JP2022130304A - Coculture apparatus, coculture system, and coculture method - Google Patents

Abstract

To provide a coculture apparatus capable of evaluating both a culture medium flowing in an anaerobic environment and a culture medium flowing in an aerobic environment over time.SOLUTION: A coculture apparatus comprises: a first sealed container; a coculture device arranged outside the first sealed container; a first culture medium source which is arranged inside the first sealed container and in which the first culture medium is stored; a second culture medium source in which second culture medium having lower dissolved oxygen concentration than the first culture medium is stored; and a first conduit connected to the coculture device and the first culture medium source. The coculture device comprises: a membrane which has a first principal plane and a second principal plane, which lies on the opposite side of the first principal plane and is used to culture cells; a first flow channel a part of which is defined by the first principal plane, and which is arranged such that the first culture medium flows in the channel; a second flow channel a part of which is defined by the second principal plane, and which is arranged such that the second culture medium flows in the channel. An inlet of the first flow channel is connected to the first conduit.SELECTED DRAWING: Figure 1

Description

The present invention relates to a co-culture device, a co-culture system and a co-culture method.

For the purpose of investigating pharmacokinetics, drug metabolism, etc., devices that simulate the intestinal environment are being developed. Patent Document 1 (International Publication No. 2018/079793) discloses a system that simulates an intestinal environment by placing a device in which intestinal epithelial cells are seeded on a porous membrane in an anaerobic chamber.

WO2018/079793

With conventional techniques including Patent Document 1, it is difficult to temporally evaluate both a medium flowing in an anaerobic environment and a medium flowing in an aerobic environment.

The present invention provides a co-cultivation device, a co-cultivation system, and a co-cultivation method capable of temporally evaluating both a medium flowing in an anaerobic environment and a medium flowing in an aerobic environment.

The co-cultivation apparatus of the present invention comprises a first closed container, a co-culture device arranged outside the first closed container, and a first medium source arranged in the first closed container and storing a first medium. , a second medium source storing a second medium having a lower dissolved oxygen concentration than the first medium, and a first conduit connected to the co-cultivation device and the first medium source. a co-culture device partially defined by a first major surface and a membrane including a second major surface opposite the first major surface and for culturing cells; and a first channel arranged so that the first culture medium flows; and a second channel partially defined by the second main surface and arranged so that the second culture medium flows. have. The inlet of the first flow path is connected to the first conduit.

A co-cultivation system of the present invention comprises an anaerobic chamber and the co-cultivation apparatus described above arranged in the anaerobic chamber.

The co-cultivation method of the present invention includes, in an anaerobic chamber, a first major surface and a membrane opposite the first major surface and including a second major surface for culturing cells, and arranging a co-cultivation device having a first channel defined by a portion and a second channel partially defined by a second major surface; and supplying a second medium having a lower dissolved oxygen concentration than the first medium to the second channel. The first culture medium supplied to the first channel is maintained in an aerobic environment within the anaerobic chamber.

According to the co-cultivation device, the co-cultivation system and the co-cultivation method of the present invention, it is possible to temporally evaluate both the medium flowing in the anaerobic environment and the medium flowing in the aerobic environment.

1 is a schematic cross-sectional view of a co-culture system according to an embodiment; FIG. 1 is an exploded perspective view of co-culture device 10. FIG. FIG. 4 is an exploded perspective view for explaining how the first holder 17a and the second holder 17b are attached to the co-culture device 10; 1 is a process chart of a co-culture method according to an embodiment; FIG. FIG. 4 is a schematic cross-sectional view of a co-culture system according to a comparative example; FIG. 4 is a schematic cross-sectional view of a co-culture system according to a first modified example; FIG. 11 is a schematic cross-sectional view of a co-culture system according to a second modified example;

Details of embodiments will be described with reference to the drawings. In the drawings below, the same or corresponding parts are denoted by the same reference numerals, and redundant description will not be repeated.

(Co-culture system according to the embodiment)
A co-culture system according to an embodiment will be described below.

<Schematic configuration of the co-culture system according to the embodiment>
FIG. 1 is a schematic cross-sectional view of a co-culture system according to an embodiment. As shown in FIG. 1, the co-culture system according to the embodiment has a co-culture device 10 and a first sealed container 20. As shown in FIG. The co-cultivation system according to the embodiment has a first medium container 30a, a second medium container 30b, a third medium container 30c, and a fourth medium container 30d. The co-culture system according to the embodiment has a first tube 40a, a second tube 40b, a third tube 40c, and a fourth tube 40d.

The co-culture system according to the embodiment has a first pump 50a, a second pump 50b, and a transepithelial electrical resistance measuring device 60.

The co-culture device 10 has a first surface 10a and a second surface 10b. The first surface 10a faces the first sealed container 20 side. The second surface 10b is opposite the first surface 10a. The co-culture device 10 has a membrane 11, a first channel 12, and a second channel 13 inside. The co-culture device 10 is arranged outside the first sealed container 20 .

The membrane 11 has a first main surface 11a and a second main surface 11b. The second major surface 11b is opposite the first major surface 11a. The second main surface 11b is the surface of the membrane 11 for culturing cells. Cells cultured on the second main surface 11b are, for example, intestinal epithelial cells that form tight junctions on the second main surface 11b. A specific example of cells cultured on the second main surface 11b is Caco-2 cells.

The membrane 11 is, for example, a polycarbonate track-etched film. The membrane 11 may be, for example, a porous membrane made of other material such as PET (polyethylene terephthalate) or a collagen vitrigel membrane. The membrane 11 is not particularly limited as long as cell culture can be performed on the second main surface 11b and oxygen and nutrients can be supplied from the first main surface 11a.

A part of the first flow path 12 is defined by the first main surface 11a. A part of the second flow path 13 is defined by the second main surface 11b. The first channel 12 is a channel through which the first culture medium 80a flows, and the second channel 13 is a channel through which the second culture medium 80b flows.

The dissolved oxygen concentration in the second medium 80b is lower than the dissolved oxygen concentration in the first medium 80a. That is, the first medium 80a is an aerobic medium, and the second medium 80b is an anaerobic medium. The second medium 80b may contain bacteria. Bacteria contained in the second medium 80b are, for example, anaerobic bacteria.

While the first medium 80a is flowing through the first channel 12, oxygen in the first medium 80a is supplied through the membrane 11 to the cells cultured on the second major surface 11b. Therefore, even if the dissolved oxygen concentration in the second medium 80b is low, the cells cultured on the second major surface 11b can be maintained.

The first sealed container 20 is a container whose internal space can be sealed. The internal space of the first sealed container 20 is maintained in an aerobic environment. More specifically, the internal space of the first closed container 20 is maintained in an atmospheric environment. The first airtight container 20 has a body portion 21 and a lid 22 . The body portion 21 is cylindrical. The lower end of the body portion 21 is closed by the bottom wall, and the upper end of the body portion 21 is open. The lid 22 is detachably attached to the upper end of the body portion 21 . A through hole 22a is formed in the lid 22 so as to penetrate the lid 22 in the thickness direction. A non-contact oxygen monitor (for example, a spot sensor capable of measuring the amount of oxygen by irradiating excitation light) may be arranged in the first sealed container 20 . Thereby, the amount of oxygen remaining in the first sealed container 20 can be confirmed.

A first medium 80a is stored in the first medium container 30a. The first culture medium container 30a and the inlet of the first channel 12 are connected by a first tube 40a. The first tube 40 a is arranged inside the first sealed container 20 . The first tube 40a connects the first culture medium container 30a and the inlet of the first channel 12 through the through hole 22a.

A second medium 80b is stored in the second medium container 30b. The second culture medium container 30b and the inlet of the second channel 13 are connected by a second tube 40b. The second tube 40b is arranged outside the first sealed container 20 . The inlet of the first channel 12 is on the first surface 10a. The inlet of the second channel 13 is on the second surface 10b.

The third medium container 30c and the outlet of the first channel 12 are connected by a third tube 40c. The fourth medium container 30d and the outlet of the second channel 13 are connected by a fourth tube 40d. The third tube 40 c and the fourth tube 40 d are arranged outside the first sealed container 20 . The outlet of the first channel 12 is on the second surface 10b. The outlet of the second channel 13 is on the second surface 10b.

The first pump 50a supplies the first medium 80a stored in the first medium container 30a to the first channel 12 via the first tube 40a. The first pump 50a supplies the first medium 80a discharged from the first channel 12 to the third medium container 30c via the third tube 40c. The first pump 50a is attached to the first tube 40a. That is, the first pump 50 a is arranged inside the first closed container 20 . However, the first pump 50a may be arranged outside the first sealed container 20 (may be attached to the third tube 40c).

The second pump 50b supplies the second medium 80b stored in the second medium container 30b to the second channel 13 via the second tube 40b. The second pump 50b supplies the second medium 80b discharged from the first channel 12 to the fourth medium container 30d via the fourth tube 40d. The second pump 50b is attached to the second tube 40b. That is, the second pump 50b is arranged outside the first sealed container 20. As shown in FIG. However, the second pump 50b may be attached to the fourth tube 40d.

The first tube 40a, the second tube 40b, the third tube 40c, and the fourth tube 40d are tubes made of, for example, PTFE (polytetrafluoroethylene), PEEK (polyetheretherketone, silicone, etc.). The first pump 50a is, for example, a ring pump, and the second pump 50b is, for example, a ring pump.

The co-culture device 10 has a first electrode 14a and a second electrode 14b. The transepithelial electrical resistance measuring device 60 is electrically connected to the first electrode 14a and the second electrode 14b. Thereby, the transepithelial electrical resistance measuring device 60 measures the electrical resistance value between the first culture medium 80 a flowing through the first channel 12 and the second culture medium 80 b flowing through the second channel 13 .

When the cells cultured on the second principal surface 11b form tight junctions, this electrical resistance value increases, and the cells cultured on the second principal surface 11b do not form tight junctions. In this case, the electrical resistance value is low, so by measuring this electrical resistance value with the transepithelial electrical resistance measuring device 60, the state of the cells cultured on the second main surface 11b can be monitored.

The inside of the anaerobic chamber 70 is maintained in an anaerobic environment. A co-culture device according to the embodiment is arranged in the anaerobic chamber 70 .

<Detailed configuration of co-culture device 10>
FIG. 2 is an exploded perspective view of the co-culture device 10. FIG. As shown in FIG. 2, the co-culture device 10 has a structure in which a membrane 11, a first glass plate 15a, a second glass plate 15b, a first sheet 16a, and a second sheet 16b are laminated. is doing. The first sheet 16a and the second sheet 16b are made of resin material, for example. A specific example of this resin material is silicone rubber.

The first glass plate 15a is arranged on the first surface 10a side. A through hole 15aa is formed in the first glass plate 15a. The through hole 15aa penetrates the first glass plate 15a in the thickness direction. The through hole 15 aa serves as an inlet of the first flow path 12 .

The second glass plate 15b is arranged on the second surface 10b side. A through hole 15ba, a through hole 15bb, and a through hole 15bc are formed in the second glass plate 15b. The through hole 15ba serves as an inlet of the second flow path 13. As shown in FIG. The through hole 15bb serves as an outlet of the first flow path 12. As shown in FIG. The through hole 15bc serves as the outlet of the second flow path 13. As shown in FIG.

The membrane 11 is sandwiched between the first sheet 16a and the second sheet 16b. The first main surface 11a of the membrane 11 faces the first sheet 16a side, and the second main surface 11b of the membrane 11 faces the second sheet 16b side. The first sheet 16a and the second sheet 16b are in contact with each other except for the portion where the membrane 11 is sandwiched.

The first sheet 16a and the second sheet 16b are sandwiched between the first glass plate 15a and the second glass plate 15b. The first sheet 16a is in contact with the first glass plate 15a. The second sheet 16b is in contact with the second glass plate 15b.

Although not shown, a first electrode 14a is formed on the surface of the first glass plate 15a facing the first sheet 16a, and a second electrode 14b is formed on the surface of the second glass plate 15b facing the second sheet 16b. is formed. The first electrode 14a and the second electrode 14b are made of platinum, for example. The first electrode 14a and the second electrode 14b are formed by sputtering, for example.

A groove 16aa is formed in the surface of the first sheet 16a on the side of the first glass plate 15a. The groove 16aa is positioned to overlap the through hole 15aa. A through hole 16ab and a through hole 16ac are formed in the first sheet 16a. The through holes 16ab and 16ac pass through the first sheet 16a in the thickness direction. The through hole 16ab and the through hole 16ac are positioned to overlap the groove 16aa.

A groove 16ba is formed in the surface of the second sheet 16b on the side of the second glass plate 15b. The groove 16ba is positioned to overlap the through hole 15ba and the through hole 15bc.

A through hole 16bb and a through hole 16bc are formed in the second sheet 16b. The through holes 16bb and 16bc penetrate the second sheet 16b in the thickness direction. The through hole 16bb is positioned to overlap with the groove 16ba. The through hole 16bb is positioned to overlap with the through hole 16ab. The through holes 16ab and 16bb are closed by the membrane 11 . The through hole 16bc is positioned to overlap with the through hole 15bb and the through hole 16ac.

The through-hole 15aa, the groove 16aa, the through-hole 16ac, the through-hole 16bc, the through-hole 15bb, and the first main surface 11a of the membrane 11 form the first flow path 12. As shown in FIG. The through hole 15ba, the groove 16ba, the through hole 15bc, and the second main surface 11b of the membrane 11 form a second flow path 13. As shown in FIG.

The bonding between the first glass plate 15a and the first sheet 16a, the bonding between the first sheet 16a and the second sheet 16b, and the bonding between the second sheet 16b and the second glass plate 15b are performed by, for example, oxygen plasma. This is done by applying pressure to the joint surfaces in an activated state.

FIG. 3 is an exploded perspective view for explaining how the first holder 17a and the second holder 17b are attached to the co-culture device 10. FIG. As shown in FIG. 3, the co-culture device 10 is sandwiched between a first holder 17a and a second holder 17b. A first rubber sheet 18 a is sandwiched between the first holder 17 a and the co-culture device 10 . A second rubber sheet 18b is sandwiched between the second holder 17b and the co-culture device 10 .

The first holder 17a and the second holder 17b are made of, for example, polyetheretherketone resin (PEEK resin). The first rubber sheet 18a and the second rubber sheet 18b are made of, for example, butyl rubber.

A through hole 17aa is formed in the first holder 17a. The through hole 17aa penetrates the first holder 17a in the thickness direction. The through-hole 17aa overlaps the through-hole 15aa when the first holder 17a is attached to the co-culture device 10 . A through hole 18aa is formed in the first rubber sheet 18a. The through-hole 18aa overlaps the through-hole 15aa when the first holder 17a is attached to the co-culture device 10 . That is, the first tube 40a passing through the through hole 22a is connected to the through hole 15aa (the inlet of the first channel 12) through the through holes 17aa and 18aa.

A through hole 17ba, a through hole 17bb, and a through hole 17bc are formed in the second holder 17b. The through hole 17ba, the through hole 17bb, and the through hole 17bc penetrate the second holder 17b in the thickness direction. A through hole 18ba, a through hole 18bb, and a through hole 18bc are formed in the second rubber sheet 18b. The through hole 18ba, the through hole 18bb, and the through hole 18bc penetrate the second rubber sheet 18b in the thickness direction.

The through-hole 17ba, the through-hole 17bb, and the through-hole 17bc overlap with the through-hole 15ba, the through-hole 15bb, and the through-hole 15bc, respectively, when the second holder 17b is attached to the co-culture device 10 . The through-hole 18ba, the through-hole 18bb, and the through-hole 18bc overlap the through-hole 15ba, the through-hole 15bb, and the through-hole 15bc, respectively, when the second holder 17b is attached to the co-culture device 10 . The second tube 40b is connected to the through hole 15ba (the inlet of the second flow path 13) through the through hole 17ba and the through hole 18ba. The third tube 40c is connected to the through hole 15bb (the outlet of the first channel 12) through the through hole 17bb and the through hole 18bb. The fourth tube 40d is connected to the through hole 15bc (the outlet of the second channel 13) through the through hole 17bc and the through hole 18bc.

The co-culture device 10 is attached to the lid 22 of the first sealed container 20 while being sandwiched between the first holder 17a and the second holder 17b. This attachment is performed, for example, by screwing.

(Co-culture method according to the embodiment)
The co-culture method according to the embodiment will be described below.

FIG. 4 is a process chart of the co-culture method according to the embodiment. As shown in FIG. 4, the co-cultivation method according to the embodiment has a preparation step S1 and a medium supply step S2. The medium supply step S2 is performed after the preparation step S1.

In the preparation step S<b>1 , the co-culture device according to the embodiment is placed inside the anaerobic chamber 70 . In the medium supply step S2, the first medium 80a stored in the first medium container 30a is supplied to the first channel 12 via the first tube 40a. In addition, in the medium supply step S2, the second medium 80b stored in the second medium container 30b is supplied to the second channel 13 via the second tube 40b. The first culture medium 80a is supplied to the first channel 12 by driving the first pump 50a. The supply of the second culture medium 80b to the second channel 13 is performed by driving the second pump 50b. As a result, co-cultivation of cells and bacteria is performed in the co-cultivation device 10 .

By driving the first pump 50a, the first medium 80a that has flowed through the first channel 12 is supplied to the third medium container 30c via the third tube 40c. By driving the second pump 50b, the second medium 80b that has flowed through the second flow path 13 is supplied to the fourth medium container 30d via the fourth tube 40d. The first medium 80a stored in the third medium container 30c and the second medium 80b stored in the fourth medium container 30d are subjected to mass spectrometry using, for example, liquid chromatography mass spectrometry.

(Effect of co-culture system according to embodiment)
The effect of the co-culture system according to the embodiment will be described below in comparison with a comparative example.

FIG. 5 is a schematic cross-sectional view of a co-culture system according to a comparative example. The co-culture system according to the comparative example does not have the first sealed container 20, as shown in FIG. Regarding other points, the co-culture system according to the comparative example is common to the co-culture system according to the embodiment.

In the co-cultivation system according to the comparative example, since the first medium container 30a and the first tube 40a are not arranged in the first closed container 20, the first medium container 30a is stored and supplied to the first channel 12. The first culture medium 80 a to be processed is exposed to the anaerobic environment within the anaerobic chamber 70 . On the other hand, in the co-culture system according to the embodiment, since the first medium container 30a and the first tube 40a are arranged inside the first sealed container 20, the first medium 80a stored in the first medium container 30a is supplied to the first channel 12 without being exposed to the anaerobic environment.

Further, in the co-cultivation system according to the embodiment, since the second medium container 30b and the second tube 40b are arranged in the anaerobic chamber 70, the second medium 80b stored in the second medium container 30b is anaerobic. It flows under the environment and is supplied to the second channel 13 . Thus, according to the co-culture system according to the embodiment, both the medium flowing in the aerobic environment (first medium 80a) and the medium flowing in the anaerobic environment (second medium 80b) are evaluated over time. It is possible.

In the co-culture system according to the embodiment, the outlet of the first channel 12 (through hole 15bb), the inlet of the second channel 13 (through hole 15ba), and the outlet of the second channel 13 (through hole 15bc) are the second While formed on the surface 10b, the inlet (through hole 15aa) of the first flow path 12 is formed on the first surface 10a facing the first closed container 20 side, so that the first culture medium container 30a The inlet of the first channel 12 and the first culture medium container 30a can be easily connected by the first tube 40a while being arranged in the first sealed container 20. As shown in FIG.

(Co-culture system according to the first modification)
A co-culture system according to the first modified example will be described below. Here, differences from the co-culture system according to the embodiment will be mainly described, and redundant description will not be repeated.

FIG. 6 is a schematic cross-sectional view of a co-culture system according to a first modified example. The culture system according to the first modified example has a second sealed container 90, as shown in FIG. The internal space of the second sealed container 90 is sealed. The co-culture device 10, the second pump 50b and the second culture medium container 30b are arranged in the second closed container 90. As shown in FIG. Regarding other points, the co-culture system according to the first modified example is in common with the co-culture system according to the embodiment. The second pump 50b and the second culture medium container 30b may be arranged outside the second sealed container 90.

When the evaluation is over a long period of time (for example, several days), it may be necessary to bring the first sealed container 20 out of the anaerobic chamber 70 in order to replace the first medium 80a stored in the first medium container 30a. be. At this time, the anaerobic environment in the co-culture device 10 cannot be maintained.

In the co-cultivation system according to the first modification, the co-cultivation device 10, the second pump 50b, and the second culture medium container 30b are arranged in the second closed container 90, so that the first culture medium container 30a is reserved. Even if the oxygen concentration in the anaerobic chamber 70 temporarily increases when the first closed container 20 is taken out of the anaerobic chamber 70 to replace the first medium 80a, the co-culture device 10, the second pump 50b and the It becomes possible to keep the second medium container 30b in the anaerobic environment.

(Co-culture system according to the second modification)
A co-culture system according to the second modified example will be described below. Here, differences from the co-culture system according to the embodiment will be mainly described, and redundant description will not be repeated.

FIG. 7 is a schematic cross-sectional view of a co-culture system according to a second modified example. In the co-culture system according to the second modification, as shown in FIG. 7, the co-culture device 10 has a culture vessel 19a and an adapter 19b, unlike the laminated structure of the co-culture device 10 in the co-culture system according to the embodiment. and a channel plate 19c.

The culture tank 19a is, for example, a cell culture insert. The culture tank 19a has a tubular portion 19aa and a membrane 11 . The tubular portion 19aa is made of, for example, a resin material. The membrane 11 closes the lower end side of the cylindrical portion 19aa. The first main surface 11a and the second main surface 11b face the outside and the inside of the culture tank 19a (cylindrical portion 19aa), respectively.

A through hole 22b is formed in the lid 22 and communicates with the inside of the first closed container 20 through the lid 22 along the thickness direction. The lower end side of the culture tank 19 a is inserted into the through hole 22 b so as to be positioned inside the first closed container 20 . A space between the culture tank 19a and the through hole 22b is sealed with a gas sealing portion 19d. Thereby, the airtightness inside the first sealed container 20 is maintained. The gas sealing portion 19d is, for example, an O-ring.

The adapter 19b is inserted into the tubular portion 19aa from the upper end side so as to be separated from the membrane 11 (second main surface 11b). A space defined by the bottom surface of the adapter 19b, the inner wall surface of the cylindrical portion 19aa, and the second main surface 11b constitutes a second flow path 13. As shown in FIG. The adapter 19b is made of rubber, for example.

A through hole 19ba and a through hole 19bb are formed in the adapter 19b. The through hole 10ba and the through hole 19bb pass through the adapter 19b along the direction from the top surface of the adapter 19b to the bottom surface of the adapter 19b. The through hole 19ba and the through hole 19bb communicate with the second channel 13 . A second tube 40b and a fourth tube 40d are inserted into the through hole 19ba and the through hole 19bb, respectively. Thereby, the second tube 40 b and the fourth tube 40 d are connected to the second flow path 13 .

The channel plate 19c is a plate-like member. The channel plate 19c is arranged inside the first sealed container 20 . A groove 19ca, a through hole 19cb, and a through hole 19cc are formed in the channel plate 19c. The groove 19ca is formed in one main surface of the channel plate 19c. One main surface of the channel plate 19c faces the culture tank 19a. The through holes 19cb and 19cc are formed in the other main surface of the channel plate 19c so as to penetrate the channel plate 19c along the thickness direction and communicate with the grooves 19ca.

The culture tank 19a is arranged on the channel plate 19c such that the first main surface 11a faces the groove 19ca. The space between the culture tank 19a and the groove 19ca is liquid-tightly sealed by a liquid sealing portion 19e. The liquid sealing part 19e is, for example, a seal or an O-ring made of PDMS (polydimethylsiloxane). The groove 19ca, the through hole 19cb, the through hole 19cc, the liquid sealing portion 19e, and the first main surface 11a constitute the first flow path 12. As shown in FIG. A first tube 40a and a third tube 40c are connected to the through hole 19cb and the through hole 19cc, respectively. Regarding other points, the configuration of the co-culture system according to the second modification is common to the configuration of the co-culture system according to the embodiment.

In the co-cultivation system according to the second modified example, since the first medium container 30a and the first tube 40a are arranged in the first sealed container 20, the first medium 80a stored in the first medium container 30a , is supplied to the first channel 12 without being exposed to the anaerobic environment. In addition, in the co-cultivation system according to the second modification, the second medium container 30b and the second tube 40b are arranged in the anaerobic chamber 70, so the second medium 80b stored in the second medium container 30b is , flows under an anaerobic environment and is supplied to the second channel 13 . In this way, even with the co-cultivation system according to the second modification, both the medium flowing in the aerobic environment (first medium 80a) and the medium flowing in the anaerobic environment (second medium 80b) are changed over time. It is possible to evaluate

Although the embodiment of the present invention has been described as above, it is also possible to modify the above-described embodiment in various ways. Moreover, the scope of the present invention is not limited to the above embodiments. The scope of the present invention is indicated by the scope of claims, and is intended to include all changes within the meaning and scope of equivalence to the scope of claims.

10 co-culture device, 10a first surface, 10b second surface, 11 membrane, 11a first main surface, 11b second main surface, 12 first channel, 13 second channel, 14a first electrode, 14b second second Electrode 15a First glass plate 15aa Through hole 15b Second glass plate 15ba, 15bb, 15bc Through hole 16a First sheet 16aa Groove 16ab Through hole 16b Second sheet 16ba Groove 16bb, 16bc Penetration hole 17a first holder 17aa through hole 17b second holder 17ba, 17bb, 17bc through hole 18a first rubber sheet 18aa through hole 18b second rubber sheet 18ba, 18bb, 18bc through hole 19a culture Tank 19aa Cylindrical part 19b Adapter 19ba Through hole 19bb Through hole 19c Channel plate 19ca Groove 19cb Through hole 19cc Through hole 19d Gas sealing part 19e Liquid sealing part 20 First sealing Container 21 Main body 22 Lid 22a Through hole 22b Through hole 30a First medium container 30b Second medium container 30c Third medium container 30d Fourth medium container 40a First tube 40b Second tube , 40c third tube, 40d fourth tube, 50a first pump, 50b second pump, 60 transepithelial electrical resistance measuring device, 70 anaerobic chamber, 80a first medium, 80b second medium, 90 second sealed container, S1 Preparation step, S2 medium supply step.

Claims (9)

a co-culture device;
a first sealed container connected to the co-culture device;
a first medium source disposed within the first closed container for supplying a first medium to the co-cultivation device;
a second medium source for supplying the co-cultivation device with a second medium having a lower dissolved oxygen concentration than the first medium;
a first conduit connected to the co-cultivation device and the first medium source;
The co-culture device comprises
a membrane comprising a first major surface and a second major surface opposite the first major surface and for culturing cells;
a first channel partially defined by the first major surface and arranged to flow the first culture medium;
a second flow path partly defined by the second main surface and arranged so that the second culture medium flows;
The co-cultivation device, wherein the inlet of the first channel is connected to the first conduit. The co-culture device is arranged outside the first sealed container,
The co-culture device has a first plate-like member and a second plate-like member that sandwich the membrane from the first main surface side and the second main surface side,
The co-cultivation device according to claim 1, wherein the first conduit is connected to the first plate member. The co-culture device has a culture tank having a bottom surface on which the membrane is arranged,
2. The co-cultivation apparatus according to claim 1, wherein said first closed container has an opening for receiving said co-cultivation device. further comprising a first driving source that supplies the first culture medium stored in the first culture medium source to the first channel via the first conduit;
The co-cultivation device according to any one of claims 1 to 3, wherein the first drive source is arranged inside the first sealed container. The co-culture device has a first surface facing the first closed container and a second surface opposite to the first surface,
The inlet of the first flow path is formed on the first surface,
5. Any one of claims 1, 2 and 4, wherein the outlet of the first channel, the inlet of the second channel and the outlet of the second channel are formed on the second surface. The co-culture device according to the item. Further comprising a second closed container,
The co-cultivation apparatus according to any one of claims 1 to 5, wherein the co-cultivation device is arranged in the second sealed container. a second conduit connecting the inlet of the second channel and the second medium source;
a second drive source that supplies the second medium stored in the second medium source to the second flow path via the second conduit,
7. The co-cultivation device according to claim 6, wherein said second drive source and said second culture medium source are arranged within said second sealed container. an anaerobic chamber;
A co-cultivation system comprising the co-cultivation device according to any one of claims 1 to 7, which is arranged in the anaerobic chamber. a membrane including a first major surface and a second major surface opposite the first major surface and for culturing cells; and a first channel partially defined by the first major surface. and a second flow path partially defined by the second major surface in an anaerobic chamber;
supplying a first culture medium to the first channel;
A step of supplying a second medium having a lower dissolved oxygen concentration than the first medium to the second flow path,
The co-cultivation method, wherein the first medium supplied to the first channel is maintained in an aerobic environment within the anaerobic chamber.

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