WO2018146943A1

WO2018146943A1 - Sparger and culture apparatus

Info

Publication number: WO2018146943A1
Application number: PCT/JP2017/045282
Authority: WO
Inventors: 貴史浅倉; 浩二中村; 博志大川
Original assignee: キリン株式会社
Priority date: 2017-02-10
Filing date: 2017-12-18
Publication date: 2018-08-16
Also published as: JPWO2018146943A1; JP7220567B2

Abstract

A sparger for introducing a gas in the form of bubbles into a culture vessel is equipped with a sparger main body of which at least a part is formed from a porous body and a gas supply tube connection section to which a gas supply tube for supplying a gas to be introduced into the culture vessel to the sparger main body is connected, wherein the gas supply tube connection section is composed of a closed-end perforated section which is formed in the sparger main body and through which the gas supply tube can be inserted, and at least a lower half part of the sparger main body is formed from a porous body when an opening in the closed-end perforated section is turned up.

Description

Sparger and culture equipment

The present invention relates to a sparger and a culture apparatus using the same.

Conventionally, as an apparatus for aerobically cultivating cultures such as microorganisms and cells, a culture tank containing a culture solution seeded with the cells and the like, and for aerating air to the culture solution in the culture tank A culture apparatus having a sparger and a stirring blade that stirs the culture solution in the culture tank is known (Patent Document 1, etc.).

In such a culture apparatus, the culture solution is agitated by mechanically moving the stirring blade in the culture tank to increase the chance of contact between the culture solution and cells or to promote cell dispersion. . However, there is a possibility that the cells and the like may be damaged by contact between the cells and the stirring blade during the cultivation of the cells and the like. In particular, cells that do not have a cell membrane like mesenchymal stem cells and are extremely weak against shearing force are greatly damaged by contact with a stirring blade and cannot be efficiently cultured. is there.

In order to solve such a problem, conventionally, as a culture apparatus that stirs a culture solution with a gas that is vented to the culture solution without using a stirring blade, a lower portion of a pair of culture tubes is connected by a bent tube to form a U A culture tank formed in a letter shape, connected to the lower part of the left and right culture cylinders, gas blowing means for alternately supplying gas to the culture solution, connected to the upper part of the left and right culture cylinders, and supplied into the culture cylinder Have been proposed that include exhaust means for alternately discharging the gas (Patent Document 2, etc.).

JP2015-142546A JP 2012-115232 A

In the culture apparatus disclosed in Patent Document 2, the culture solution can be moved left and right in a U-shaped culture tank by alternately supplying gas to the left and right culture tubes, There is a problem that a sufficient agitation effect cannot be obtained from the viewpoint of increasing the chance of contact between the culture solution and cells and promoting cell dispersion.

When the gas supply amount is increased in order to obtain a sufficient stirring effect, liquid splashing of the culture solution occurs, and the culture solution comes into contact with the filter provided in the exhaust means, so that contamination of the filter, There is a risk of clogging or the like, or the culture solution may be contaminated by backflow.

In view of the above problems, the present invention provides a sparger that can provide a sufficient stirring effect by supplying gas to a culture solution and that does not cause splashing of the culture solution, and a culture apparatus using the same. The purpose is to provide.

In order to solve the above-mentioned problems, the present invention provides a sparger for introducing a gas into a culture vessel in the form of bubbles, the sparger main body at least partially comprising a porous body, and the culture vessel A gas supply pipe connecting part to which a gas supply pipe for supplying gas introduced into the sparger main body is connected, and the gas supply pipe connecting part is formed on the sparger main body, It is composed of a bottomed hole part into which the gas supply pipe can be inserted, and in a state where the opening of the bottomed hole part faces upward, at least the lower half of the sparger body is composed of the porous body. Provide the featured sparger.

In the above invention, the entire sparger body is preferably composed of the porous body, the average pore diameter of the porous body is preferably 20 to 50 μm, and the porous body is 20 to 50 μm. It is preferable to have pores with a pore size distribution of

In the above invention, the bottom surface of the bottomed hole is preferably positioned below the center of the sparger body in a state where the opening of the bottomed hole is positioned upward. The main body is preferably substantially spherical, and the diameter of the sparger main body is preferably 10 to 20 mm.

The present invention also includes a culture vessel capable of containing a culture, a sparger according to the invention provided in the culture vessel, one end inserted into the bottomed hole, and the other end supplied with gas. And a sparger provided in the culture vessel so that the porous body faces the bottom of the culture vessel.

In the above invention, the flow rate of the gas supplied to the sparger through the gas supply pipe is preferably 300 to 700 mL / min, and the bottom of the culture vessel is preferably substantially hemispherical.

According to the present invention, it is possible to provide a sparger that can obtain a sufficient stirring effect by supplying gas to the culture solution and does not cause the liquid splash of the culture solution, and a culture apparatus using the sparger. it can.

FIG. 1 is a side view showing a schematic configuration of a culture apparatus according to an embodiment of the present invention. FIG. 2 is a side view showing a schematic configuration of the culture apparatus in one embodiment of the present invention. FIG. 3A is a perspective view illustrating a schematic configuration of a cap according to an embodiment of the present invention. FIG. 3B is a top view illustrating a schematic configuration of the cap according to the embodiment of the present invention. FIG. 4 is a perspective view showing a schematic configuration of a sparger in one embodiment of the present invention. FIG. 5 is a cross-sectional view showing a schematic configuration of a sparger in one embodiment of the present invention. FIG. 6 is a side view for explaining the operational effects of the culture apparatus according to the embodiment of the present invention.

Embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a side view illustrating a schematic configuration of a culture apparatus according to the present embodiment, FIG. 2 is a side view illustrating a schematic configuration of the culture apparatus according to the present embodiment, and FIG. 3A is a cap according to the present embodiment. FIG. 3B is a top view showing the schematic configuration of the cap, FIG. 4 is a perspective view showing the schematic configuration of the sparger in the present embodiment, and FIG. It is sectional drawing which shows schematic structure of the sparger in this embodiment, FIG. 6 is a side view for demonstrating the effect in the culture apparatus which concerns on this embodiment.

As shown in FIG. 1, a culture apparatus 1 according to this embodiment includes a culture container 2 that can accommodate a culture object, a sparger 3 provided in the culture container 2, and a gas supply pipe attached to the sparger 3. 4. Note that a gas supply unit (not shown) is connected to the gas supply tube 4, and gas (air) is supplied in the form of bubbles from the gas supply unit to the culture solution via the gas supply tube 4 and the sparger 3. it can.

Examples of the culture medium that can be cultured in the culture apparatus 1 according to the present embodiment include animal cells, plant bodies, plant tissues, plant cells, algae, fungi, yeasts, aerobic bacteria, and the like. Moreover, the culture solution 8 (see FIG. 6) that can be accommodated in the culture vessel 2 together with the culture object is suitable for the culture object. For example, Dulbecco's modified Eagle (DMEM) medium, Roswell Park Memorial Institute ( (RPMI) 1640 medium, Murashige scoog (MS) medium, Gamborg B5 medium, Luria Bertani (LB) medium, YPD Broth (Yeast Extract-Peptone-Dextrose) medium, PD Broth (Potato Dextrose) medium and the like.

The culture vessel 2 is made of polyethylene terephthalate (PET), polybutylene terephthalate, polyethylene naphthalate, polyester resin such as polytrimethylene terephthalate, polytrimethylene naphthalate, olefin resin such as polyethylene or polypropylene, polycarbonate resin, polyacrylate resin. Further, it may be made of a resin material such as a polyamide resin, glass or the like. The capacity of the culture vessel 2 is, for example, 500 mL to 200 L, preferably 1 L to 20 L.

As shown in FIG. 2, the culture container 2 includes a mouth portion 21, a neck portion 22 that continues to the mouth portion 21, a shoulder portion 23 that continues to the neck portion 22, and a trunk portion 24 that continues to the lower end of the shoulder portion 23, And a bottom portion 25 continuous with the lower end of the body portion 24. The mouth portion 21 has a substantially cylindrical shape, and a thread for attaching the cap 5 (see FIG. 3) is formed on the outer surface of the mouth portion 21 on the opening end side. Thereby, after accommodating to-be-cultured material and the culture solution 8 (refer FIG. 6) in the culture container 2, the culture container 2 can be sealed by attaching the cap 5 to the opening | mouth part 21. FIG.

The shoulder portion 23 is configured by a curved structure that curves from the continuous portion with the neck portion 22 toward the outside of the culture vessel 2 and gradually increases in diameter toward the trunk portion 24. The bottom portion 25 has a convex curved shape toward the outside of the culture vessel 2, that is, a substantially hemispherical shape (so-called round bottom). Since the bottom 25 of the culture vessel 2 has a round bottom, the objects to be cultured that settle in the culture vessel 2 can be collected below the sparger 3, so that the culture is performed by gas (air) discharged from the sparger 3. By stirring the liquid 8, the culture object can be efficiently dispersed.

The cap 5 can be made of a resin material such as polyethylene or polypropylene, or a metal material such as aluminum. The diameter (outer diameter) of the cap 5 can be appropriately set according to the size of the mouth portion 21 of the culture vessel 2, but is, for example, about 28 to 52 mm, and preferably about 28 to 38 mm.

As shown in FIGS. 3A and 3B, the top surface 51 of the cap 5 has a first hole 52 through which the gas supply pipe 4 (see FIG. 1) can be inserted, and a culture solution into the culture vessel 2. A second hole 53 through which the culture medium introduction pipe 6 (see FIG. 1) can be inserted, and a third hole 54 through which the exhaust pipe 7 (see FIG. 1) for exhausting from the inside of the culture vessel 2 can be inserted. Is formed. An exhaust filter 55 is provided inside the top surface 51 of the cap 5 so as to cover at least the third hole 54. On the inner surface of the cap 5, a thread that can be screwed into the mouth portion 21 of the culture vessel 2 is formed.

As shown in FIGS. 4 and 5, the sparger 3 provided in the culture vessel 2 is formed along a substantially spherical sparger body 31 and the radial direction of the sparger body 31, and the gas supply pipe 4 ( 1) and a bottomed hole 32 into which one end is inserted. In the state where the opening 33 of the bottomed hole portion 32 of the sparger 3 is directed upward, it is sufficient that at least the lower half of the sparger body 31 is made of a porous body, and the entire sparger body 31 is made of a porous body. It may be constituted by. Since at least the lower half or the whole of the sparger body 31 is made of a porous body, the gas (air) is bubbled through the gas supply pipe 4 inserted into the bottomed hole 32 and the culture vessel 2 While being able to be introduced into the inside, bubbles are discharged from the sparger body 31 toward the bottom 25 of the culture vessel 2. When the bubbles are discharged toward the bottom 25 of the culture vessel 2, a flow of the culture solution 8 (see FIG. 6) is generated upward from the bottom of the culture vessel 2, and the culture solution 8 convects in the entire culture vessel 2. Therefore, the culture solution 8 in the culture vessel 2 can be agitated by supplying gas (air) from the sparger 3.

Examples of the material constituting the sparger body 31 include resin materials such as polyethylene, polypropylene, polymethyl methacrylate, polystyrene, and fluororesin, and ceramic materials such as alumina and SiC.

The porous body constituting at least the lower half or the whole of the sparger body 31 with the opening 33 of the bottomed hole portion 32 of the sparger body 31 facing upward has pores having an average pore diameter of 20 to 50 μm. It is particularly preferable to have pores having an average pore diameter of 20 to 30 μm. If the average pore diameter of the pores of the porous body is less than 20 μm, there is a risk of liquid splashing on the surface of the culture solution, and if it exceeds 50 μm, the effect of stirring the culture solution in the culture vessel 2 is insufficient. There is a risk of becoming.

The pores of the porous body constituting at least the lower half or the whole of the sparger body 31 preferably have random pore diameters within a predetermined pore diameter distribution range, and randomly within a pore diameter distribution range of 20 to 50 μm. It is more preferable to have a fine pore diameter. Since the pores in the porous body have random pore diameters within a predetermined pore diameter distribution range, particularly within a pore diameter distribution range of 20 to 50 μm, the stirring effect of the culture medium in the culture vessel 2 can be improved. . The average pore size and pore size distribution in the porous body can be measured by a laser microscope (for example, VK-X200 (manufactured by Keyence Corporation)).

The bottomed hole portion 32 plays a role as a gas supply pipe connection portion to which the gas supply pipe 4 is connected, and has an inner diameter into which one end of the gas supply pipe 4 can be inserted. The inside diameter of the tube 4 is such that it cannot be easily pulled out. The bottom surface 34 of the bottomed hole portion 32 is positioned below the center C of the sparger body 31 in a state where the opening 33 of the bottomed hole portion 32 is positioned upward. Since the bottom surface 34 of the bottomed hole portion 32 is positioned below the center C of the sparger body 31, the gas supply tube 4 is made to have a bottomed hole so that one end of the gas supply tube 4 is brought into contact with the bottom surface 34. If inserted into the portion 32, one end of the gas supply pipe 4 can be positioned below the center of the sparger body 31. As a result, gas (air) can be discharged from below the sparger body 31 toward the bottom 25 of the culture vessel 2. The center C of the sparger body 31 means the center of gravity when the sparger body 31 is spherical.

The length D between the bottom surface 34 of the bottomed hole 32 and the center C of the sparger body 31 (the length in the radial direction of the sparger body 31) is 10 to 40% of the diameter of the sparger body 31. Can be set to a degree.

The inner diameter of the bottomed hole 32 can be appropriately set such that the gas supply pipe 4 can be inserted and the gas supply pipe 4 cannot be easily removed, and can be set to about 6 to 8 mm, for example.

The size of the sparger body 31 is not particularly limited, and can be set as appropriate according to the capacity (size) of the culture vessel 2. The culture vessel 2 has a capacity of about 500 mL to 5000 mL. In some cases, the diameter of the sparger body 31 is preferably about 10 to 20 mm. If the diameter of the sparger body 31 is about 10 to 20 mm, the culture solution 8 can be sufficiently stirred by the gas (bubbles) discharged from the sparger body 31.

The position of the sparger 3 in the culture vessel 2 is not particularly limited. For example, the distance T from the bottom 25 of the culture vessel 2 to the lower end of the sparger 3 is determined by the amount of the object to be cultured that stays in the bottom 25 by gas (air) discharged from the sparger 3 toward the bottom 25 of the culture vessel 2 Any distance that can be dispersed in the culture solution 8 may be used. The distance T is preferably about 5 mm or less, and more preferably about 0 to 3 mm. If the distance T exceeds 5 mm, culturing caused by bubbles discharged from the sparger 3 may cause the culture object to stay in the bottom 25 of the culture vessel 2 without being stirred.

In addition, the sparger 3 in this embodiment uses an upper mold and a lower mold having cavities in accordance with the shape of the sparger main body 31, and fills the cavities with resin particles having a predetermined particle diameter, and heats them. Then, it can be produced by partially fusing adjacent resin particles. The sparger 3 composed of a porous body having a predetermined pore size distribution can be produced in the same manner as described above using resin particles having a predetermined particle size distribution. The sparger 3 whose lower half is made of a porous body is formed by applying a resin material such as an epoxy resin to the upper half of the sparger body 31 manufactured as described above, and the upper half of the pores in the upper half. It can be made by filling with material.

In the culture apparatus 1 having the above-described configuration, the culture object and the culture solution are accommodated in the culture vessel 2, and gas (air) is supplied to the culture solution from the gas supply unit (not shown) via the gas supply pipe 4. Supply. The gas (air) that has passed through the gas supply pipe 4 is discharged in the form of bubbles through the pores of the porous body of the sparger body 31. At this time, the bottom surface 34 of the bottomed hole portion 32 of the sparger body 31 is positioned below the center of the sparger body 31 (on the bottom 25 side of the culture vessel 2), so that one end of the gas supply pipe 4 is It is located below the center of the sparger body 31.

The gas (air) that has passed through the gas supply pipe 4 is discharged from the porous body of the sparger body 31, but one end of the gas supply pipe 4 (that is, the gas discharge port) is the center C of the sparger body 31. If it is located above, the gas (air) discharged from the sparger body 31 (porous body) will be directed upward from the sparger body 31. However, as in the present embodiment, the gas (air) discharged from the sparger body 31 is positioned below the center C of the sparger body 31 so that the sparger is directed toward the bottom of the culture vessel 2. Gas (air) is discharged from the main body 31 (arrow A), and a culture fluid flow (arrow B) is generated so as to move upward from between the sparger main body 31 and the bottom 25 of the culture vessel 2 (see FIG. 6). ). With this flow (arrow B), the culture solution 8 can be agitated without causing the culture object to stay in the bottom 25 of the culture vessel 2.

The flow rate of the gas (air) supplied to the culture solution via the gas supply pipe 4 is preferably set to about 300 to 700 mL / min, more preferably about 400 to 500 mL / min. If the flow rate of the gas (air) is less than 300 mL / min, there is a possibility that at least a part of the culture object will stay in the bottom 25 of the culture vessel 2. If the flow rate exceeds 700 mL / min, the liquid of the culture solution Liquid splashes may occur on the surface, and the exhaust filter 55 may be contaminated.

As described above, according to the sparger 3 and the culture apparatus 1 of the present embodiment, a sufficient stirring effect can be obtained without increasing the amount of gas (air) supplied to the culture solution. It is possible to prevent the exhaust filter 55 from being contaminated without causing splashing or the like.

The embodiment described above is described for facilitating understanding of the present invention, and is not described for limiting the present invention. Therefore, each element disclosed in the above embodiment is intended to include all design changes and equivalents belonging to the technical scope of the present invention.

Hereinafter, the present invention will be described in more detail with reference to examples and comparative examples, but the present invention is not limited to the following examples.

[Example 1]
A sparger 3 having a bottomed hole portion 32 having a diameter of 10 mm and an average pore diameter of 20 μm and having an inner diameter of 6 mm, a gas supply pipe 4, a culture vessel 2 having an internal volume of 3000 mL, and a cap 5 And the culture apparatus 1 having the structure shown in FIG. 1 was assembled.

[Example 2]
A gas supply tube 4, a culture vessel 2 and a cap 5 were prepared in the same manner as in Example 1 except that the sparger 3 was composed of a porous body having a diameter of 15 mm, and the culture device 1 was assembled.

Example 3
A gas supply tube 4, a culture vessel 2, and a cap 5 were prepared in the same manner as in Example 1 except that the sparger 3 was composed of a porous body having a diameter of 20 mm, and the culture device 1 was assembled.

Example 4
A gas supply tube 4, a culture vessel 2 and a cap 5 were prepared in the same manner as in Example 1 except that the sparger 3 was composed of a porous body having a diameter of 25 mm, and the culture device 1 was assembled.

Example 5
Example 1 except that an epoxy resin is applied to the surface of the upper half of the sparger body 31 with the opening of the bottomed hole portion 32 facing upward, and the pores of the upper half are filled with the epoxy resin. In the same manner as above, a sparger 3, a gas supply pipe 4, a culture vessel 2 and a cap 5 were prepared, and the culture apparatus 1 was assembled.

Example 6
Example 2 except that an epoxy resin was applied to the upper half surface of the sparger body 31 with the bottomed hole 32 facing upward, and the upper half pores were filled with the epoxy resin. In the same manner as above, a sparger 3, a gas supply pipe 4, a culture vessel 2 and a cap 5 were prepared, and the culture apparatus 1 was assembled.

Example 7
Example 3 except that the upper half surface of the sparger body 31 with the opening of the bottomed hole portion 32 facing upward is coated with epoxy resin and the upper half pores are filled with the epoxy resin. In the same manner as above, a sparger 3, a gas supply pipe 4, a culture vessel 2 and a cap 5 were prepared, and the culture apparatus 1 was assembled.

Example 8
Example 4 except that an epoxy resin was applied to the surface of the upper half of the sparger body 31 with the opening of the bottomed hole portion 32 facing upward, and the pores of the upper half were filled with the epoxy resin. In the same manner as above, a sparger 3, a gas supply pipe 4, a culture vessel 2 and a cap 5 were prepared, and the culture apparatus 1 was assembled.

Example 9
A gas supply tube 4, a culture vessel 2 and a cap 5 were prepared in the same manner as in Example 6 except that the sparger 3 was composed of a porous material having an average pore diameter of 50 μm, and the culture device 1 was assembled.

Example 10
A gas supply tube 4, a culture vessel 2 and a cap 5 were prepared in the same manner as in Example 7 except that the sparger 3 was composed of a porous material having an average pore diameter of 50 μm, and the culture device 1 was assembled.

Example 11
The gas supply tube 4, the culture vessel 2 and the cap 5 were the same as in Example 2 except that the sparger 3 was composed of a porous body having pores with random pore sizes in the pore size distribution range of 20 to 50 μm. And the culture apparatus 1 was assembled.

Example 12
The gas supply tube 4, the culture vessel 2, and the cap 5 are the same as in Example 3 except that the sparger 3 is composed of a porous material having pores with random pore sizes in the pore size distribution range of 20 to 50 μm. And the culture apparatus 1 was assembled.

[Comparative Example 1]
Implementation was performed except that epoxy resin was applied to the surface of the lower half of the sparger body 31 with the opening of the bottomed hole portion 32 facing upward, and the pores of the lower half were filled with the epoxy resin. In the same manner as in Example 1, a sparger 3, a gas supply pipe 4, a culture vessel 2 and a cap 5 were prepared, and the culture apparatus 1 was assembled.

[Comparative Example 2]
Implementation was performed except that epoxy resin was applied to the surface of the lower half of the sparger body 31 with the opening of the bottomed hole portion 32 facing upward, and the pores of the lower half were filled with the epoxy resin. In the same manner as in Example 2, a sparger 3, a gas supply pipe 4, a culture vessel 2 and a cap 5 were prepared, and the culture apparatus 1 was assembled.

[Comparative Example 3]
Implementation was performed except that epoxy resin was applied to the surface of the lower half of the sparger body 31 with the opening of the bottomed hole portion 32 facing upward, and the pores of the lower half were filled with the epoxy resin. In the same manner as in Example 3, a sparger 3, a gas supply pipe 4, a culture vessel 2 and a cap 5 were prepared, and the culture apparatus 1 was assembled.

[Comparative Example 4]
Implementation was performed except that epoxy resin was applied to the surface of the lower half of the sparger body 31 with the opening of the bottomed hole portion 32 facing upward, and the pores of the lower half were filled with the epoxy resin. In the same manner as in Example 4, a sparger 3, a gas supply pipe 4, a culture vessel 2 and a cap 5 were prepared, and the culture apparatus 1 was assembled.

[Test Example 1]
In the culture apparatus 1 of Example 3, when the gas supply pipe 4 is connected to the sparger 3 so that one end of the gas supply pipe 4 is positioned in the vicinity of the opening of the bottomed hole portion 32, one end of the gas supply pipe 4 When the gas supply pipe 4 is connected to the sparger 3 so as to be positioned above the center C of the sparger body 31, one end of the gas supply pipe 4 is positioned below the center of the sparger body 31. Thus, the test which evaluates stirring efficiency was done about three aspects at the time of connecting the gas supply pipe | tube 4 to the sparger 3. FIG.

In this test, 2500 mL of a glucose solution as a culture solution 8 was accommodated in the culture vessel 2, and 100 nylon resin beads having a diameter of 3 mm were placed in place of the culture object. Then, the gas flow rate was set to 600 mL / min, and the number of beads suspended and agitated in the culture vessel 2 was counted 5 minutes after the start of aeration, and the agitation rate (= the number of suspended beads / the number of charged beads). The number x 100 (%)) was determined, and the stirring efficiency was evaluated based on the indices shown in Table 1. The results are shown in Table 2.

As shown in Table 2, a sufficient agitation effect can be obtained by bubbles discharged from the sparger 3 by positioning one end of the gas supply pipe 4 below the center C of the sparger body 31. confirmed.

[Test Example 2]
In the culture apparatus 1 of Examples 1 to 4, the gas supply pipe 4 was connected to the sparger 3 so that one end of the gas supply pipe 4 was located below the center of the sparger body 31, and Similarly, a test for evaluating the stirring efficiency was conducted. In Test Example 2, the gas flow rate was varied in the range of 300 to 900 mL / min, and the stirring efficiency at each flow rate was evaluated. The results are shown in Table 3.

As shown in Table 3, it was confirmed that the smaller the diameter of the sparger 3, the better the stirring effect. In addition, when the gas flow rate was 800 mL / min or more, it was confirmed that although the stirring effect was excellent, liquid splashing occurred on the liquid surface of the culture solution. Therefore, it was confirmed that by setting the gas flow rate to 300 to 700 mL / min, the culture solution can be sufficiently stirred without causing splashing.

[Test Example 3]
In the culture apparatuses 1 of Examples 1 to 12 and Comparative Examples 1 to 4, the gas supply pipe 4 is connected to the sparger 3 so that one end of the gas supply pipe 4 is located below the center of the sparger body 31. In the same manner as in Test Example 1, a test for evaluating the stirring efficiency was conducted. In Test Example 3, the gas flow rate was varied in the range of 300 to 700 mL / min, and the stirring efficiency at each flow rate was evaluated. The results are shown in Table 4.

As shown in Table 4, in Comparative Examples 1 to 4 in which the upper half of the sparger body is composed of a porous body, the stirring effect is insufficient, and the lower half or the whole of the sparger body is a porous body. In Examples 1 to 12 constituted by the above, it was confirmed that a sufficient stirring effect was obtained by the bubbles discharged from the sparger.

Further, from the results of Examples 1 to 8, Example 5 in which the lower half of the sparger body is made of a porous body, compared to Examples 1 to 4 in which the entire sparger body is made of a porous body. It was confirmed that ˜8 was superior in stirring effect.

Further, Examples 6 and 7 in which the average pore diameter of the porous body is relatively smaller than those in Examples 9 and 10 in which the average pore diameter of the porous body is relatively large are more effective for stirring. It was confirmed to be excellent.

Furthermore, even in Example 11 and Example 12 in which the entire sparger body is composed of a porous body, the pores of the porous body have random pore sizes within a predetermined pore size distribution range. As a result, turbulent flow can be generated in the culture solution in the culture vessel, and it was confirmed that the stirring effect was excellent.

DESCRIPTION OF SYMBOLS 1 ... Culture apparatus 2 ... Culture container 3 ... Sparger 31 ... Sparger main body 32 ... Bottomed hole part 4 ... Gas supply pipe 5 ... Cap

Claims

A sparger for introducing gas into the culture vessel in the form of bubbles,
A sparger body at least partially composed of a porous body;
A gas supply pipe connecting portion to which a gas supply pipe for supplying the gas introduced into the culture vessel to the sparger body is connected;
The gas supply pipe connecting portion is formed by a bottomed hole portion into which the gas supply pipe can be inserted, formed in the sparger body.
In the state where the opening of the bottomed hole portion is directed upward, at least the lower half of the sparger body is constituted by the porous body.
The sparger according to claim 1, wherein the whole sparger body is constituted by the porous body.
The sparger according to claim 1 or 2, wherein the porous body has an average pore diameter of 20 to 50 µm.
The sparger according to any one of claims 1 to 3, wherein the porous body has pores having a pore size distribution of 20 to 50 µm.
5. The bottom surface of the bottomed hole portion is located below the center of the sparger body in a state where the opening of the bottomed hole portion is positioned upward. The sparger according to any one.
The sparger according to any one of claims 1 to 5, wherein the sparger body is substantially spherical.
The sparger according to claim 6, wherein the sparger body has a diameter of 10 to 20 mm.
A culture vessel capable of accommodating a culture,
The sparger according to any one of claims 1 to 7, provided in the culture vessel;
One end portion is inserted into the bottomed hole portion, and the other end portion includes a gas supply pipe continuing to the gas supply portion,
The sparger is provided in the culture vessel so that the porous body faces the bottom of the culture vessel.
The culture apparatus according to claim 8, wherein the flow rate of the gas supplied to the sparger through the gas supply pipe is 300 to 700 mL / min.
The culture apparatus according to claim 8 or 9, wherein the bottom of the culture vessel has a substantially hemispherical shape.