JP6744161B2 - Separation device, culture device and separation method - Google Patents

Description

The present invention relates to a separation device, a culture device and a separation method for separating cells from a culture solution of living cells.

When culturing living cells, it is necessary to maintain the culture environment, that is, the inside of the culture tank under optimal conditions for culturing, and to provide the nutrients necessary for survival and growth of living cells from the medium as inorganic or organic compounds. There is. Therefore, in addition to controlling the dissolved oxygen concentration, pH, temperature, stirring speed, etc., substances required by living cells are supplemented during the culture. Fed-Bach culture, continuous culture, and perfusion culture are available as culture methods for supplementing a medium in which a substance required by living cells is dissolved during the culture. Fed-batch culture is a method of culturing while supplying a liquid in which a substance required by living cells is dissolved to a culture tank during culturing. The continuous culture is a method in which a part of the culture solution is withdrawn from the culture tank during the culture and the culture is performed while supplying a supplemental solution corresponding to the withdrawn amount. The perfusion culture is a method in which cells of the culture solution are separated from the culture tank during the culture, only the liquid component is drawn, and the culture is performed while supplying a supplemental liquid corresponding to the drawn amount. The culture solution means a solution containing cultured cells cultured in a liquid medium. Cultured cells refer to cells cultured in a liquid medium.

Among these culturing methods, Patent Documents 1 and 2 describe culturing devices equipped with a separator suitable for perfusion culturing, which is provided with a separator for separating a liquid content from a living cell culture solution.
Patent Document 1 describes (illustrates) that the flat sheet membrane element is arranged in the middle of the culture tank.
Further, in Patent Document 2, one or more stationary, vertically-oriented baffles are arranged in the liquid recovery space to dampen the liquid motion in the liquid recovery space, thus allowing cells to gather on the inward facing spin filter surface. It is stated that the above is substantially prevented. Further, in this Patent Document 2, in order to minimize the tendency of protein debris to collect on the filter surface facing the outside, the spin filter is rotated in a direction opposite to the impeller for stirring the contents of the culture vessel. Has been done.

JP, 2008-206430, A Japanese Patent Publication No. 03-505041

However, according to the flow analysis of the culture solution by computer simulation, it is suggested that the flow that is concentrically generated around the stirring blade described in Patent Document 1 is generated radially from the stirring shaft. It was Therefore, according to the invention described in Patent Document 1, since the flow of the culture solution hits the surface of the flat sheet membrane element perpendicularly, the cleaning effect of the filtration surface may not be obtained. Therefore, the invention described in Patent Document 1 may not prevent clogging of the filtration filter.
Further, as described in Patent Document 1, when the flat sheet membrane element is arranged in the middle of the culture tank, the flat sheet membrane element becomes an obstacle and the culture solution is not sufficiently stirred, and the cleaning effect of the filtration surface is obtained. Can't get enough. Therefore, in the invention described in Patent Document 1, the effect of cleaning the filtration surface is not sufficiently obtained, and the filtration filter may be clogged.

Further, in the invention described in Patent Document 2, since the installation position of the filtration filter is not considered at all, the effect of cleaning the filtration surface may not be sufficiently obtained. Therefore, the invention described in Patent Document 2 may not be able to sufficiently prevent clogging of the filtration filter.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a separation device, a culture device, and a separation method that can prevent clogging of a filtration filter.

The separation device according to the present invention, which has solved the above-mentioned problems, is provided in a culture tank, a stirring blade for stirring the culture solution to generate a stirring flow, and a filtering surface in the culture tank in parallel with the stirring flow. And a filtration filter for separating a liquid content of the culture solution from the filtration surface, wherein the filtration filter is provided in an area within 70% of a maximum flow rate of the agitated flow in the culture tank. In addition, the filtration filter is provided on a part of the side wall of the culture tank so as to be rotatable in the circumferential direction, and the filtration filter supports the stirring shaft to which the stirring blade is attached. It is fixed integrally by a member .
Further, the separation apparatus according to the present invention is provided in a culture tank, a stirring blade for stirring a culture solution to generate a stirring flow, and a filtration surface in the culture tank in parallel with the stirring flow. And a filtration filter that separates the liquid content of the culture solution from the filtration surface, and the filtration filter is provided in a region within 70% of the maximum flow rate of the stirring flow in the culture tank. The filtration filter is provided in a part of the side wall of the culture tank so as to be rotatable in the circumferential direction, and the filtration filter is provided separately from a stirring shaft to which the stirring blade is attached. Further, the filtration filter is integrally fixed by a rotating member and a supporting member capable of rotating in the circumferential direction of the culture tank.

The culture apparatus according to the present invention is a culture tank for culturing a culture solution, a stirring blade provided in the culture tank for stirring the culture solution to generate a stirring flow, and a filtration surface in the culture tank. A filtration filter that is provided in parallel with the agitated flow and that separates the liquid content of the culture solution from the filtration surface, and a collection container that is connected to the filtration filter and that collects the separated liquid content, and the culture. And an addition device that is connected to the tank and that adds the same amount of the supplemented liquid as the separated liquid component into the culture tank, and the filtration filter has the maximum flow rate of the stirring flow in the culture tank. Is provided in a region of 70% or less, the filtration filter is provided in a part of the side wall portion of the culture tank so as to be rotatable in the circumferential direction, and the filtration filter includes the stirring blade. It is integrally fixed by the attached stirring shaft and a supporting member .
Further, the culture device according to the present invention, a culture tank for culturing a culture solution, a stirring blade provided in the culture tank for stirring the culture solution to generate a stirring flow, and a filtration surface in the culture tank. Is provided in parallel with the agitated flow, a filtration filter for separating a liquid content of the culture solution from the filtration surface, and a collection container connected to the filtration filter for collecting the separated liquid content, An addition device that is connected to the culture tank and has the same amount of the supplemented liquid as the separated liquid component is added to the culture tank, and the filtration filter of the agitated flow in the culture tank. The filtration filter is provided in a region within 70% from the maximum flow velocity, and the filtration filter is provided in a part of the side wall of the culture tank so as to be rotatable in the circumferential direction, and the filtration filter is agitated. It is integrally fixed by a support member and a rotating member, which is provided separately from the stirring shaft to which the blades are attached and can rotate the filtration filter in the circumferential direction of the culture tank.

The separation method according to the present invention is provided in a region within 70% of the maximum flow rate of the stirred flow in the culture tank while stirring the culture solution in the culture tank with a stirring blade to generate a stirred flow. A stirring shaft having a filtering surface parallel to the stirring flow , rotatably provided in a part of the side wall of the culture tank in the circumferential direction, and having the stirring blade attached thereto, and a supporting member. separating the liquid component from the culture solution Llorca filter are integrally fixed by.
Further, the separation method according to the present invention is provided in a region within 70% of the maximum flow rate of the agitation flow in the culture tank while agitating the culture solution in the culture tank by the agitation blade to generate agitation flow. Also, a stirring shaft having a filtering surface parallel to the stirring flow, and rotatably provided in a part of a side wall portion of the culture tank in a circumferential direction, and having the stirring blade attached thereto, The liquid component is separated from the culture liquid by a separately provided filter that is integrally fixed by a rotating member that can rotate in the circumferential direction of the culture tank and a supporting member.

The separation device, the culture device and the separation method according to the present invention can prevent clogging of the filtration filter.

It is a schematic structure figure explaining composition of a separation device concerning this embodiment. It is a schematic block diagram explaining the structure of the separator which concerns on other embodiment. It is a schematic block diagram explaining the structure of the separator which concerns on other embodiment. It is a schematic block diagram explaining the structure of the separator which concerns on other embodiment. It is a schematic block diagram explaining the structure of the culture apparatus which concerns on this embodiment. It is a graph which shows the relationship between the number of culture days and the number of viable cells concerning an example and a comparative example. In the figure, the horizontal axis shows the number of days of culture (day), and the vertical axis shows the number of viable cells (10 ⁶ cells/mL).

Hereinafter, one embodiment of a separation device, a culture device, and a separation method according to the present invention will be described in detail with reference to the drawings as appropriate.
The separation device, the culture device, and the separation method according to the present embodiment can be applied, for example, when culturing cells that produce a substance as a main raw material such as a drug. Examples of the substance to be produced in the present embodiment include proteins such as antibodies and enzymes, physiologically active substances such as low molecular weight compounds and high molecular weight compounds, but are not limited thereto. Examples of cells to be cultured include, but are not limited to, animal cells, plant cells, insect cells, bacteria, yeasts, fungi and algae. In particular, the separation device, the culture device, and the separation method according to the present embodiment can be suitably applied when culturing animal cells that produce proteins such as antibodies and enzymes. The culture solution may be appropriately prepared or commercially available depending on the cells to be cultured, and is not limited to a specific one.

First, the separation method and the separation apparatus according to the present embodiment will be described, and then the culture apparatus will be described.
(Separation method and separation device)
The separation method according to the present embodiment, while stirring a culture solution in a culture tank to generate a stirring flow, with respect to the stirring flow provided in a region within 70% from the maximum flow rate of the stirring flow in the culture tank, Then, the liquid content is separated from the culture liquid by a filtration filter having parallel filtration surfaces.
This separation method is preferably carried out by the separation device according to this embodiment described below. Hereinafter, the separation method according to this embodiment will be described instead of the description of the separation method according to this embodiment.

FIG. 1 is a schematic configuration diagram illustrating the configuration of the separation device according to the present embodiment.
As shown in FIG. 1, the separation device 1 according to the present embodiment includes a stirring blade 2 and a filtration filter 3.
The stirring blade 2 is provided in the culture tank 4, stirs the culture solution 5 to generate a stirring flow A, and uniformly mixes the culture solution 5 in the culture tank 4. As shown in FIG. 1, the stirring blade 2 is attached to one end of a stirring shaft 6, and the other end of the stirring shaft 6 is connected to a drive motor (not shown). That is, the stirring blade 2 is rotated by the driving motor, and the rotational force is transmitted to the stirring blade 2 via the stirring shaft 6 to stir the culture solution 5. The stirring blade 2 allows the stirring flow A generated by stirring to circulate in the vertical direction, and specifically, the stirring flow A rises along the wall surface of the culture tank 2 and moves along the stirring shaft 6. The thing which can be lowered by this can be used suitably. The flow of the stirring flow A causes a shearing force on the adhered matter adhering to the film surface to remove the adhered matter and prevent clogging. As such a stirring blade 2, for example, a Supermix HS-600 series stirring blade manufactured by Satake Chemical Machinery Co., Ltd. can be used.

As the culture tank 4, any one can be used as long as the filtration filter 3 can be provided as described later.
The filtration filter 3 has a filtration surface 31 provided in parallel with the agitated flow A in the culture tank 4, and separates the liquid content of the culture solution 5 from the filtration surface 31. The liquid separated from the filtration surface 31 contains lactic acid, ammonia and the like which are cell growth inhibiting substances generated in the process of cell culture. That is, since the filtration filter 3 removes these cell growth inhibitory substances from the culture medium 5, it is possible to prevent the growth and proliferation of cells from being inhibited. In addition, as shown in FIG. 1, the filtration filter 3 preferably separates the liquid component of the culture liquid 5 from the filtration surface 31 in the direction B perpendicular to the stirring flow A. By doing so, it is possible to make it difficult for the filtration filter 3 to be clogged.

As the filtration filter 3, for example, any commonly used filter cloth, membrane filter or the like can be preferably used as long as it can prevent passage of cells. Since a large number of fine cell fragments produced by the decay of dead cells are present in the culture medium 5, a filter having a filtering characteristic that prevents the passage of healthy cells and allows the passage of fine cell fragments is used. Is particularly preferable. Therefore, it is preferable to use the filtration filter 3 having a pore size that is substantially equal to or smaller than the average size of the cells to be cultured. Generally, since the floating animal cells have an average diameter of 5 to 30 μm, the pore diameter is set to the same level or less. In the present embodiment, as the filtration filter 3, for example, a commercially available microfiltration membrane having a pore size of 0.01 μm to 10 μm can be used. In addition, in the present embodiment, as the filtration filter 3, the purpose is to remove lactic acid and ammonia, which are cell growth inhibitory substances generated in the process of cell culture, while leaving the antibody, which is a product of cells, with the cells in the culture solution 5. Then, an ultrafiltration membrane having a finer pore size can be used. Further, in the present embodiment, as the filtration filter 3, it is also possible to use a combination of a microfiltration membrane and an ultrafiltration membrane in different areas.

Then, in the present embodiment, the filtration filter 3 is provided in the region 71 within 70% of the maximum flow rate of the agitated flow A in the culture tank 4. By doing so, the effect of cleaning the filtration surface 31 by the stirring flow A can be obtained, and the clogging of the filtration filter 3 can be prevented. From the viewpoint of more reliably preventing clogging of the filtration filter 3, the filtration filter 3 is preferably provided in the region 72 where the maximum flow velocity of the stirring flow A in the culture tank 4 is provided. The flow velocity of the culture solution 5 can be determined by measurement using a commercially available velocity meter or analysis using commercially available computational fluid dynamics software such as Fluent manufactured by ANSYS. A region 71 within 70% of the maximum flow velocity of the agitated flow A and a region 72 having the maximum flow velocity of the agitated flow A will be described later.

Here, in the separation device 1 in the present embodiment, the filtration filter 3 is installed so that the filtration surface 31 that filters the culture solution 5 is parallel to the flow velocity vector of the culture solution 5. Therefore, the extracting direction of the culture solution 5 is perpendicular to the filtration surface 31, that is, perpendicular to the flow velocity vector of the culture solution 5. In general, the specific gravity of cells and microorganisms is very close to that of water, and the size of floating animal cells is as small as about 5 to 30 μm. Therefore, the behavior of cells and microorganisms in the culture medium 5 is almost the same. Follow the average flow of 5. Here, the average flow refers to a velocity vector obtained by combining a flow velocity vector of the culture solution 5 generated by the stirring blade 2 and a withdrawal velocity vector in the withdrawal direction of the culture solution 5.

As described above as the stirring blade 2, when the Supermix HS-600 system stirring blade manufactured by Satake Chemical Machinery Co., Ltd. is used, the flow velocity vector of the culture solution 5 is approximately in the direction indicated by the arrow in FIG. A). In this case, the region 71 within 70% from the maximum flow rate of the agitated flow A is the dimension from the bottom of the tank to the liquid surface in the side wall portion 41 of the culture tank 4, which is 20% to 80%, preferably 30%. To 70% corresponds.

In the present embodiment, it is preferable that the filtration filter 3 be provided on the side wall portion 41 of the culture tank 4 in a region 71 within 70% of the maximum flow rate of the agitated flow A. With this configuration, for example, the flow of the culture solution 5 is less likely to be obstructed as compared with the case where the filtration filter 3 is provided at a position separated from the side wall portion 41 of the culture tank 4, so that the clogging of the filtration filter 3 can be more reliably performed. Can be prevented. When the filtration filter 3 is provided on the side wall portion 41 of the culture tank 4 as described above, it is more preferable that the filtration surface 31 and the side wall portion 41 of the culture tank 4 are flush with each other. In this way, the flow of the culture solution is not obstructed, so that clogging of the filtration filter 3 can be prevented more reliably.

(Other embodiments)
Here, a separation device according to another embodiment will be described.
FIG. 2 is a schematic configuration diagram illustrating a configuration of a separation device according to another embodiment. The separator 1A shown in FIG. 2 differs from the separator 1 shown in FIG. 1 in the type of the stirring blade 2 provided and the location where the filtration filter 3 is attached. Other configurations of the separating apparatus 1A shown in FIG. 2 are the same as those of the separating apparatus 1 shown in FIG. Therefore, the same components are denoted by the same reference numerals and detailed description thereof will be omitted.

As shown in FIG. 2, the stirring blade 2 provided in this separation device 1A can also circulate the stirring flow A generated by stirring in the vertical direction, specifically, the stirring blade 2 as described above. It is possible to preferably use the one in which the flow A can be raised along the stirring shaft 6 and can be lowered along the side wall portion 41 of the culture tank 4. Further, in the present embodiment, the filtration filter 3 is preferably provided around the stirring shaft 6 to which the stirring blade 2 is attached. More specifically, the filtration filter 3 is preferably provided adjacent to the stirring shaft 6 from the upper part of the culture tank 4 by a mounting member 61. In this way, since the filtration filter 3 can be provided in the area 71 within 70% of the maximum flow rate of the agitated flow A, the clogging of the filtration filter 3 can be prevented as in the mode shown in FIG. ..

The mode shown in FIG. 1 or the mode shown in FIG. 2 can be arbitrarily selected. Further, the aspect shown in FIG. 1 and the aspect shown in FIG. 2 can be used together. That is, the filtration filter 3 can be provided on at least one of the side wall 41 of the culture tank 4 and the periphery of the stirring shaft 6 to which the stirring blade 2 is attached. In this case, if one of the filtration filters 3 is provided in the region 71 within 70% of the maximum flow rate of the agitated flow A, the filtration filter 3 can prevent clogging, and thus perfusion culture should be performed for a long time. It is possible to culture a large number of viable cells, that is, to perform high-density culture. Further, in this case, since another filtration filter 3 is provided, it is possible to provide a separator having a high ability to separate a liquid component from the culture liquid 5.

Furthermore, in the present embodiment, the following modes can be adopted.
3 and 4 are schematic configuration diagrams illustrating the configuration of a separation device according to another embodiment.
In the separation device 1B shown in FIG. 3, the filtration filter 3 is provided on the side wall portion 41 of the culture tank 4 so as to be rotatable in the circumferential direction, and the filtration filter 3 is integrated with the stirring shaft 6 by the support member 8. 1 is different from the separating device 1 shown in FIG. The other configuration of the separating apparatus 1B shown in FIG. 3 is the same as that of the separating apparatus 1 shown in FIG. Therefore, the same components are denoted by the same reference numerals and detailed description thereof will be omitted.
With such a configuration, in the separation device 1B shown in FIG. 3, the rotation of the stirring shaft 6 causes the stirring filter 2 and the filtration filter 3 to rotate in the circumferential direction of the culture tank 4. Therefore, in the separation device 1B shown in FIG. 3, since the filtration filter 3 is rotating, the cultured cells are less likely to adhere to the filtration surface 31.

The separation device 1C shown in FIG. 4 is similar to the separation device 1B shown in FIG. 3 in that the filtration filter 3 is provided on the side wall portion 41 of the culture tank 4 so as to be rotatable in the circumferential direction. However, in the separation device 1C shown in FIG. 4, the filtration filter 3 is a hollow tube that is provided separately from the stirring shaft 6 and is a rotating member that can rotate the filtration filter 3 in the circumferential direction of the culture tank 4. It is different from the separating device 1B shown in FIG. 3 in that it is integrally fixed by the shaft 62 and the supporting members (the first supporting member 63 and the second supporting member 81). The hollow tube shaft 62 is a shaft member of a hollow tube provided so as to surround the stirring shaft 6. The hollow tube shaft 62 is connected to a rotary motor (not shown) different from a drive motor (not shown) for rotating the stirring shaft 6. Therefore, the separator 1C shown in FIG. 4 can rotate the filtration filter 3 in the circumferential direction of the culture tank 4 separately from the stirring shaft 6 and the stirring blade 2. Therefore, the separator 1C shown in FIG. 4 can not only rotate the stirring shaft 6 and the stirring blade 2 and the filtration filter 3 in the same direction, but also disassemble the stirring shaft 6 and the stirring blade 2 and the filtration filter 3. It can also be rotated in the opposite direction. When rotating the stirring shaft 6 and the stirring blade 2 and the filtration filter 3 in the same direction, they may be rotated at the same rotation speed or may be rotated at different rotation speeds. These modes can be arbitrarily selected, but in any case, in the separation device 1C shown in FIG. 4, since the filtration filter 3 is rotating, the cultured cells are more difficult to adhere to the filtration surface 31.

According to the separation method and the separation device 1 according to the present embodiment described above, the filtration surface 31 of the filtration filter 3 is provided in parallel with the stirring flow A in the culture tank 4, and the filtration filter 3 is cultured. It is provided in a region 71 within 70% of the maximum flow velocity of the stirring flow A in the tank 4. Therefore, in the separation method and the separation apparatus 1 according to the present embodiment, the effect of cleaning the filtration surface by the stirring flow A can be obtained, and the clogging of the filtration filter 3 can be prevented.

(Incubator)
Next, the culture device according to the present embodiment will be described.
FIG. 5 is a schematic configuration diagram illustrating the configuration of the culture device according to the present embodiment.
The culture device 10 shown in FIG. 5 illustrates an example in which the separation device 1 shown in FIG. 1 is applied, but the separation devices 1A to 1C shown in FIGS. 2 to 4 can be applied instead of the separation device 1. Needless to say.

In the culture device 10 shown in FIG. 5, the same components as those in the separation device 1 shown in FIG. 1 are designated by the same reference numerals, and detailed description thereof will be omitted.
As shown in FIG. 5, the culture device 10 has a culture tank 4, a stirring blade 2, a filtration filter 3, a collection container 11, and an addition device 14. In the culturing apparatus 10, like the separating apparatus 1, the filtration filter 3 has an area 71 within 70% of the maximum flow rate of the agitated flow A in the culture tank 4, preferably an area 72 where the agitated flow A has the maximum flow rate. It is provided in. Since the culture tank 4, the stirring blade 2, and the filtration filter 3 have been described in detail, the description thereof will be omitted.

The collection container 11 is connected to the filtration filter 3 and collects the liquid component separated by the filtration filter 3. As the recovery container 11, any container can be used as long as it can store the liquid content of the culture liquid 5.
The filtration filter 3 and the collection container 11 are connected by a pipe 12 such as a flexible tube, for example. An infusion device 13 such as a suction pump or a peristaltic infusion device is provided at an arbitrary position of the pipe 12, and the culture solution 5 by the filtration filter 3 is activated by operating the infusion device 13 to make the inside of the pipe 12 negative pressure. It facilitates the filtration of the liquid component of.

The adding device 14 is connected to the culture tank 4 and adds the same amount of the supplementary liquid as the liquid content of the culture liquid 5 separated by the filtration filter 3 into the culture tank 4. The replenishing liquid may be the liquid medium itself, an aqueous solution containing only components that are consumed in large amounts by the cultured cells, or a composition liquid containing a large amount of such components. The adding device 14 supplies the replenishing liquid of the same amount as the replenishing liquid into the culture tank 4 based on the liquid amount of the liquid component separated by the filtration filter 3 measured by a measuring means (not shown), for example. The addition device 14 may supply a predetermined amount of replenisher solution after a predetermined time has elapsed from the start of culture, according to the contents programmed in advance. The supply amount of the replenishing solution can be experimentally determined from the culture solution 5 used, the pore diameter of the filtration filter 3, the degree of negative pressure in the pipe 12, and the like.

In addition to these elements, the culture device 10 according to the present embodiment includes a gas supply facility 15 for air, oxygen, nitrogen, carbon dioxide gas, a hot water/cooled water supply facility (not shown), and a steam supply facility (not shown). ) And water supply/drainage equipment (not shown). Although the gas supply equipment 15 can be provided for each type of gas, they are collectively shown in FIG.
Further, the culture device 10 according to the present embodiment is equipped with a measuring means 16 for measuring the properties of the culture solution 5, and the dissolved oxygen concentration, dissolved carbon dioxide concentration, pH, temperature, ammonia concentration, lactate concentration of the culture liquid are included. , Get glutamine concentration and other measured values. Although the measuring means 16 can be provided with a measuring device for each measuring object, they are collectively shown in FIG.
Further, the culture device 10 according to the present embodiment has a liquid surface of the culture medium 5 and an aeration means (not shown) arranged in the culture medium 5, and oxygen necessary for the culture is aerated. It is supplied into the liquid through the means.

According to the culture apparatus 10 according to the present embodiment described above, the filtration surface 31 of the filtration filter 3 is provided in parallel with the agitated flow A in the culture tank 4, and the filtration filter 3 is in the culture tank 4. It is provided in a region 71 within 70% of the maximum flow rate of the agitated flow A in FIG. Therefore, in the culture device 10 according to the present embodiment, the effect of cleaning the filtration surface 31 by the agitated flow A can be obtained, and the clogging of the filtration filter 3 can be prevented.

Next, a confirmation experiment confirming the effect of the present invention will be described.
As an example, cells were cultured using the culture device 10 shown in FIG. 5 to which the separation device 1 shown in FIG. 1 was applied, and the number of viable cells in a predetermined culture period was measured. In addition, as a comparative example, ordinary batch culture was performed using a conventional culture device, that is, a general culture tank without a filtration filter, and the number of viable cells was similarly measured.

Specifically, CRL-1606 cells (purchased from American Type Culture Collection) that are mouse-mouse hybridomas were used as the cells. This cell is a floating cell that secretes an anti-fibronectin antibody.
For culturing, a medium was prepared by adding Fetal bovine serum (FBS) to 5% concentration in Iscove's Modified Dulbecco's Medium (IMDM medium).
As the culture device, a glass cylindrical culture tank having an inner diameter of 150 mm and a culture volume of 5 L was used. A rubber heater for heating, a stirring blade driven by a magnet, a temperature measuring electrode, a pH electrode, a DO electrode, and a controller for measuring and adjusting these are connected.
A sintered metal sparger having an average pore diameter of 100 μm was incorporated for aeration in the liquid, and the culture liquid was aerated.
The pH of the culture solution was automatically adjusted by increasing or decreasing the carbon dioxide concentration in the mixed gas (air, nitrogen, oxygen, carbon dioxide) supplied to the gas phase part of the culture tank.
The temperature of the culture solution was adjusted to 37°C. When the culture broth was acidified, a 2% sodium hydroxide solution was added to adjust the concentration.
The dissolved oxygen concentration was adjusted by increasing or decreasing the oxygen partial pressure of the gas on the liquid surface and the gas supplied into the liquid.
The stirring blade was rotated at a rotation speed of 100 rpm (rotation/minute) using a drive motor directly connected to a stirring shaft equipped with the stirring blade.
The liquid content of the culture solution separated by the filtration film was stored in a collection container. Also, the same amount of the above-mentioned medium as the collected liquid was added by an addition device.

The culture solution in the culture tank was aseptically sampled 2-3 times a day from the start of the culture, and the number of viable cells was measured. The number of viable cells was measured by using a viable cell counter BioProfile 100Plus manufactured by Beckman Coulter, which was distinguished from the dead cells stained with trypan blue and counted by image processing.

The result is shown in FIG. FIG. 6 is a graph showing the relationship between the number of culture days (day) and the number of viable cells (10 ⁶ cells/mL) according to Examples and Comparative Examples.
As shown in FIG. 6, the culture device to which the separation device according to the example was applied showed a good culture result in the growth of cultured cells (solid line in FIG. 6 ).
On the other hand, in the culture device according to the comparative example, the cell growth was suppressed when the ammonia accumulated in the culture reached a concentration at which the growth of living cells was inhibited (broken line in FIG. 6 ).
As described above, the culture device (separation device) according to the embodiment enables continuous culture for a long period of time by removing cell growth and growth inhibitory substances such as ammonia, thereby improving productivity. be able to.
After completion of the culture, the presence or absence of clogging of the filtration filter was confirmed, and the filtration film of the culture device (separation device) according to the example was not clogged.

As described above, the separation device, the culture device and the separation method according to the present invention have been described in detail with reference to the embodiments and examples. However, the present invention is not limited to the above-described embodiments and examples, and various modifications can be made. included. For example, the above-described embodiments and examples have been described in detail in order to explain the present invention in an easy-to-understand manner, and are not necessarily limited to those having all the configurations described. Further, a part of the configuration of an embodiment or example can be replaced with the configuration of another embodiment or example, and the configuration of a certain embodiment or example can be replaced with that of another embodiment or example. It is also possible to add a configuration. Further, it is possible to add/delete/replace other configurations with respect to a part of the configurations of the respective embodiments and examples.

DESCRIPTION OF SYMBOLS 1 Separation device 2 Stirring blade 3 Filtration filter 31 Filtration surface 4 Culture tank 41 Side wall 5 Culture solution 6 Stirring shaft 71 Area within 70% of maximum flow velocity of stirred flow 72 Region where maximum flow velocity of stirred flow A A Stirred flow

Claims (8)

A stirring blade provided in the culture tank for stirring the culture solution to generate a stirring flow,
A filtration surface is provided in parallel with the stirring flow in the culture tank, and has a filtration filter for separating a liquid content of the culture solution from the filtration surface,
The filtration filter is provided in a region within 70% of the maximum flow rate of the stirring flow in the culture tank ,
The filtration filter is provided in a part of the side wall of the culture tank so as to be rotatable in the circumferential direction, and
A separation device , wherein the filtration filter is integrally fixed by a stirring shaft to which the stirring blade is attached and a supporting member . A stirring blade provided in the culture tank for stirring the culture solution to generate a stirring flow,
A filtration surface is provided in parallel with the stirring flow in the culture tank, and has a filtration filter for separating a liquid content of the culture solution from the filtration surface,
The filtration filter is provided in a region within 70% of the maximum flow rate of the stirring flow in the culture tank,
The filtration filter is provided in a part of the side wall of the culture tank so as to be rotatable in the circumferential direction, and
The filtration filter is integrally fixed by a rotation member and a support member that are provided separately from the stirring shaft to which the stirring blade is attached and that can rotate the filtration filter in the circumferential direction of the culture tank. separation and wherein the are. In claim 1 or claim 2 ,
The separation device, wherein the filtration filter is provided in a region where the stirring flow has a maximum flow velocity in the culture tank . A culture tank for culturing a culture solution,
A stirring blade provided in the culture tank and stirring the culture solution to generate a stirring flow,
In the culture tank, a filtration surface is provided in parallel with the agitated flow, and a filtration filter for separating the liquid content of the culture solution from the filtration surface,
A collection container that is connected to the filtration filter and collects the separated liquid component,
An addition device that is connected to the culture tank, and that adds the same amount of replenisher liquid as the separated liquid component into the culture tank,
Have
The filtration filter is provided in a region within 70% of the maximum flow rate of the stirring flow in the culture tank,
The filtration filter is provided in a part of the side wall of the culture tank so as to be rotatable in the circumferential direction, and
The culture device , wherein the filtration filter is integrally fixed by a stirring shaft to which the stirring blade is attached and a supporting member . A culture tank for culturing a culture solution,
A stirring blade provided in the culture tank and stirring the culture solution to generate a stirring flow,
In the culture tank, a filtration surface is provided in parallel with the agitated flow, and a filtration filter for separating the liquid content of the culture solution from the filtration surface,
A collection container that is connected to the filtration filter and collects the separated liquid component,
An addition device that is connected to the culture tank, and that adds the same amount of replenisher liquid as the separated liquid component into the culture tank,
Have
The filtration filter is provided in a region within 70% of the maximum flow rate of the stirring flow in the culture tank,
The filtration filter is provided in a part of the side wall of the culture tank so as to be rotatable in the circumferential direction, and
The filtration filter is integrally fixed by a rotation member and a support member that are provided separately from the stirring shaft to which the stirring blade is attached and that can rotate the filtration filter in the circumferential direction of the culture tank. and culturing and wherein the are. In claim 4 or claim 5 ,
The filtration filter, culture apparatus characterized by being provided in the region of maximum flow rate of the stirring flow in the culture tank. While causing stirred flow by stirring the culture solution in the culture Yoso by stirring blade, provided in a region within 70% from a maximum flow rate of the stirring flow in the culture tank, filtration to the stirred flow The surfaces are parallel to each other, and a part of the side wall of the culture tank is rotatably provided in the circumferential direction, and is integrally fixed by a stirring shaft to which the stirring blade is attached and a supporting member. Separation method characterized in that a liquid content is separated from the culture liquid by a filtration filter . While stirring the culture solution in the culture tank with a stirring blade to generate a stirring flow, a filtering surface for the stirring flow is provided in a region within 70% of the maximum flow rate of the stirring flow in the culture tank. Are parallel to each other, and are provided rotatably in a circumferential direction on a part of the side wall of the culture tank, and are provided separately from a stirring shaft to which the stirring blade is attached. A method of separation, wherein the liquid content is separated from the culture solution by a filtration filter that is integrally fixed by a rotating member that can be rotated in the circumferential direction and a supporting member .

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