JP7231425B2 - Culture system, culture method and method for producing cultured product - Google Patents

Description

The present invention provides a culture system, a culture method, and a method for producing a culture product.

Biopharmaceuticals such as antibody drugs can be produced by culturing cells that produce proteins that serve as active ingredients. In this case, biopharmaceuticals can be produced by purifying proteins secreted from cells.

Cell culture methods are classified into, for example, batch culture, fed-batch culture, and continuous culture. Among these, batch culture is a method in which a new medium is prepared each time, a cell line is planted in the prepared medium, and no medium is added until harvest. With batch culture, the quality tends to vary from culture to culture, but the risk of contamination can be dispersed or reduced. Fed-batch culture, also called semi-batch culture, is a culture method in which the medium itself or specific components in the medium are added during the culture and the product is not withdrawn until the end of the culture. Fed-batch culture is usually performed using large production facilities.

Continuous culture, also called perfusion culture, is a culture method in which a culture medium is continuously supplied to a culture system and the same amount of culture solution is withdrawn at the same time. In continuous culture, when extracting the culture medium, not only the target substance but also waste products such as ammonia and lactic acid that hinder cell growth are extracted. Therefore, cells can be cultured at high density. Further, in continuous culture, it is easy to keep the culture environment constant, and productivity can be stabilized.

In recent years, attention has been focused on continuous culture, which allows downsizing of production equipment. As a technique related to continuous culture, the technique described in Patent Document 1 is known. US Pat. No. 5,400,003 discloses an apparatus for growing cells, comprising at least one bioreactor for cell culture, at least one vessel for culture medium, and a culture medium in fluid communication between the bioreactor and the vessel. and/or comprising means for circulating the cell culture and at least one means for supplying oxygen.

Japanese Patent Application Publication No. 2003-521877 (especially see FIG. 1)

By the way, the oxygen consumption per unit culture solution varies depending on the density (concentration) of culture objects such as cells. Specifically, for example, if the culture target has a low density, the oxygen consumption per unit culture solution is small, and if the culture target has a high density, the oxygen consumption per unit culture solution is high. Therefore, it is preferable to control the dissolved oxygen concentration so that the dissolved oxygen concentration is suitable for the culture object density.

However, especially when the object to be cultured has a high density, when the culture solution containing the object to be cultured is brought into contact with a dissolved oxygen concentration measuring device (usually an electrode unit provided in the dissolved oxygen concentration measuring device), the electrode portion may cause the culture It becomes easier for objects to adhere. As a result, it becomes difficult for the dissolved oxygen in the culture solution to come into contact with the electrode portion due to the adhering culture target, and the measurement accuracy of the dissolved oxygen concentration decreases. As a result, the dissolved oxygen concentration of the culture solution containing the culture target cannot be properly evaluated, and the culture of the culture target may be affected due to inappropriate dissolved oxygen concentration control.

An object of the present invention is to provide a culture system, a culture method, and a method for producing a culture product that can appropriately culture an object to be cultured.

The present invention provides a culture vessel for culturing a culture object that produces a culture product in a medium, a separation device for separating the culture object from a culture solution containing the culture object cultured in the culture container, and A dissolved oxygen concentration measuring device that measures the dissolved oxygen concentration of the culture medium after the separation of the culture object in the separation device, and a dissolved oxygen concentration measuring device formed between the culture vessel and the dissolved oxygen concentration measuring device, Based on a medium system formed of a gas-permeable material while one medium flows, the dissolved oxygen concentration measured by the dissolved oxygen concentration measuring device, and the flow conditions of the medium flowing through the medium system, the culture vessel and an arithmetic device having an oxygen supply amount determination unit that determines the oxygen supply amount by the culture medium supplied to the culture system. Other solutions will be described later in the detailed description.

According to the present invention, it is possible to provide a culture system, a culture method, and a method for producing a culture product that can appropriately culture an object to be cultured.

1 is a system diagram of a culture system according to a first embodiment; FIG. It is a graph which shows the saturated dissolved oxygen concentration with respect to the oxygen content rate in oxygen-containing gas. FIG. 2 is a block diagram of an arithmetic unit provided in the culture system shown in FIG. 1; It is a figure which shows the content of a flow condition database. 4 is a flow chart showing the culture method of the first embodiment. 1 is a system diagram of a culture system used in a simulated culture test; FIG. It is a graph which shows the amount of air supply with respect to the amount of nitrogen supply. It is a graph showing changes in dissolved oxygen concentration with respect to test time, and is a graph when the nitrogen gas flow rate to the culture vessel is 30 mL/min. It is a graph showing changes in dissolved oxygen concentration with respect to test time, and is a graph when the nitrogen gas flow rate to the culture vessel is 50 mL/min. 4 is a graph showing changes in dissolved oxygen concentration (medium and culture solution after separation of culture objects) and biological cell density with respect to the number of culture days. FIG. 4 is a system diagram of a culture system according to a second embodiment; FIG. FIG. 10 is a system diagram of a culture system according to a third embodiment; FIG. FIG. 10 is a system diagram of a culture system according to a fourth embodiment; FIG. FIG. 11 is a system diagram of a culture system according to a fifth embodiment;

EMBODIMENT OF THE INVENTION Hereinafter, the form for implementing this invention is demonstrated, referring drawings. However, the present invention is not limited to the following examples, and can be arbitrarily modified within the scope of the present invention. Moreover, each embodiment can be combined arbitrarily and implemented. Also, the same devices and systems are denoted by the same reference numerals, and overlapping descriptions are omitted.

FIG. 1 is a system diagram of the culture system 100 of the first embodiment. The culture system 100 is, for example, a system for culturing a culture object containing at least one type of cells selected from human cells and animal cells. At least one of human cells and animal cells includes, for example, Chinese hamster ovary cells (CHO cells). However, the culture object is not limited to these cells, and may include, for example, microorganisms (bacteria, yeast, etc.).

In the culture system 100, a culture target is cultured in a medium. By culturing the culture object, the culture object produces a culture product. Any medium can be used as long as the object to be cultured can be cultured. When the object to be cultured is, for example, CHO cells, the medium includes, for example, Dulbecco's Modified Eagle Medium (DMEM medium). Culture products also include, for example, proteins of interest produced by culture subjects. For example, biopharmaceuticals (antibody drugs) can be produced by producing the target protein.

In this specification, a medium containing a culture object (a medium containing a cultured object) is referred to as a “culture solution”. Strictly speaking, the liquid obtained by removing the object to be cultured from the culture solution will be called a "medium" for the sake of convenience, though strictly speaking, the composition is different from that of the medium before use for culture due to the nutritional consumption by the object to be cultured.

A culture system 100 includes a culture container 1 , a separator 2 , and a dissolved oxygen concentration measuring device 3 . The culture system 100 also includes a culture fluid system 31 , medium systems 32 , 34 , 35 , a culture target system 33 , and a medium system 36 . Furthermore, the culture system 100 is a system for performing perfusion culture, and includes a culture medium system 34 (first culture medium supply system) that supplies culture medium after separation of the culture target in the separation device 2 to the culture container 1 . The culture medium system 34 can use the nutrients of the culture medium without waste, reduce the amount of culture medium discharged to the outside, and reduce the waste liquid treatment cost.

The culture vessel 1 is for culturing an object to be cultured in a medium. The culture vessel 1 is capable of continuous culture. The culture vessel 1 is supplied with the medium from the medium storage container 4 through the medium system 35 . In addition, the culture solution containing the object to be cultured in the culture container 1 is supplied to the separator 2 through the culture solution system 31 .

In the illustrated example, the culture vessel 1 includes a stirring tank type culture vessel (for example, a stainless steel vessel) provided with a stirring blade 7 . However, the culture vessel 1 is not limited to a stirring tank type culture vessel, and may be, for example, a shaking agitation type culture vessel (for example, a resin culture bag) that can be agitated by shaking, rocking, or the like. may

The culture container 1 includes a gas supply device 5 for supplying an oxygen-containing gas to the culture solution in the culture container 1, and a stirring blade 7 for stirring the culture solution. During culturing, the culture solution is stirred by driving the stirring blade 7 while oxygen-containing gas is supplied by the gas supply device 5.

The gas supply device 5 includes, for example, a submerged aeration device, and more specifically includes, for example, a sparger. The sparger can increase the efficiency of gas dissolution into the solution. As the sparger, for example, any one or more of a ring sparger, a sintered sparger, and the like can be used. However, the gas supply device 5 is not limited to a sparger, and may be, for example, an air diffuser. Further, the gas supply device 5 is not limited to the submerged aeration device, and may be a liquid surface aeration device. As the liquid surface aeration device, for example, any one or more of a device capable of supplying oxygen to the liquid surface of the medium, a membrane surface aeration device equipped with a gas exchange membrane, and the like can be used.

The amount of oxygen-containing gas supplied by the gas supply device 5 (aeration amount per unit time) is, for example, the amount of oxygen-containing gas supplied by the gas supply device 6 in the culture medium storage container 4 described later (per unit time airflow). In other words, the medium can be supplied mainly by the gas supply device 6 (the second gas supply device), and the culture solution can be supplementarily supplied by the gas supply device 5 . By doing so, it is possible to reduce the number of air bubbles that come into contact with the culture solution, and to reduce the influence on the culture object. In addition, a sufficient amount of oxygen can be supplied to the culture medium by supplying gas to the culture medium containing no culture target in the culture medium storage container 4 .

The oxygen-containing gas supplied by the gas supply devices 5 and 6 may be, for example, a gas partially containing oxygen, such as air, or a gas consisting only of oxygen (pure oxygen). As the oxygen-containing gas, air is preferable from the viewpoint of the production cost of the culture product, but pure oxygen is preferable from the viewpoint that the dissolved oxygen concentration can be increased by a high partial pressure.

FIG. 2 is a graph showing the saturated dissolved oxygen concentration with respect to the oxygen content ratio in the oxygen-containing gas. This graph shows the results at 37° C., which is suitable for culturing human and animal cells, for example. The dashed line in FIG. 2 is the saturated dissolved oxygen concentration (6.86 mg/L) when air is dissolved in water. A gas with an oxygen content of 100% on the horizontal axis is pure oxygen.

As indicated by the black dot plot, the saturated dissolved oxygen concentration increases in proportion to the oxygen content in the oxygen-containing gas. Therefore, by supplying (dissolving) an oxygen-containing gas having a high oxygen content (for example, pure oxygen), the dissolved oxygen concentration can be effectively increased. In particular, the dissolved oxygen in the culture solution is consumed due to respiration in the culture solution by the culture object, and the dissolved oxygen concentration decreases. In particular, as the culture target density increases, the dissolved oxygen consumption rate increases. Therefore, by using an oxygen-containing gas with a high oxygen content rate, the dissolved oxygen concentration can be recovered quickly even when the consumption rate is high, and effective control becomes possible.

Returning to FIG. 1, in the culture system 100, the amount of oxygen-containing gas supplied by the gas supply devices 5 and 6 is not controlled based on the density of the culture object, culture time, and the like. However, the amount supplied by the gas supply devices 5 and 6 (only one of them may be sufficient) is controlled based on the density of the object to be cultured, the culture time, etc., as shown in FIGS. can be

The culture vessel 1 is supplied with the medium stored in the medium storage container 4 through the medium system 35 . Therefore, the medium storage container 4 is provided in the culture system 100 and serves to store the medium supplied to the culture container 1 . The culture system 100 also includes a gas supply device 6 (second gas supply device) that supplies an oxygen-containing gas to the culture medium stored in the culture medium storage container 4 . The culture medium storage container 4 is equipped with a stirring blade 8, and by driving the stirring blade 8 in the culture medium, oxygen in the oxygen-containing gas supplied from the gas supply device 6 quickly dissolves in the entire culture medium. Then, an oxygen-enriched medium is obtained by oxygen dissolution, and the oxygen-enriched medium is supplied to the culture vessel 1 .

Since the medium stored in the medium storage container 4 does not contain the object to be cultured, the gas supply in the medium storage container 4 can suppress the influence of air bubbles on the object to be cultured. In addition, since the oxygen-containing gas is supplied to the medium containing no culture target, the oxygen-containing gas can be supplied to the medium without considering the influence on the culture target. This makes it easier to control the dissolved oxygen concentration of the medium to the set value.

The amount of medium supplied to the culture container 1 is adjusted by controlling the rotational speed of the inverter-controlled pump 13 . In addition, for example, usually, the medium is mainly circulated through the medium system 36 (described later), and only when the culture time in the culture container 1 becomes long and the nutrients in the culture solution in the culture container 1 become insufficient, A culture medium can be supplied to the culture container 1 .

The separation device 2 is for separating the culture object from the culture medium containing the culture object cultured in the culture vessel 1 . That is, the separation device 2 separates the culture object and the medium containing the culture product from the culture medium containing the culture object and the culture product produced by the culture object. The culture solution is supplied from the culture container 1 to the separator 2 by driving the pump 11 provided in the culture solution system 31 . The cultured product is usually soluble in the medium, while the cultured object is usually in slurry form. Therefore, in the separation device 2, the object to be cultured and the medium containing the cultured product can be separated. The separated culture object is returned to the culture container 1 through the culture object system 33 . On the other hand, the separated medium is liquid, and the liquid medium is supplied to the dissolved oxygen concentration measuring device 3 (described later) through the medium system 32 .

The separation device 2 comprises a separation membrane in the example shown. Separation by a separation membrane may be performed using an apparatus using one or more of the tangential flow filtration method (TFF method), the alternative tangential flow filtration method (ATF method), and the like. However, the separation device 2 is not limited to a separation membrane, and may be one or more of, for example, a gravity sedimentation device that sediments the culture object by gravity, a centrifugal separator that separates the culture object from the medium by centrifugal force, a spin filter, and the like. There may be.

The dissolved oxygen concentration measuring device 3 is for measuring the dissolved oxygen concentration of the culture medium after the culture object is separated by the separation device 2 . The dissolved oxygen concentration measuring device 3 is provided in the culture medium system 32 . That is, the culture medium system 32 is equipped with the dissolved oxygen concentration measuring device 3 . By measuring the dissolved oxygen concentration in the culture medium instead of the culture medium, the dissolved oxygen concentration in the culture medium can be measured without being affected by the culture target. In particular, the dissolved oxygen concentration measuring device 3 (specifically, the dissolved oxygen concentration measuring device 3 Dirt easily adheres to the electrode part that is normally provided. As a result, the measurement accuracy of the dissolved oxygen concentration is lowered. However, by measuring the dissolved oxygen concentration in the medium that does not contain the culture target, instead of the culture solution containing the culture target, the dissolved oxygen concentration of the medium can be measured without being affected by the contamination caused by the culture target, waste products, etc. Oxygen concentration can be measured with high accuracy.

After measuring the dissolved oxygen concentration, the medium is taken out of the system together with the cultured product through the medium system 32 . The medium containing the cultured product is continuously taken out by driving the pump 12 provided in the medium system 32 .

As the dissolved oxygen concentration measuring device 3, any type of dissolved oxygen concentration measuring device such as a polarographic method or a galvanic cell method can be applied. The dissolved oxygen concentration measured by the dissolved oxygen concentration measuring device 3 is input to the computing device 21 described later.

Part of the medium separated by the separation device 2 is supplied to the culture vessel 1 through a medium system 34 (first supply system) that supplies the culture medium after the separation of the object to be cultured by the separation device 2 to the culture vessel 1. be done. The culture medium system 34 is formed by branching from the branch A of the culture medium system 32 . The culture medium system 34 includes a gas supply device 15 (first gas supply device) that supplies an oxygen-containing gas to the culture medium after separation of the culture object. Specifically, oxygen is dissolved in the flowing medium by supplying oxygen-containing gas from the gas supply device 15 , and the oxygen-dissolved medium flows through the medium system 34 and is supplied to the culture vessel 1 .

For the gas supply device 15, for example, the same system as the gas supply devices 5 and 6 can be applied. The amount of oxygen-containing gas supplied by the gas supply device 15 can be adjusted, for example, so that the concentration of dissolved oxygen in the medium flowing through the medium system 34 becomes saturated. Therefore, the dissolved oxygen concentration of the culture medium in the culture container 1 can be adjusted, for example, by controlling the rotational speed of the pump 14 (dissolved oxygen concentration adjusting device) provided in the culture medium system 34 and controlled by an inverter. That is, the culture medium system 34 (first supply system) includes a pump 14 (dissolved oxygen concentration adjusting device) that adjusts the dissolved oxygen concentration of the culture medium in the culture vessel 1 . The dissolved oxygen concentration of the culture medium in the culture container 1 can be adjusted by the pump 14 . A specific adjustment method will be described later with reference to FIG.

The culture system 100 includes a medium system 36 formed between the culture container 1 and the dissolved oxygen concentration measuring device 3 and through which either a culture solution or a culture medium flows. Here, in the culture system 100, the medium system 36 is formed so that the medium circulates. Therefore, the medium system 36 includes a system that flows from the culture container 1 toward the dissolved oxygen concentration measuring device 3 and a system that flows from the dissolved oxygen concentration measuring device 3 toward the culture container 1 .

The medium system 36 includes a culture solution system 31 formed between the culture container 1 and the separation device 2, a medium system 32 formed between the separation device 2 and the dissolved oxygen concentration measuring device 3, and a medium system 32. Among them, the culture medium system 32 formed between the dissolved oxygen concentration measuring device 3 and the branch A, and the culture medium system 34 formed between the branch A and the culture vessel 1 are included. The medium system 36 is formed, for example, by piping (a tube may be used) made of a gas-permeable material.

Gas permeable materials include, for example, resins. Resins include, for example, one or more of polystyrene, polysulfone, polytrimethylene terephthalate, polyetheretherketone, polypropylene, and the like. Since resin is easy to incinerate, when changing the type of culture object in the culture system 100, the used medium system 36 can be incinerated as it is, and a new gas-permeable material can be used to restore the The risk of contamination due to residue can be reduced.

By the way, the dissolved oxygen concentration of the culture solution in the culture container 1 can be adjusted by the pump 14 as described above. Specifically, as described above, the dissolved oxygen concentration of the culture solution can be adjusted by controlling the rotational speed of the pump 14 . Rotational speed control of the pump 14 is performed based on a signal input from the control device 22 through an electric signal line indicated by a dashed arrow in FIG. The control device 22 generates a drive signal for the pump 14 based on the calculation result of the calculation device 21 . The drive signal for the pump 14 is generated by the control device 22 based on the signal input from the arithmetic device 21 through the electric signal line indicated by the dashed arrow in FIG. Here, the arithmetic device 21 will be described with reference to FIG.

FIG. 3 is a block diagram of the computing device 21 provided in the culture system 100 shown in FIG. The computing device 21 includes a measurement unit 21a, an oxygen supply amount determination unit 21b, a signal transmission unit 21c, and a flow condition database 21d. The measurement unit 21a is for measuring the dissolved oxygen concentration of the culture medium using the dissolved oxygen concentration measuring device 3 (see FIG. 1). The measured dissolved oxygen is input to the oxygen supply amount determination unit 21b.

The oxygen supply amount determination unit 21b determines the oxygen supply amount from the medium supplied to the culture vessel 1 based on the dissolved oxygen concentration measured by the dissolved oxygen concentration measuring device 3 and the flow conditions of the medium flowing through the medium system 36. It is for decision making. As described above, the medium system 36 is made of, for example, a gas permeable material from the viewpoint of contamination suppression. Therefore, oxygen in the outside air may be absorbed by the medium between the culture vessel 1 and the dissolved oxygen concentration measuring device 3, and the dissolved oxygen concentration of the medium may fluctuate.

Therefore, in the culture system 100, the amount of oxygen supplied to the medium in the culture vessel 1 (the amount of medium with saturated dissolved oxygen concentration supplied) is adjusted in consideration of such fluctuations. For example, if the medium flowing through the medium system 36 is susceptible to the outside air (easily absorbs gas), a medium having a dissolved oxygen concentration lower than the set value of the dissolved oxygen concentration for the culture solution is supplied. On the other hand, when it is difficult to be affected by the outside air (difficult to absorb gas), a medium having a dissolved oxygen concentration close to the set value of the dissolved oxygen concentration for the culture solution (may be exactly the set value) is supplied. Then, whether or not it is likely to be affected by outside air is determined based on the following flow conditions. By determining the amount of oxygen to be supplied based on the flow conditions, the dissolved oxygen concentration can be controlled in consideration of the influence of the outside air, and the dissolved oxygen concentration can be controlled with high accuracy.

The flow conditions include the material (which may include the thickness) of the piping that constitutes the medium system 36, the internal dimensions, the system length (pipe length) of the piping that constitutes the medium system 36, and the medium flowing through the medium system 36. flow rate. The inner dimension referred to here is the inner dimension of the cross section in the direction perpendicular to the medium flow direction in the medium system 36 . The inner dimension corresponds to the inner diameter of the pipe, for example, when the pipe is a circular pipe, and corresponds to the length of each side of the pipe, for example, when the pipe is a square pipe. In addition, the inner dimension of the pipe determines the cross-sectional area (passage cross-sectional area) in the pipe in the cross section in the direction perpendicular to the medium flow direction.

Regarding the above conditions, for example, among gas-permeable materials, there are constituent materials that allow gas to permeate relatively easily, and constituent materials that allow gas to permeate relatively poorly. Furthermore, the amount of gas permeation also varies depending on the inner dimensions of the piping that constitutes the medium system 36 . That is, the gas concentration in the medium also changes depending on the cross-sectional area of the flow path calculated from the inner dimensions. Therefore, the amount of oxygen supply is adjusted in consideration of the material and inner dimensions of the medium system 36 .

Further, if the system length of the medium system 36 is relatively long, the oxygen absorption amount increases, and if the system length of the medium system 36 is relatively short, the oxygen absorption amount decreases. Therefore, the oxygen supply amount is adjusted in consideration of the system length of the medium system 36 . The gas concentration in the medium varies depending on the internal volume of the medium system 36 determined based on the internal dimensions and system length. Furthermore, if the flow rate of the medium flowing through the medium system 36 is high, the amount of oxygen absorbed increases, and if the flow rate of the medium flowing through the medium system 36 is low, the amount of oxygen absorbed decreases. Therefore, the oxygen supply amount is adjusted in consideration of the flow rate of the medium flowing through the medium system 36 .

The flow conditions are determined in advance through test runs, experiments, etc., and stored as, for example, the flow condition database 21d. The flow condition database 21d will be described with reference to FIG.

FIG. 4 is a diagram showing the contents of the flow condition database 21d. In the flow condition database 21d, the materials of the pipes forming the medium system 36, the inner dimensions of the pipes forming the medium system 36, the system length of the medium system 36, and the flow rate of the medium flowing through the medium system 36 are stored. The difference from the set value of the dissolved oxygen concentration of the culture medium in 1 is stored. For example, as an example, the constituent material of the medium system 36 is X1, the inner dimension of the medium system 36 is Y1, the system length of the medium system 36 is Z1, and the flow rate of the medium flowing through the medium system 36 is P1. Occasionally, the rotation speed of the pump 14 is controlled so that the amount of oxygen supplied is the dissolved oxygen concentration set value for the culture solution in the culture container 1 reduced by Q1. Similarly, when the flow rate is P2, the rotational speed of the pump 14 is controlled so that the dissolved oxygen concentration is reduced by Q2 from the dissolved oxygen concentration target value. Furthermore, when the flow rate is P3, the rotation speed of the pump 14 is controlled so that the dissolved oxygen concentration is reduced by Q3 from the dissolved oxygen concentration target value.

Also, if there is no applicable condition, the difference corresponding to the closest flow condition in the flow condition database 21d is used. Instead of using the flow condition database 21d, for example, a numerical formula based on statistical processing (multivariate analysis, etc.) of experimental data relating to flow conditions may be calculated, and the rotation speed of the pump 14 may be controlled based on the calculated formula. .

Returning to FIG. 3, the signal transmission unit 21c is for generating a rotational speed signal of the pump 14 that will provide the oxygen supply amount determined by the oxygen supply amount determination unit 21b, and transmitting the signal to the pump 14. FIG. Sending the rotational speed signal to the pump 14 drives the pump 14 at the desired rotational speed.

The computing device 21 includes a CPU, a ROM, a RAM, and an I/F, although none of them are shown. Then, the arithmetic device 21 is embodied by executing the program recorded in the ROM by the CPU.

FIG. 5 is a flow chart showing the culture method of the first embodiment. The flowchart shown in FIG. 5 is performed in the culture system 100 shown in FIG. 1 above. Therefore, FIG. 5 will be described with appropriate reference to FIG.

The culture method of the first embodiment includes a culture step S1, a separation step S2, a dissolved oxygen concentration measurement step S3, and a control step S4. The culturing step S1 is to culture an object to be cultured to produce a culture product in a medium. The culture product and the culture object are synonymous with the culture product and the culture object explained with reference to FIG. 1, so the explanation is omitted. Culturing in the culturing step S1 is performed in the culture container 1 .

The separation step S2 separates the culture object from the culture solution containing the culture object cultured in the culture step S1. The separation step S2 is performed in the separation device 2 . The separated culture object is returned to the culture container 1 . In addition, the culture solution after the separation of the culture object, that is, the remaining culture medium is supplied to the dissolved oxygen concentration measuring device 3 as described above.

The dissolved oxygen concentration measurement step S3 measures the dissolved oxygen concentration of the culture medium after the culture object is separated in the separation step S2. The dissolved oxygen concentration measurement step S<b>3 is performed by the dissolved oxygen concentration measurement device 3 .

The control step S4 controls the amount of oxygen supplied to the culture vessel 1 (that is, the dissolved oxygen concentration of the culture solution in the culture vessel 1) based on the dissolved oxygen concentration measured by the dissolved oxygen concentration measuring device 3. The amount of oxygen supplied to the culture vessel 1 is adjusted by controlling the rotational speed of the pump 14 . The oxygen supply amount adjustment is performed based on the dissolved oxygen concentration measured by the dissolved oxygen concentration measuring device 3 and the flow conditions of the medium flowing through the medium system 36 . Since the flow conditions are the same as those described with reference to FIG. 4 above, description thereof is omitted.

In addition, by culturing the object to be cultured by the above culture method, a culture product can be produced using the object to be cultured. That is, the method for producing the cultured product of the first embodiment includes at least the culture step S1, the separation step S2, and the dissolved oxygen concentration measurement step S3. The culture product can then be removed from culture system 100 through medium system 32 along with the medium.

Here, in order to make the dissolved oxygen concentration in the culture vessel 1 constant, the culture system 100 is used to evaluate how the amount of oxygen supplied to the medium supplied to the culture vessel 1 changes. A simulated culture test was performed using the culture system 50 that was used.

FIG. 6 is a system diagram of the culture system 50 used in the simulated culture test. In the simulated culture test according to Experimental Example 1, instead of oxygen consumption by the culture target, nitrogen was supplied to the medium to expel oxygen from the medium, thereby simulating the culture of the culture target. Therefore, the culture vessel 1 is provided with a gas supply device 5 for supplying an oxygen-containing gas and a gas supply device 9 for supplying nitrogen (having the same configuration as the gas supply device 5).

Phosphate buffer solution (PBS) was used instead of the medium. Furthermore, since the culture target was not included, the separation device 2 was not provided, and the culture solution (PBS) extracted from the culture container 1 was returned to the medium storage container 4 as it was after measuring the dissolved oxygen concentration with the dissolved oxygen concentration measuring device. For simplification of the experiment, the medium system 35 and the medium system 36 were made of gas-impermeable material (stainless steel).

First, 700 mL of PBS was placed in the culture vessel 1, and the culture vessel 1 was connected by the medium system 35 to the medium storage vessel 4 in which a sufficient amount of PBS was stored. The culture medium storage container 4 was provided with a gas supply device 6, and air was supplied by the gas supply device 6 to make the PBS in the culture medium storage container 4 into an oxygen-enriched state (saturated dissolved oxygen concentration). The rotational speeds of the pumps 11 and 13 were set to be the same, and the PBS was circulated at a constant flow rate. Then, the nitrogen supply from the gas supply device 9 in the culture vessel 1 is performed at a constant flow rate, and the dissolved oxygen concentration measured by the dissolved oxygen concentration measurement device 3 is kept constant (3.2 mg / L as a set value). , air was supplied to the PBS from the gas supply device 6 .

As Experimental Example 2, a simulated culture test was conducted in the same manner as in Experimental Example 1 except that the medium system 35, the medium system 36 and the medium storage container 4 were not provided, that is, the medium was not circulated.

FIG. 7 is a graph showing the amount of air supplied versus the amount of nitrogen supplied. A black circle indicates Experimental Example 1, and a black triangle indicates Experimental Example 2. The air supply amount on the vertical axis is shown as a relative value when the nitrogen supply amount in Experimental Example 2 is set to 1. Also, the air supply amount is the total amount of air supply when a constant amount of nitrogen is supplied. In both Experimental Examples 1 and 2, when the amount of nitrogen supply increased, that is, the density of the culture object increased, the amount of air supplied to keep the dissolved oxygen concentration constant also increased. However, in Example 1, compared to Experimental Example 2, the amount of air supplied was smaller when the amount of nitrogen supplied was the same.

In particular, when the nitrogen supply rate was 30 mL/min, a significant difference was observed in the air supply rate. Specifically, in Experimental Example 1 in which the oxygen-enriched culture medium was circulated by the medium system 36, the air supply amount was 0.5, whereas in Experimental Example 2 in which the oxygen-enriched culture medium was not circulated, The air supply rate was 0.84. Therefore, it was found that by circulating the oxygen-enriched culture medium, the amount of air supply can be reduced by about 0.6 times. Therefore, it was found that the dissolved oxygen concentration can be easily controlled because the dissolved oxygen concentration can be adjusted to the set value with a small amount of air by circulating the culture medium.

FIG. 8 is a graph showing changes in dissolved oxygen concentration with respect to test time, and is a graph when the nitrogen gas flow rate to the culture vessel 1 is 30 mL/min. In both Experimental Examples 1 and 2, the dissolved oxygen concentration remained around the set value of 3.2 mg/L. Therefore, it was found that when the nitrogen gas flow rate was 30 mL/min, the dissolved oxygen concentration in the medium could be brought close to the set value regardless of whether the medium was circulated or not.

FIG. 9 is a graph showing changes in dissolved oxygen concentration with respect to test time, and is a graph when the nitrogen gas flow rate to the culture vessel 1 is 50 mL/min. In both Experimental Examples 1 and 2, the dissolved oxygen concentration remained slightly lower than the set value of 3.2 mg/L. However, in Experimental Example 1 in which the medium was circulated, unlike Experimental Example 2 in which the medium was not circulated, the dissolved oxygen concentration could sometimes rise to the set value (for example, B, C, and D in FIG. 9 , E). Normally, a variation of about 10% to 20% with respect to the set value is permissible, so Experimental Example 2, which is 2.5 mg/L or more, which is 20% smaller than the set value, is sufficiently permissible. However, it was found that circulation of the medium allowed the dissolved oxygen concentration of the medium to approach sufficiently close to the set point.

As shown in Figures 8 and 9, there is variation in the dissolved oxygen concentration of the medium. When the culture object is actually cultured, if the dissolved oxygen concentration of the culture solution is to be directly measured, the measured value of the dissolved oxygen concentration will be large due to the influence of the culture object on the dissolved oxygen concentration measuring device 3. expected to fluctuate. As a result, the dissolved oxygen concentration tends to deviate greatly from the set value (described later with reference to FIG. 10). However, by measuring the dissolved oxygen concentration of the culture solution after the separation of the culture object, that is, the medium, the influence of the culture object can be suppressed. As a result, as shown in FIGS. 8 and 9, which measure the dissolved oxygen concentration of the culture medium, the dissolved oxygen concentration of the culture solution can be stably measured for a long period of time. As a result, the dissolved oxygen concentration in the medium can be controlled with high accuracy, and appropriate culture of the culture target and efficient production of culture products are possible.

Next, the culture object was actually cultured, and the influence of the culture object on the dissolved oxygen concentration measuring device 3 was evaluated.

Culture of the culture target was performed using the culture system 100 shown in FIG. However, for simplification of evaluation, all systems including the medium system 36 were made of a gas-impermeable material (stainless steel). CHO cells were used as culture objects, and DMEM medium (containing 10% serum, pH 7.2) was used as the medium. A culture medium of 700 mL was placed in the culture vessel 1, and the seeding density of the object to be cultured in the culture vessel 1 was set to 0.2 cells/mL.

By controlling the rotational speed of the pump 14 provided in the medium system 36, the flow rate of the medium flowing through the medium system 36 (circulation amount of medium) was set to 10 mL/min. As the separation device 2, a TFF separation device using a membrane was used. Cultivation of the object to be cultured was performed at 37° C., and the dissolved oxygen concentration in the culture container 1 was set to 2.7 mg/L, and the perfusion rate was set to 1 vvd. "vvd" represents the amount of culture fluid inside the culture vessel 1 that is replaced per day, and 1 vvd means that the entire culture fluid inside the culture vessel 1 is replaced with new medium once.

Cultivation was carried out continuously for 30 days while circulating the medium. During the culture, the dissolved oxygen concentration (measured by the dissolved oxygen concentration measuring device 3) of the culture medium after the culture target was separated was measured at regular intervals. For comparison, the dissolved oxygen concentration of the culture solution inside the culture vessel 1 (measured by a dissolved oxygen concentration measuring device (not shown)) was also measured during the measurement by the dissolved oxygen concentration measuring device 3 . Furthermore, the live culture object number density (live culture object (living cell) number density) at the time of dissolved oxygen concentration measurement was also measured. The results are shown in FIG.

FIG. 10 is a graph showing changes in dissolved oxygen concentration (medium and culture solution after separation of culture objects) and live culture object number density with respect to culture days. In the graph, the horizontal axis indicates the number of culture days, the left vertical axis indicates the dissolved oxygen concentration, and the right vertical axis indicates the number density of living cultured objects (sperm cell number density). The graph indicated by the thick solid line indicates the dissolved oxygen concentration in the culture medium after the culture object was separated (Example), and the graph indicated by the thin solid line indicates the dissolved oxygen concentration in the culture solution (Comparative Example). The plot also shows the live culture object number density. In FIG. 10, when the number of days of culture exceeds 25 days, the number density of living cultured objects tends to decrease, so the dissolved oxygen concentration and the number density of living cultured objects after 25 days are partially omitted. ing.

The number density of living cultured objects increased with the passage of culture days, reaching a high density state by about 25 days. However, the number density of live culture objects reached an upper limit at approximately 25 days and then decreased. In addition, the dissolved oxygen concentration in the medium after the separation of the culture object (example indicated by the thick solid line) was measured as a value near the set value of 2.7 mg/L during the entire culture period. However, the dissolved oxygen concentration of the culture solution (comparative example indicated by a thin solid line) was measured as a value close to the set value of 2.7 mg/L when the number of culture days was short (for example, about 0 to 15 days). However, for example, it turned to decrease after 15 days, and after 15 days, the dissolved oxygen concentration was measured as a value greatly deviated from the set value. In particular, on the 25th day when the number density of living cultured objects was the highest, the measured dissolved oxygen concentration in the culture medium was 1.3 mg/L, which was less than half of the set value. Therefore, it can be seen that the concentration of dissolved oxygen in the culture solution is affected particularly when the number density of the living cultured objects becomes high, and the accuracy of the measured value decreases.

In particular, in this evaluation, the entire system is made of a gas-impermeable material as described above, and oxygen in the outside air is not absorbed by the culture medium. Since the dissolved oxygen concentration in the culture medium remained near the set value throughout the entire period, it can be said that the oxygen supplied by the gas supply device 15 was approximately the same as the amount of oxygen consumed by respiration of the culture object. Therefore, it is possible to suppress the influence of excessive air supply on the object to be cultured. In addition, it is possible to suppress the operating cost by supplying the air with little excess or deficiency. Moreover, these effects become greater as the number of culture days elapses, and stable culture can be carried out over a long period of time.

According to the culture system 100 described above and, for example, the culture method and culture product production method using the culture system 100 described above, the dissolved oxygen concentration can be measured in the culture medium after the culture target is separated. As a result, the dissolved oxygen concentration can be stably measured regardless of the density of culture objects in the culture solution. As a result, the dissolved oxygen concentration can be measured with high accuracy, and the appropriate amount of oxygen supply to the culture solution can be controlled. In addition, the object to be cultured can be appropriately cultured, and the culture product can be efficiently produced.

FIG. 11 is a system diagram of the culture system 200 of the second embodiment. In the culture system 100 (see FIG. 1) described above, the computing device 21 controls the rotation speed of the pump 14 via the control device 22 . However, in the culture system 200 shown in FIG. 11 , the computing device 21 controls the amount of oxygen-containing gas supplied by the gas supply device 15 via the control device 22 . Therefore, the culture medium system 34 (first culture medium supply system) includes a gas supply device 15 (dissolved oxygen concentration adjusting device) that adjusts the dissolved oxygen concentration of the culture medium in the culture vessel 1 . The pump 14 in the culture system 200 is driven at a constant rotational speed (that is, constant flow rate). Therefore, in the culture system 200 , the amount of oxygen supplied to the medium supplied to the culture vessel 1 through the medium system 36 is controlled based on the dissolved oxygen concentration of the medium measured by the dissolved oxygen concentration measuring device 3 .

In the culture system 200, when the dissolved oxygen concentration of the culture solution in the culture vessel 1 is desired to be increased, control is performed to increase the amount of oxygen-containing gas supplied by the gas supply device 15. FIG. On the other hand, when it is desired to lower the dissolved oxygen concentration of the culture solution in the culture container 1, control is performed to reduce the amount of oxygen-containing gas supplied by the gas supply device 15. FIG.

Also in this way, the dissolved oxygen concentration of the culture medium in the culture vessel 1 can be controlled.

FIG. 12 is a system diagram of the culture system 300 of the third embodiment. The culture system 100 described above includes a medium system 36 that circulates the culture medium inside and outside the culture vessel 1 . Therefore, the culture medium separated by the separator 2 was supplied to the culture container 1 through the medium system 36 . However, the culture system 300 shown in FIG. 12 includes a medium system 37 (second medium supply system) that supplies the culture medium after the separation of the object to be cultured in the separation device 2 to the culture medium storage container 4 instead of the medium system 36. . Therefore, the culture medium after separation by the separation device 2 is supplied to the culture vessel 1 through the medium system 37, the medium storage container 4, and the culture medium system 35.

Regarding the dissolved oxygen concentration control of the culture solution in the culture vessel 1 , in the culture system 300 , oxygen-containing gas is directly supplied to the culture medium in the culture medium storage vessel 4 . That is, in the culture system 300 , the dissolved oxygen concentration of the culture solution in the culture vessel 1 is controlled by the gas supply device 6 provided in the culture medium storage vessel 4 . Since the culture medium in the medium storage container 4 is supplied to the culture container 1, the culture solution in the culture container 1 can be improved by directly supplying the oxygen-containing gas to the culture medium in the culture container 4. Dissolved oxygen concentration can be controlled.

FIG. 13 is a system diagram of the culture system 400 of the fourth embodiment. In the culture systems 100 to 300 described above, oxygen-containing gas is supplied to the medium in the medium system 36 or the medium storage container 4 . However, the culture system 400 is provided with a gas supply container 41 for supplying an oxygen-containing gas to the culture medium. In the culture system 400, the gas supply device 42 (third gas supply device). The gas supply device 42 is provided inside the gas supply container 41 in the culture system 400 . However, the gas supply device 42 may be provided in at least one of the medium system 45 (described later) and the medium system 46 (described later).

The gas supply container 41 and the medium storage container 4 are connected by a medium system 45 . Also, the gas supply container 41 and the culture container 1 are connected by a culture medium system 46 . Therefore, the medium is supplied from the medium storage container 4 to the gas supply container 41 through the medium system 45 . Then, in the gas supply container 41, an oxygen-containing gas is supplied to the supplied culture medium. An oxygen-containing gas is supplied, and an oxygen-dissolved medium is supplied to the culture vessel 1 through a medium system 46 . The medium is supplied to the culture vessel 1 through the medium system 46 by a pump 47 provided in the medium system 46 .

The gas supply container 41 is provided with a stirring blade 43, and the culture medium is stirred by driving the stirring blade 43 while the oxygen-containing gas is being supplied. As a result, oxygen can be evenly dissolved in the entire culture medium inside the gas supply container 41 . In addition, the internal volume of the gas supply container 41 is smaller than the internal volume of the culture medium storage container 4 . By supplying the oxygen-containing gas from the gas supply container 41 having a relatively small internal volume, the concentration of dissolved oxygen in the culture medium is rapidly increased compared to supplying the oxygen-containing gas from the medium storage container 4 having a relatively large internal volume. can be made As a result, it is possible to improve the ability to follow changes in the dissolved oxygen concentration based on the dissolved oxygen concentration measuring device 3 .

FIG. 14 is a system diagram of the culture system 500 of the fifth embodiment. In the culture systems 100 to 400 described above, oxygen supply via the gas supply device 5 provided in the culture vessel 1 was not particularly controlled. Specifically, the gas supply device 5 only supplies the oxygen-containing gas at a flow rate that can suppress damage to the culture object regardless of the measured value by the dissolved oxygen concentration measurement device 3. . However, in the culture system 500 shown in FIG. 14, in addition to controlling the amount of oxygen supplied to the culture vessel 4 via the gas supply device 6, the amount of oxygen supplied to the culture vessel 1 via the gas supply device 5 is also controlled. . However, as described above, the amount of oxygen-containing gas per unit time via the gas supply device 5 in the culture vessel 1 is the same as the amount of oxygen-containing gas per unit time via the gas supply device 6 in the culture vessel 4. less than

In this way, the medium in the medium storage container 4 can be enriched with oxygen by the gas supply device 6, while the culture solution can be directly oxygenated by the gas supply device 5 in the culture vessel 1. Gas can be supplied. By directly supplying oxygen to the culture solution by the gas supply device 5, it is possible to improve the ability to follow changes in dissolved oxygen concentration based on the dissolved oxygen concentration measuring device 3. FIG. Furthermore, since the amount of oxygen-containing gas supplied to the culture solution is smaller than the amount of oxygen-containing gas supplied to the culture medium, damage to the object to be cultured in the culture solution can be suppressed.

1 culture vessel 100 culture system 11 pump 12 pump 13 pump 14 pump (dissolved oxygen concentration adjusting device)
15 gas supply device (first gas supply device, dissolved oxygen concentration adjustment device)
2 Separation device 200 Culture system 21 Arithmetic device 21a Measurement unit 21b Oxygen supply amount determination unit 21c Signal transmission unit 21d Flow condition database 22 Control device 3 Dissolved oxygen concentration measurement device 300 Culture system 31 Culture solution system 32 Medium system 34 Medium system (No. 1 medium supply system)
35 medium system 36 medium system 37 medium system (second medium supply system)
4 culture medium storage container 400 culture system 41 gas supply container 42 gas supply device (third gas supply device)
43 stirring blade 45 culture medium system 46 culture medium system 47 pump 5 gas supply device 500 culture system 6 gas supply device (second gas supply device)
7 Stirring blade 8 Stirring blade 9 Gas supply device A Branch part S1 Culture step S2 Separation step S3 Dissolved oxygen concentration measurement step S4 Control step

Claims (13)

a culture vessel for culturing a culture object producing a culture product in a medium;
a separation device for separating the culture object from a culture solution containing the culture object cultured in the culture container;
a dissolved oxygen concentration measuring device for measuring the dissolved oxygen concentration of the culture medium after the separation of the culture object in the separation device ;
a medium system formed between the culture container and the dissolved oxygen concentration measuring device, through which one of the medium of the culture solution or the medium flows and formed of a gas permeable material;
an oxygen supply amount determination unit that determines the amount of oxygen supplied by the culture medium supplied to the culture vessel based on the dissolved oxygen concentration measured by the dissolved oxygen concentration measuring device and the flow conditions of the medium flowing through the medium system; A culture system, comprising : a computing device . The culture system according to claim 1, further comprising a first culture medium supply system that supplies the culture medium after the separation of the culture object by the separation device to the culture vessel. The culture system according to claim 2, wherein the first medium supply system includes a first gas supply device that supplies an oxygen-containing gas to the medium after the separation of the culture object. The culture system according to claim 2 or 3, wherein the first medium supply system includes a dissolved oxygen concentration adjusting device that adjusts the dissolved oxygen concentration of the medium in the culture vessel. The flow conditions are
Constituent materials for piping that constitutes the medium system,
inner dimensions of the piping that constitutes the medium system;
System length of the medium system, and
a flow rate of the medium flowing through the medium system;
The culture system according to any one of claims 1 to 4 , comprising at least one condition of The culture system according to any one of claims 1 to 5 , wherein the gas permeable material contains resin. a medium storage container for storing the medium supplied to the culture container;
The culture system according to any one of claims 1 to 6 , further comprising a second gas supply device that supplies an oxygen-containing gas to the culture medium stored in the culture medium storage container. a culture vessel for culturing a culture object producing a culture product in a medium;
a separation device for separating the culture object from a culture solution containing the culture object cultured in the culture container;
a dissolved oxygen concentration measuring device for measuring the dissolved oxygen concentration of the culture medium after the separation of the culture object in the separation device;
a medium storage container for storing the medium supplied to the culture container;
A third gas supply device for supplying an oxygen-containing gas to the medium supplied to the culture vessel is provided after the medium flow of the medium storage container and before the medium flow of the culture vessel. nourishment system. 9. The culture system according to claim 7 or 8 , further comprising a second medium supply system for supplying the medium after the separation of the object to be cultured by the separation device to the medium storage container. The culture system according to any one of claims 1 to 9 , wherein the culture object contains at least one kind of human cells and animal cells. a culturing step of culturing a culture object producing a culture product in a medium;
a separation step of separating the culture object from a culture solution containing the culture object cultured in the culturing step;
a dissolved oxygen concentration measuring step of measuring the dissolved oxygen concentration of the culture medium after the culture object is separated in the separating step ;
The dissolved oxygen concentration measured in the dissolved oxygen concentration measuring step, the culture solution or the culture medium formed between a culture vessel for culturing the culture object and a dissolved oxygen concentration measuring device for measuring the dissolved oxygen concentration an oxygen supply amount determination step of determining the oxygen supply amount by the medium supplied to the culture vessel based on the flow conditions of the medium flowing through the medium system formed by the gas-permeable material while one medium flows; including
A culture method characterized by: a culturing step of culturing a culture object producing a culture product in a medium;
a separation step of separating the culture object from a culture solution containing the culture object cultured in the culturing step;
a dissolved oxygen concentration measuring step of measuring the dissolved oxygen concentration of the culture medium after the culture object is separated in the separating step ;
The dissolved oxygen concentration measured in the dissolved oxygen concentration measuring step, the culture solution or the culture medium formed between a culture vessel for culturing the culture object and a dissolved oxygen concentration measuring device for measuring the dissolved oxygen concentration an oxygen supply amount determination step of determining the oxygen supply amount by the medium supplied to the culture vessel based on the flow conditions of the medium flowing through the medium system formed by the gas-permeable material while one medium flows; including
A method for producing a cultured product, characterized by: a culturing step of culturing a culture object producing a culture product in a medium;
a separation step of separating the culture object from a culture solution containing the culture object cultured in the culturing step;
a dissolved oxygen concentration measuring step of measuring the dissolved oxygen concentration of the culture medium after the culture object is separated in the separating step;
An oxygen-containing gas is supplied to the culture medium supplied to the culture vessel after the medium flow of the medium storage container for storing the medium to be supplied to the culture vessel for culturing the culture object and before the medium flow of the culture vessel. and a third gas supply step of
A method for producing a cultured product, characterized by:

Images