easily, and when S/V≦4 [cm ^-1 ], the number of viable cells increases. , the maximum number of viable cells was obtained.

These experimental results show that in static culture, the culture performance is determined by S/V, regardless of the size and shape of the culture container. From the viewpoint of keeping the specific growth rate and survival rate of cells high and achieving good culture efficiency, it is preferable that S/V satisfies S/V≦8 [cm ⁻¹ ], and S/V≦4 [cm −1 ]. cm ⁻¹ ] is more preferable.

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

Therefore, in the lower space 110 of the culture tank 1, the volume of the culture medium placed in the lower space 110 is V [cm ³ ], and the area of the interface between the culture medium placed in the lower space 110 and the gas phase is S [cm ² ]. In this case, it is preferable that the liquid surface area-volume ratio S/V satisfies the relationship expressed by the following formula (I).
S/V≦8[cm ^-1 ]...(I)

If the lower space 110 of the culture tank 1 has a shape that satisfies the relationship of 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 in which the size is increased stepwise. In addition, the survival rate of cells is increased and impurities are less likely to be generated in the culture solution, so 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 culture efficiency, S/V≦4 [cm ⁻¹ ] is more preferable.

<Example of shape of culture tank>
The lower space 110 of the culture tank 1 can be provided in any shape as long as it has an inner width narrower than the upper space 120 and projects downward from the culture tank 1. The lower space 110 of the culture tank 1 preferably has a shape that satisfies the relationship of formula (I), but may also have a shape that satisfies the relationship of formula (I) only when a predetermined amount of culture solution is put therein. However, it may have a shape that satisfies the relationship of formula (I) in all states containing an arbitrary amount of culture solution.

The lower space 110 of the culture tank 1 can be provided, for example, in a shape in which the inner width becomes narrower toward the bottom of the culture tank 1. Examples of such a shape include, for example, a stepped shape in which the inner width gradually narrows toward the bottom of the culture tank 1, or a stepped shape in which the inner width narrows continuously as it moves toward the bottom of the culture tank 1. Examples include a tapered shape.

FIG. 5, FIG. 6, and FIG. 7 are diagrams showing examples of the shape of the lower space of the culture tank provided in the culture apparatus.
As shown in FIGS. 5, 6, and 7, the lower space 110 of the culture tank 1 can be provided in the shape of a cone. FIG. 5 is a diagram illustrating an inverted conical pyramid shape, FIG. 6 is a diagram illustrating an inverted quadrangular pyramid shape, and FIG. 7 is a diagram illustrating an inverted triangular pyramid shape. It is a figure which illustrates. 5, FIG. 6, and FIG. 7, reference numeral 50 indicates the culture solution placed in the lower space 110, and reference numeral S indicates the area of the interface between the culture solution 50 placed in the lower space 110 and the gas phase. 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 solution 50 placed in the lower space 110 is V=S·h/3, and the S/V of the culture solution 50 is 3/h. Since it is preferable to satisfy S/V≦8 [cm ⁻¹ ], and more preferable to satisfy S/V≦4 [cm ⁻¹ ], when the lower space 110 is provided in a conical shape under this condition The height h of the culture solution 50 placed in the lower space 110 is preferably greater than 3/8 cm, and more preferably greater 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 a given height and a section below that height, similar to the culture solution 50. It is preferable that the volume v [cm ³ ] satisfies s/v≦8 [cm ⁻¹ ], and more preferably satisfies s/v≦4 [cm ⁻¹ ]. Therefore, when the lower space 110 is provided in a conical shape under this condition, 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 apparatus.
As shown in FIG. 8, the lower space 110 of the culture tank 1 can also be provided in a columnar shape. FIG. 8 is a diagram illustrating a rectangular parallelepiped column shape. In FIG. 8, the reference numeral 50 indicates the culture solution placed in the lower space 110, the reference numeral S indicates the area of the interface between the culture solution 50 entered in the lower space 110 and the gas phase, and the reference numeral h indicates the area of the interface between the culture solution 50 placed in the lower space 110 and the gas phase. The height of the culture solution 50 placed in the container is shown.

When the lower space 110 is provided in a columnar shape, the volume V of the culture solution 50 placed in the lower space 110 is V=S·h, and the S/V of the culture solution 50 is 1/h. It is preferable to satisfy S/V≦8 [cm ⁻¹ ], and it is more preferable to satisfy S/V≦4 [cm ⁻¹ ], so when the lower space 110 is provided in a columnar shape under this condition The height h of the culture solution 50 placed in the lower space 110 is preferably greater than 1/8 cm, and more preferably greater 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 a given height and a section below that height, similar to the culture solution 50. It is preferable that the volume v [cm ³ ] satisfies s/v≦8 [cm ⁻¹ ], and more preferably satisfies s/v≦4 [cm ⁻¹ ]. Therefore, when the lower space 110 is provided in a columnar shape 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 apparatus.
As shown in FIG. 9, the lower space 110 of the culture tank 1 can also be provided in a partially spherical shape. FIG. 9 is a diagram illustrating a spherical cutout 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 entered in the lower space 110 and the gas phase, and reference numeral r indicates the area of the interface between the culture solution 50 placed in the lower space 110 and the gas phase. The symbol h indicates the height of the culture solution 50 placed in the lower space 110, and the symbol c indicates the radius of the interface between the culture solution 50 placed 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 volume V of the culture solution 50 placed in the lower space 110 is The area S of the interface between and the gas phase is S=π·h(2r−h). It is preferable to satisfy S/V≦8 [cm ⁻¹ ], and it is more preferable to satisfy S/V≦4 [cm ⁻¹ ], so 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 relationship between the culture solution 50 placed in the lower space 110 and the gas phase. When the ^area of the interface of In addition to formula (I), it is preferable that the following formulas (II) and (III) are satisfied.
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 the shape of a cone, in addition to the illustrated example of the shape, it may be formed into other polygonal pyramid shapes such as an inverted pentagonal pyramid, an inverted hexagonal pyramid, or an inverted elliptical cone. It is also possible to provide one. Further, it can also be provided in the shape of a polygonal pyramid with rounded corners or in the shape of a polygonal pyramid with slightly curved sides, which are 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.

In addition, when the lower space 110 of the culture tank 1 is provided in a columnar shape, in addition to the illustrated shape, other polygonal columnar shapes such as a cube, a triangular columnar shape, a pentagonal columnar shape, a hexagonal columnar shape, a cylindrical shape, an elliptical columnar shape, etc. It can also be provided in, etc. Further, it can also be provided in the shape of a polygonal column with rounded corners or in the shape of a polygonal column with slightly curved sides, which are 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 apparatus.
As shown in FIGS. 10 and 11, the lower space 110 of the culture tank 1 can be provided in other discontinuous shapes other than the illustrated cone shape, column shape, and spherical shape. FIG. 8 is a diagram illustrating a discontinuous shape in the shape of an inverted truncated cone, in which a part of an inverted cone is cut out. FIG. 8 is a diagram illustrating a multi-stage convex discontinuous shape that is a combination of rectangular parallelepipeds of different sizes.

In addition, the lower space 110 of the culture tank 1 may be shaped like a truncated sphere with two parallel faces cut out, an inverted polygonal pyramid, an inverted elliptical cone, etc. It can also be provided in a discontinuous shape. In addition, the lower space 110 of the culture tank 1 has a multi-stage convex discontinuous shape that is a combination of polygonal prisms, cylinders, elliptical cylinders, inverted polygonal truncated pyramids, inverted truncated cones, inverted elliptic truncated cones, spheres, etc. of different sizes. It can also be provided in

<Example of structure of culture tank>
The culture tank 1 is of an upper mechanical stirring type in which a stirrer 2 is disposed on the upper side in FIG. 2 . The upper mechanical stirring type stirrer 2 is installed such that all the stirring blades 2a are located in the upper space 120 and the lowest ends of the stirring blades 2a are located above the lower space 110. Further, the rotating 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 down 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, during stationary culture using the lower space 110, cells are less likely to adhere or aggregate to the agitation blade 2a or the rotating shaft 2b. Therefore, it is possible to prevent strong mechanical stress from being applied to the cells during transition to agitation culture and from inaccurate counting of the number of cells.

However, instead of the upper mechanical stirring type as shown in FIG. 2, other stirring methods may be applied to the culture tank 1. For example, in addition to a mechanical stirring type that includes a rotating shaft and a motor, a magnetic stirring type in which stirring blades are rotated by magnetic force may also be used. Alternatively, a shaking stirring type without a stirring blade or an air flow stirring type may be used.

FIG. 12 is a diagram showing an example of the structure of a culture tank provided in the culture apparatus.
As shown in FIG. 12, the culture tank 1 can also be of a lower mechanical stirring type in which the stirrer 2 is disposed on the lower side. The lower mechanical stirring type stirrer 2 is installed such that all the stirring blades 2 a are located in the upper space 120 and the lowest ends of the stirring blades 2 a are 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 center axis of the culture tank 1, extends from the outside separated from the center of the bottom of the culture tank 1, penetrates the lower space 110, and extends to the center side of the upper space 120, and is connected to the stirring blade 2a. is fixed to the lower part near the center of the upper space 120.

According to such a structure of the lower mechanical stirring type, even if the motor 2c is not fixed to the upper part, cells are difficult to adhere and aggregate to the stirring blades 2a and the rotating shaft 2b during static culture using the lower space 110. . Therefore, it is possible to prevent strong mechanical stress from being applied to the cells during transition to agitation culture and from inaccurate counting of the number of cells.

FIG. 13 is a diagram showing an example of the structure of a culture tank provided in the culture apparatus.
The culture tank 1 can also be of a shaking and stirring type without stirring blades. The shaking agitation type stirrer 2 shown in FIG. 13 includes a motor 2c that rotates a rotating shaft 2b, a shaking table 2d that supports the culture tank 1, and a hub (rotating member) that drives the shaking movement of the shaking table 2d by rotational movement. 2e, an eccentric pin (conversion member) 2f that converts the rotational motion of the hub 2e into a shaking motion, and a belt 2g (transmission member) that transmits the rotational motion of the rotating shaft 2b to the hub 2e. The hub 2e is located below the shaking table 2d, and is rotatably supported by a bearing (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 in the same manner as the hub 2e and eccentric pin 2f.

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

In such a shaking and stirring type structure, the center axis of the culture tank 1 and the rotation axis of the shaking table 2d do not match, and the position and direction are shifted, causing the culture tank 1 to become eccentric, causing the inside of the culture tank 1 to become eccentric. liquids are mixed. Since there is no need to install a stirring blade or a rotating shaft in the culture tank 1, cells will not adhere or aggregate to the stirring blade or rotating shaft. Therefore, it is possible to prevent strong mechanical stress from being applied to the cells during transition to agitation culture and from inaccurate counting of the number of cells. Furthermore, when a single-use type culture bag is used as the culture tank 1, attachment and detachment to the stirrer 2 and sealing of the culture tank 1 are facilitated.

Note that the shaking-stirring type stirrer 2 shown in FIG. 13 is designed to shake and stir the culture tank 1 using a hub 2e, an eccentric pin 2f, etc.; As a mechanism for converting the rotational motion of the rotating member that drives the rotary member into a shaking motion, other mechanisms such as a slider crank mechanism, a cam mechanism, etc., or a combination thereof may be used. Further, the shaking agitation type stirrer 2 may be equipped with a device that performs other shaking movements such as horizontal reciprocating motion, vertical reciprocating motion, two-dimensional or three-dimensional circular motion, and tilting rocking such as a seesaw type. good.

<Culture method (batch culture method)>
Next, a culture method according to an embodiment of the present invention will be described with reference to the drawings, as well as processing when the culture apparatus 100 is used.

FIG. 14 is a flowchart showing a culture method according to an embodiment of the present invention.
FIG. 14 exemplifies the flow of expanding and culturing cells that produce a target substance using the culture device 100. In the culture apparatus 100, the amount of culture solution in the culture tank 1 is expanded stepwise by replenishing fresh medium, and a plurality of culture steps with different amounts of culture solution are performed stepwise. During this process, the culture method is switched from static culture to agitation culture, and static culture and agitation culture in a culture solution whose volume has been increased by replenishing fresh medium are performed in stages in a single culture tank 1.

First, culture conditions necessary for culturing cells are set in the culture apparatus 100 (step S10). The culture device 100 performs expansion culture in which the culture scale is expanded in stages according to a predetermined culture schedule such that the size is increased a specified number of times and the culture method is switched at a specified time. .

The culture conditions to be set include the culture schedule, for example, the target number of cell passages (target number of size increases) for each stationary culture or agitation culture, and the culture in culture tank 1 for each stepwise culture process. Examples include liquid volume (culture scale). Other factors include culture temperature, gas composition in the gas phase during stationary culture, rotational speed of the stirrer 2 during agitation culture, dissolved oxygen concentration, pH, and the like. In addition, in order to judge the progress of each culture process, the target state amount of cells (target value such as cell number density) for each culture process, the target culture time for each culture process, or the culture solution for each culture process. Target concentrations of ingredients can be set.

Subsequently, cells to be expanded and cultured are seeded in the medium (step S20). A fresh culture medium is poured into the culture tank 1 until the liquid level reaches a predetermined height in the lower space 110. Then, the aeration device 3, temperature control device 4, etc. are controlled so that the set culture conditions are achieved, and a cell suspension diluted to an appropriate cell number density is aseptically introduced. It is preferable that the culture tank 1 is provided with an inlet for seeding cells at a predetermined height in the lower space 110.

Subsequently, cells are statically cultured in a culture solution in the lower space 110 of the culture tank 1 (step S30). Static culture is performed while the agitator 2 is stopped and a culture scale is maintained where the liquid level is at a predetermined height in the lower space 110.

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

Subsequently, during static culture, an index value representing the proliferation state of the cells is acquired (step S40). As index values representing the proliferation state of cells, state quantities that have a correlation with cell proliferation, such as cell number density, specific growth rate, survival rate, metabolic reaction rate of cells, cumulative metabolic amount of cells, cell cycle, etc. , can be obtained by actual measurement using the analyzer 8. The actual measurement may be performed online using a sensor, or may be performed offline by sampling the culture fluid.

Alternatively, the culture time for each culture step may be acquired by a timer as an index value representing the state of cell proliferation. Alternatively, the concentration of the culture solution components for each culture step may be obtained by actual measurement using the analyzer 8. If the relationship with the cell growth state has been confirmed in advance, not only the cell state quantity but also the passage of culture time after starting the culture process at a certain culture scale and changes in the concentration of culture solution components can be It can be used as an indicator of the growth status of.

During static culture, when an index value representing the cell proliferation state is acquired, it is determined whether the statically cultured cells have reached the target proliferation state (step S50). The control device 9 receives the analysis data acquired by the analyzer 8, and performs preset index values regarding the state quantity of the cells, the culture time, or the concentration of the culture solution components, as well as the index values acquired through actual measurements. Compare with the target value. The progress of the culture process can be determined based on whether the index value obtained through actual measurement exceeds the target value.

As a result of the determination, if the target growth state has not been reached (step S50; No), static culture at 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, if the target growth state has been reached (step S50; Yes), a determination is made as to whether the switching conditions necessary for switching the culture method are satisfied (step S60). The control device 9 compares the actual number of passages (number of times of size increase) in static culture and the target number of passages determined as a culture schedule. The culture method can be switched depending on whether the parameter representing the actual passage number has reached the target passage number.

For example, when the height of the liquid level in the lower space 110 is rounded up as shown by lines I to IV in FIG. 2, the number of passages during static culture is four. If the relationship with the number of passages has been confirmed in advance, the index value obtained by actual measurement and the preset target value in static culture for the cell state quantity or the concentration of culture solution components. , may be compared. It is also possible to switch the culture method by determining the progress of subculture based on whether the index value obtained by actual measurement exceeds the target value in static culture.

As a result of the determination, if the target passage number has not been reached (step S60; No), the supply pump 7 is activated to replenish a predetermined amount of fresh medium from the medium tank 5 into the culture tank 1, and then proceed to the next stage of culture scale. 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, static culture on the expanded culture scale is continued in the lower space 110 of the culture tank 1 (step S30).

On the other hand, if the target passage number has been reached as a result of the determination (step S60; Yes), the culture method is switched from static culture to agitation culture, the agitator 2 is operated, and the lower space 110 and upper space 120 of the culture tank 1 are In step S80, cells are agitated and cultured in a culture solution whose volume has been increased by replenishing fresh culture medium. The agitation culture is performed by stirring the culture solution using the agitation blade 2a located in the upper space 120 in the culture tank 1, and by maintaining a culture scale in which the liquid level is at a predetermined height in the upper space 120.

During the agitation culture, the culture state is monitored using a temperature sensor, dissolved oxygen sensor, carbon dioxide sensor, pH sensor, etc. The culture temperature is controlled within a predetermined range by a temperature control device 4. Further, 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 aeration device 2. Further, the pH of the culture solution is controlled by adding an alkaline solution or aerating carbon dioxide.

Subsequently, during the agitation culture, an index value representing the proliferation state of the cells is obtained (step S90). As the index value representing the proliferation state of the cells, the state quantity of the cells, the culture time, or the concentration of the culture solution components can be acquired, similarly to the process in static culture (see step S40).

During agitation culture, when an index value representing the proliferation state of the cells is acquired, it is determined whether the cells being agitated and cultured have reached the target proliferation state (step S100). The control device 9 receives the analysis data acquired by the analyzer 8, and performs preset index values regarding the state quantity of the cells, the culture time, or the concentration of the culture solution components, as well as the index values acquired through actual measurements. Compare the target value for each culture step. The progress of the culture process can be determined based on whether the index value obtained through actual measurement exceeds the target value for each culture process.

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

On the other hand, as a result of the determination, if the target proliferation state has been reached (step S100; Yes), a determination is made as to whether or not the termination conditions required to terminate the culture are satisfied (step S110). The control device 9 compares the actual number of passages (number of times of size increase) in the agitation culture and the target number of passages set as the culture schedule. Continuation or termination of expansion culture is determined depending on whether the parameter representing the actual passage number reaches the target passage number.

For example, when the height of the liquid level in the upper space 120 is rounded up as shown by lines V to VI in FIG. 2, the number of passages during agitation culture is two. If the relationship with the passage number has been confirmed in advance, the index value obtained by actual measurement and the preset target value in agitation culture for the state quantity of cells or the concentration of culture solution components, You can also compare. It is also possible to determine whether to continue or terminate expansion culture by determining the progress of subculture based on whether the index value obtained through actual measurement exceeds the target value in agitation culture.

As a result of the determination, if the target passage number has not been reached (step S110; No), the supply pump 7 is activated, a predetermined amount of fresh medium is replenished from the medium tank 5 into the culture tank 1, and the next stage of culture scale is started. 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, in the lower space 110 and upper space 120 of the culture tank 1, the agitation culture on the expanded culture scale is continued (step S80).

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

According to the culture apparatus 100 and the culture method described above, it is possible to replenish a fresh medium in a culture tank with a predetermined shape and structure, so the amount of culture solution is increased stepwise, and during the process, the culture method is changed to static culture. Expansion culture in which the culture is switched to agitation culture can be performed using a single culture tank 1. Common culture vessels and bioreactors often have flat bottoms, and the shape and structure are not optimized, but the lower space 110 of the culture tank 1 is designed to have a shape that satisfies the relationship of formula (I), An appropriate agitation structure increases the specific growth rate and survival rate of cells. Therefore, a plurality of culture steps using different amounts of culture solution and culture methods can be carried out in stages in one culture tank while maintaining good culture efficiency.

In particular, according to the culture apparatus 100 and the culture method described above, it is possible to eliminate the need for transferring the culture solution, so that problems related to the complexity of the work of transferring the culture solution and problems related to the handling of the culture solution and cells can be solved. , it is possible to solve problems related to contamination when transferring the culture solution, etc. According to the culture device 100, it is possible to automate the entire culture process in a completely closed system, which reduces the need for manual intervention, thereby improving the reproducibility of culture and the production amount and quality of the target substance. be able to. Furthermore, since there is no need to prepare separate bioreactors for each culture scale, culture apparatuses and culture facilities can be downsized. Furthermore, since batch culture type expansion culture is performed, the culture environment can be more easily stabilized than in perfusion culture type.

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

FIG. 15 is a diagram schematically showing an example of a culture device according to an embodiment of the present invention.
As shown in FIG. 15, the culture apparatus 200 according to the present embodiment includes a culture tank 1, an agitator 2, an aeration device 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 tube 20, extraction tube 22, return tube 23, recovery tube It is equipped with 24.

The culture device 200, like the culture device 100 described above, is a device for expanding and culturing cells, and has the function of expanding the culture scale by replenishing the culture tank 1 with fresh medium (culture solution) during expansion culture. have. In the culture apparatus 200, by replenishing the culture tank 1 with fresh medium, the culture scale is expanded in stages, and a multi-stage culture process is performed. The culture apparatus 200 shown in FIG. 15 has a configuration in which each culture process is performed in a perfusion culture (continuous culture) format in which a medium is continuously supplied during culture and the same amount of medium is continuously discharged. has been done.

In the culture apparatus 200, the culture method can be switched from static culture to agitation culture while expanding the culture scale step by step. Therefore, perfusion culture type expansion culture with the ultimate goal of large-scale cultivation can be performed in a single culture tank 1. The configuration and main operating method of the culture device 200 are substantially the same as those of the culture device 100 described above, except for the cell separation device 11 and the corresponding pumps and piping.

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

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

The cell separation device 11 uses, for example, a tangential flow membrane separation (TFF) method in which a liquid flows in a direction perpendicular to a separation membrane, or a tangential flow membrane separation (TFF) method in which a liquid flows in a direction perpendicular to a separation membrane to perform suction and discharge to the separation membrane. Various methods can be used, such as Alternating Tangential Flow Filtration (ATF) method, gravity sedimentation separation method, and sonic separation method, which uses ultrasonic vibration to separate differences in size and density. can.

FIG. 16 is a diagram illustrating the shape and structure of the internal space of the culture tank provided in the culture apparatus.
As shown in FIG. 16, the culture tank 1 provided in the culture apparatus 200 has an internal width that is not uniform in the height direction, and a lower space 110 having a relatively narrow inner width and a lower space 110 that has a relatively narrow inner width. The upper space 120 has a wide inner width. The pull-out pipe 22 is connected to the bottom of the lower space 110 that projects downward of the culture tank 1 .

<Culture method (perfusion culture method)>
Next, a culture method according to an embodiment of the present invention will be described with reference to the drawings, as well as processing when the culture apparatus 200 is used.

In the culture apparatus 200, similarly to the above-mentioned culture apparatus 100, the amount of culture solution in the culture tank 1 is expanded in stages by replenishing fresh medium, and a plurality of culture steps with different amounts of culture solution are performed in stages. During this process, the culture method is switched from static culture to agitation culture, and static culture and agitation culture in a culture solution whose volume has been increased by replenishing fresh medium are performed in stages in a single culture tank 1. Expansion culture using perfusion culture using the culture device 200 can be performed in the same manner as the culture device 100 described above, according to the flow shown in FIG. 14 .

In the culture apparatus 200, perfusion culture may be performed only in the culture step of static culture (see step S30 in FIG. 14) or only in the culture step of agitation culture (see step S80 in FIG. 14). However, both of these may be used. Further, among the culture process of static culture and the culture process of agitation culture, only the culture process of a predetermined culture scale may be performed. A culture process that does not involve perfusion culture can be operated in a batch culture manner.

The culture conditions necessary for culturing cells can be set in the culture device 200 similarly to the culture device 100 described above. Items of culture conditions to be set include, in addition to the items of the culture apparatus 100 described above, a culture schedule that defines a culture process in which perfusion culture is performed, and a perfusion amount for each culture process.

During perfusion culture, the withdrawal pump 12 is operated to withdraw the culture solution and cultured cells from the culture tank 1. In addition, the supply pump 7 is operated to replenish the culture solution from the culture medium tank 5 into the culture tank 1. Then, the cell separation device 11 separates the culture solution and cells that have been drawn out from each other. Thereafter, the separated cells are returned to the culture tank, and the suction pump 13 is activated to collect the culture solution containing the substances produced by the cells into the collection tank 10.

When perfusion culture is performed in the culture process of static culture (see step S30 in FIG. 14), the agitator 2 is stopped and the culture scale is maintained so that the liquid level is at a predetermined height in the lower space 110. , perfusion culture is performed under constant and controlled culture conditions. When perfusion culture is used as the culture process for static culture, depletion of nutrients and accumulation of waste products secreted by cells are less likely to occur. Therefore, cells can be grown to a higher cell density per predetermined culture scale. Since the final number of cells increases, it is also possible to downsize the culture tank 1 itself.

On the other hand, when perfusion culture is performed in the culture process of agitation culture (see step S80 in FIG. 14), the culture solution is stirred and the culture scale is maintained such that the liquid level is at a predetermined height in the upper space 120. , perform perfusion culture under variably controlled culture conditions. When perfusion culture is used as the culture step of agitation culture, not only can cells be grown to a higher density on a predetermined culture scale, but also substances secreted by cells into the culture solution can be continuously collected. Since the target substance can be continuously produced in a state where cell proliferation is not easily inhibited, high productivity can be achieved.

According to the culture apparatus 200 and culture method described above, it is possible to replenish a fresh medium into a culture tank having a predetermined shape and structure. The single culture tank 1 can be used to expand culture in which the number of cells is increased stepwise and the culture method is switched from static culture to agitation culture during the expansion. If the lower space 110 of the culture tank 1 has a shape that satisfies the relationship of formula (I) and has a structure with an appropriate stirring system, the specific growth rate and survival rate of cells will increase. Therefore, a plurality of culture steps using different amounts of culture solution and culture methods can be carried out in stages in one culture tank while maintaining good culture efficiency.

In particular, according to the above culture device 200 and culture method, since perfusion culture type expansion culture is performed, it is possible to grow cells to a higher density compared to a batch culture type, and the culture device and culture facility can be expanded. , it can be more easily miniaturized. Even in cases where cells secrete large amounts of substances that inhibit proliferation, it is possible to obtain large amounts of cells and target substances by setting a culture schedule that reduces the number of times of size-up.

Although the present invention has been described above, the present invention is not limited to the embodiments and modified examples described above, and various changes can be made without departing from the spirit of the present invention. For example, the present invention is not necessarily limited to all the configurations of the embodiments and modifications described above. Replace a part of the configuration of a certain embodiment or modification example with another configuration, add a part of the configuration of a certain embodiment or modification example to another form, or replace a part of the configuration of a certain embodiment or modification example with another configuration. You can omit part of it.

For example, the culture apparatuses 100 and 200 described above are not limited in the number of culture medium tanks 5, arrangement of other equipment, connection of piping, etc. The culture tank 1 can have an appropriate form as shown in the shape examples and structure examples, and the arrangement of not only the agitator 2 but also the aeration device 3 and the connection position of the piping are the same as the agitator 2. Can be optimized.

In addition to the aeration device 3, the culture apparatuses 100 and 200 described above can also be provided with an aeration device (not shown) for aerating a predetermined gas into the gas phase portion. In such a case, the air diffuser 3 that diffuses air into the culture solution may be opened to either the lower space 110 or the upper space 120.

Further, in the culture apparatuses 100 and 200 described above, the culture tank 1 and the replenishment apparatus (5, 7) may be connected in advance, or may be connected during culture. When a single-use culture bag is used as the culture tank 1, the stirrer 2 may be fixed in advance in a predetermined arrangement, or may be fixed in a predetermined position during culturing.

Further, the culture apparatuses 100 and 200 described above include a culture medium tank 5 and a supply pump 7 as replenishment devices. However, instead of the supply pump 7, gravity or a pressure difference may be used to supply the fresh culture medium. In such a case, the replenishment device may include only the culture medium tank 5 that replenishes fresh culture medium using gravity or pressure difference. The culture medium tank 5 may include various containers, flexible bags, and the like.

Furthermore, in the culture method using the culture apparatuses 100 and 200 described above (see FIG. 14), production culture is performed after expansion culture in the culture tank 1 is completed. However, the production culture process may be performed using the culture tank 1, or may be performed by transferring from the culture tank 1 or the recovery tank 10 to another culture tank.

Further, the culture method using the culture apparatuses 100 and 200 (see FIG. 14) may have a culture schedule in which the culture process of stationary culture and the culture process of agitation culture are each one-stage culture process. In the above culture method, the timing of starting and stopping the supply pump 7 and the stirrer 2 may be changed to appropriate conditions, or the expanded culture may be carried out in a fed-batch culture manner within a range where the culture scale does not change significantly. Good too.

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

During the step-by-step expansion of the culture scale, expansion culture is performed in which the culture method is changed from static culture to agitation culture using a single culture tank. The culture results were compared.

(experimental method)
As the test cells, CHO cells, which are a floating cell system, were used. Moreover, a liquid medium of DMEM medium was used as the medium. Frozen CHO cells were thawed in a 37° C. water bath and fresh medium was added to prepare a cell suspension with 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 increasing the size, the timing of switching the stirring method, etc. were the same as in the conventional example shown in FIG. 17. A summary of the culture schedule for expansion culture is shown in Table 2.

As shown in Table 2, from culture step I to culture step III, static culture was performed. From culture step IV to culture step IX, stirring culture was performed. Expansion of the culture scale was carried out by replenishing the culture medium with a fresh medium at 37° C. so that the amount of the culture solution was as set after the preset culture time had elapsed. After the completion of culture step IX, the process moved to a separation and purification step in which the substances produced by the cells were collected and purified.

At the end of the culture process for each culture scale, the number of viable cells and cell survival rate were determined. Then, the culture results were compared between cases where the entire culture process was performed using a batch culture method and cases where the entire culture process was performed using a perfusion culture method.

FIG. 18 is a diagram showing the results of expanded culture in a single culture tank.
In FIG. 18, the left axis is the number density of living cells confirmed in the culture tank [×10 ⁵ cells/mL], the right axis is the survival rate [%] of cells confirmed in the culture tank, and the horizontal axis is the number density of living cells confirmed in the culture tank. indicates culture time [days]. The ▲ plot is the result of the number density of living cells in the batch culture method, the △ plot is the result of the survival rate in the batch culture method, the ● plot is the result of the number density of live cells in the perfusion culture method, and the ○ plot is the result of the number density of living cells in the perfusion culture method. The plot is the survival results in a perfusion culture format.

As shown in FIG. 18, it was confirmed that a high survival rate and a certain number of viable cells could be obtained in both the batch culture method and the perfusion culture method. Comparing the results of the batch culture method and the perfusion culture method, it was confirmed that the maximum number density of living cells was five times higher or more in the case of the perfusion culture method.

This result means that when performing expanded 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 culture tank, which is smaller than that in the conventional method, can be used as the culture tank. Furthermore, in the case of the perfusion culture method, no decrease in survival rate was observed during subculture, and it can be said that superior culture efficiency can be achieved compared to the batch culture method.

100 Culture device 200 Culture device 1 Culture 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 Aeration device 4 Temperature control device 5 Culture medium tank 6 Valve 7 Supply pump 8 Analyzer 9 Control device 10 Recovery tank 11 Cell separation device 12 Pulling pump 13 Suction pump 20 Supply pipe 21 Discharge pipe 22 Drawing pipe 23 Return pipe 24 Collection tube 50 Culture solution 52 Vial 53 Flask 54 Spinner flask 55 Bioreactor 110 Lower space 120 Upper space

Claims (5)

A culture method using a culture device for expansion culture that performs agitation culture after static culture ,
The culture device includes:
a culture tank for culturing cells in a culture solution;
a stirrer for stirring the culture solution in the culture tank;
a replenishment device for replenishing the culture tank with fresh culture medium,
The lower space in the culture tank has a shape that protrudes downward and has an inner width narrower than the upper space,
The lower space in the culture tank has a volume of the culture solution placed in the lower space as V [cm ³ ], and an area of the interface between the culture solution placed in the lower space and the gas phase as S [cm ² ]. Then, the following formula (I);
S/V≦8[cm ^-1 ]...(I)
It is a shape that satisfies the relationship,
The agitator has a stirring blade and a rotating shaft that rotates the stirring blade,
The stirring blade is located only in the upper space within the culture tank,
In the culture method,
The cells are statically cultured in a culture solution in the lower space of the culture tank,
After the static culture, replenishing the culture tank with a fresh medium,
A culture method in which the cells are agitated and cultured in a culture solution whose volume is increased by supplementing the fresh medium. The culture method according to claim 1 ,
In the stationary culture, the culture method comprises controlling the gas concentration in the gas phase part in the culture tank to be constant. The culture method according to claim 1 ,
In the stirring culture, the culture solution is stirred by a stirring blade located in an upper space in the culture tank. The culture method according to claim 1 ,
In at least one of the static culture and the agitation culture, the amount of culture solution in the culture tank is expanded by replenishing a fresh medium, and culture is performed with different amounts of culture solution. The culture method according to claim 1 ,
In at least one of the static culture and the agitation culture, the culture solution and the cells are pulled out from the culture tank, and the culture solution is replenished into the culture tank, and the culture solution and the cells that have been pulled out are removed. A culture method that performs perfusion culture in which the cells are separated from each other and the separated cells are returned to the culture tank.

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