Cultivation device, culture production method, and drug evaluation method

Abstract

To provide a culture device, a method for producing a culture, and a method for evaluating a medicine, which are excellent in versatility.SOLUTION: Provided is a culture device for co-cultivating a first cell culture and a second cell culture. The culture device comprises a main body in which a culture space capable of culturing under anaerobic conditions is formed. The main body has an opening that opens from the culture space to the outside of the main body, and a porous body provided in the opening. The first cell culture is held in the porous body so as to cover the culture space side of the porous body. The second cell culture is cultured in the culture space. The main body is configured such that the culture space can be closed from the outside of the main body except for the opening.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] FIELD Embodiments of the present invention relate to a culture device, a culture production method, and a drug evaluation method. [Background technology]

[0002] It is known that intestinal bacteria living in the intestinal tract of living organisms have a significant impact on intestinal function and the maintenance of the intestinal environment. Patent Document 1 proposes a culture system that reproduces the anaerobic environment of the intestinal tract and co-cultures anaerobic intestinal bacteria with intestinal epithelial cells in order to investigate the behavior and characteristics of intestinal bacteria.

[0003] The culture system of Patent Document 1 has an anaerobic culture tank and a closed aerobic culture tank connected to the anaerobic culture tank in order to supply oxygen to intestinal epithelial cells while reproducing the anaerobic environment inside the intestine, and the entire system is formed within an anaerobic chamber. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2018 / 079793 Summary of the Invention [Problem to be solved by the invention]

[0005] However, in the culture system of Patent Document 1, the aerobic culture tank is a closed system, making it difficult to supply oxygen to the aerobic culture tank from the outside. Furthermore, the entire culture system must be constructed inside an anaerobic chamber, making it impossible to use in a regular CO2 incubator. Furthermore, it is not easy to culture cells connected to cells from other organs that require an oxygen supply.

[0006] Therefore, an object of the present invention is to provide a versatile culture device, a culture production method, and a drug evaluation method. [Means for solving the problem]

[0007] The present invention includes the following aspects. [1] A culture device for co-culturing a first cell culture medium and a second cell culture medium, comprising a main body having a culture space formed therein that allows culture under anaerobic conditions, the main body having an opening that opens from the culture space to the outside of the main body and a porous body provided in the opening, the first cell culture medium being held in the porous body so as to cover the culture space side of the porous body, the second cell culture medium being cultured within the culture space, and the main body being configured so that the culture space can be closed from the outside of the main body except for the opening. [2] The culture device described in [1], wherein the porous body has holes that supply oxygen to the first cell culture from outside the body, and the first cell culture consumes oxygen from outside the body, thereby maintaining the closed culture space in an anaerobic state. [3] The culture device according to [1] or [2], wherein the main body has a culture tank having the culture space and a cap that is detachably attached to the culture tank and closes the upper side of the culture space, and the opening is formed on the lower side of the culture space. [4] A culture device according to any one of [1] to [3], comprising a well having a storage space for storing a culture solution under aerobic conditions, and the main body being inserted into the well so that the porous body is exposed to the storage space. [5] The culture device described in [4], wherein the well has a gas-permeable substrate that is impermeable to liquids but permeable to gases, so as to supply oxygen to the storage space from outside the storage space. [6] The culture device according to any one of [1] to [5], further comprising an atmosphere replacement unit that replaces the atmosphere in the culture space, the atmosphere replacement unit having a gas flow path formed inside the main body and communicating with the culture space. [7] The culture device according to any one of [1] to [6], further comprising a culture medium exchange unit that exchanges the culture medium in the culture space, the culture medium exchange unit having a culture medium exchange passage formed inside the main body, communicating with the culture space, and into which a culture medium supply pipe can be inserted. [8] The culture device according to [7], wherein the main body has a retraction section that accommodates the tip of the culture medium supply pipe so that the tip does not point toward the perforated body within the culture space. [9] The culture device according to any one of [1] to [8], further comprising an oxygen sensor for measuring the oxygen concentration in the culture space.

[10] The culture device according to any one of [1] to [9], wherein the first cell culture is a cell layer or tissue containing aerobic cells.

[11] The culture device according to any one of [1] to

[10] , wherein the first cell culture is a cell layer of intestinal epithelial cells, and the second cell culture is intestinal bacteria.

[12] A culture production method for producing a culture by co-culturing a first cell culture and a second cell culture using the culture device described in any one of [1] to

[11] , the culture production method comprising the steps of culturing the first cell culture on the porous body, closing the culture space from the outside of the main body and making the atmosphere in the culture space anaerobic, introducing the second cell culture into the culture space, and co-culturing the second cell culture with the first cell culture in the culture space.

[13] A drug evaluation method using a culture produced by the culture production method described in

[12] , comprising the steps of contacting a drug with the culture, and measuring the response of the culture to stimulation caused by contact with the drug, or the permeability or permeability of the drug through the culture. [Effects of the Invention]

[0008] According to the embodiments of the present invention, it is possible to provide a versatile culture device, a culture medium manufacturing method, and a drug evaluation method. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a perspective view showing a culture device according to one embodiment. [Figure 2] FIG. 1 is an exploded perspective view showing a culture device according to one embodiment. [Figure 3] FIG. 1 is a front view showing a culture device according to one embodiment. [Figure 4] 4 is a cross-sectional view taken along line IV-IV in FIG. 3, showing a culture device according to one embodiment. [Figure 5] 4 is a cross-sectional view taken along line IV-IV in FIG. 3, showing a culture device according to one embodiment. [Figure 6] 1 is a graph showing the change over time in oxygen concentration in the culture device in Experimental Example 1. [Figure 7] 10 is a graph showing the change over time in oxygen concentration in the culture device in Experimental Example 2. [Figure 8] 10 is a graph showing the ratio of lactate production to glucose consumption before and after sealing the culture device in Experimental Example 2. [Figure 9] 10 is a graph showing the TEER values ​​before and after sealing of the culture device in Experimental Example 2. [Figure 10] 10 is a graph showing the change over time in oxygen concentration in the culture device in Experimental Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, a culture device, a culture medium manufacturing method, and a drug evaluation method according to embodiments will be described with reference to the drawings. The following embodiment shows one aspect of the present invention, does not limit the present invention, and can be modified as desired within the scope of the technical concept of the present invention. In addition, in the following drawings, the scale and number of each structure may differ from the actual structure in order to make each configuration easier to understand.

[0011] For the purpose of explanation, a Cartesian coordinate system consisting of x, y, and z axes is defined. The x and y axes are parallel to the horizontal plane, and the z axis is parallel to the vertical direction. Here, the +z direction is defined as upward (i.e., the direction opposite to the direction of gravity), and the -z direction is defined as downward (i.e., the direction of gravity). However, the above coordinate system is set merely for the convenience of explanation and does not limit the invention in any way.

[0012] [Culture device] The culture device of this embodiment is a culture device for co-culturing a first cell culture body and a second cell culture body, and includes a main body having a culture space formed therein that allows culture under anaerobic conditions, the main body having an opening that opens from the culture space to the outside of the main body and a porous body provided in the opening, the first cell culture body is held in the porous body so as to cover the culture space side of the porous body, the second cell culture body is cultured within the culture space, and the main body is configured so that the culture space can be closed from the outside of the main body except for the opening.

[0013] The culture device 1 according to this embodiment will be described below with reference to FIGS. Fig. 1 is a perspective view showing a culture device 1 according to this embodiment. Fig. 2 is an exploded perspective view showing the culture device 1 according to this embodiment. Fig. 3 is a front view showing the culture device 1 according to this embodiment. Figs. 4 and 5 are cross-sectional views taken along line IV-IV in Fig. 3, showing the culture device 1 according to this embodiment.

[0014] The culture device 1 is used to culture one or more types of cells, and is preferably used to co-culture multiple types of cells. The culture device 1 includes a main body 10, a well 60, and an oxygen sensor 70. The lower part of the main body 10 is housed in the upper part of the well 60, and the oxygen sensor 70 is attached to the main body 10 so as to penetrate through the main body 10. The well 60 and the oxygen sensor 70 may be commercially available general-purpose products.

[0015] (Main body 10) The main body 10 includes a culture vessel 20 and a cap 40. As shown in FIG. 2, the main body 10 is assembled by inserting the lower part of the cap 40 into the culture vessel 20. The culture vessel 20, the cap 40, the well 60, and the oxygen sensor 70 are each configured as separate bodies and are detachably assembled together to form the culture device 1 as shown in FIG. 1. The components can be fixed to each other using any locking mechanism or the like. However, some or all of these may also be formed integrally.

[0016] (Culture tank 20) The culture vessel 20 has an internal culture space 28 where cells are cultured. As shown in FIG. 4 , the culture vessel 20 is a cylindrical member that is open in the vertical direction (z direction) and has a peripheral wall 22 and a flange 24 that protrudes radially outward from the peripheral wall 22. An upper open section 26 is formed at the top of the culture vessel 20, which accommodates the lower part of a cap 40. An opening 30 is formed at the bottom of the culture vessel 20, and a perforated body 32 is provided to cover the opening 30. That is, the main body 10 has the culture vessel 20, which has the culture space 28, and the cap 40 that is detachably attached to the culture vessel 20 and closes the upper side of the culture space 28, and the perforated body 32 is formed below the culture space 28. Because the cap 40 is detachable from the culture vessel 20, it is possible to insert electrodes from above and below the cell layer cultured in the culture vessel 20 to measure transepithelial electrical resistance (TEER).

[0017] 4, the outer diameter of the peripheral wall 22 is approximately equal to the outer diameter of the upper part of the cap 40. The flange 24 is used to place the culture vessel 20 on the well 60.

[0018] When the cap 40 is inserted into the upper open portion 26 of the culture vessel 20, the culture space 28 is the internal space of the culture vessel 20 defined by the peripheral wall 22, the underside of the cap 40, and the opening 30. A first culture medium 300 is stored in the culture space 28, and co-culture of the first cell culture medium 100 on the perforated body 32 and the second cell culture medium 200 seeded in the culture space 28 can be performed. As shown in FIG. 4 , when the cap 40 is attached to the culture vessel 20, the main body 10 can close the culture space 28 from the outside of the main body 10, except for the opening 30.

[0019] The porous body 32 is attached to the lower end of the peripheral wall 22 so as to cover the entire opening 30. The porous body 32 has one or more holes through which liquids such as culture fluid (medium) and gas components dissolved in the culture fluid can pass. For example, the porous body 32 is a membrane or thin wall having a large number of holes formed therein. The size of the holes in the porous body 32 is, for example, 0.05 μm to 50 μm, preferably 0.1 μm to 30 μm, more preferably 0.2 μm to 20 μm, and even more preferably 0.5 μm to 10 μm. The thickness of the porous body 32 is, for example, 1 μm to 100 μm, preferably 2 μm to 50 μm, and more preferably 5 μm to 30 μm. The porous body 32 can be made of a material such as polycarbonate, polyethylene terephthalate, polytetrafluoroethylene, or polylactic acid, but is not limited to these.

[0020] The porous body 32 can hold cells on one or both surfaces. For example, as shown in Fig. 4, the porous body 32 can hold the first cell culture 100 in the form of a cell layer or tissue on the surface facing the culture space 28, and preferably the entire surface of the porous body 32 facing the culture space 28 is covered with the cell layer or tissue of the first cell culture 100. When the opening 30 is blocked by the first cell culture 100, the culture space 28 can be closed from the outside of the main body 10.

[0021] As shown in Fig. 5, a recess is provided on the inner surface of the peripheral wall 22, forming a retraction section 34 that accommodates a tip end 94A of a medium supply pipe 94 of a medium supply device 90. Furthermore, a guide section 36 is provided on the inner surface of the peripheral wall 22 for guiding the tip end 94A of the medium supply pipe 94 of the medium supply device 90 to the retraction section 34. The guide section 36 protrudes from the inner surface of the peripheral wall 22 toward the inside of the culture tank 20, and a passage through which the medium supply pipe 94 passes is formed obliquely in the xy-plane view inside the guide section 36. The passage of the guide section 36 is formed in a direction from the lower end of a medium exchange passage 54 (described later) toward the retraction section 34. The retraction section 34 and the guide section 36 will be described later.

[0022] The culture vessel 20 may be made of any gas-impermeable material commonly used in cell culture applications, including, but not limited to, acrylic resin, polyethylene terephthalate, polystyrene, and polycarbonate.

[0023] (Cap 40) The cap 40 is a solid member that is attached to the culture tank 20 and closes the upper side of the culture space 28. As shown in Fig. 4, the outer diameter of the lower part of the cap 40 is slightly smaller than the inner diameter of the peripheral wall 22 of the culture tank 20 so that the cap 40 can be inserted into the upper open part 26 of the culture tank 20. In order to airtightly connect the cap 40 to the culture tank 20 and seal the culture space 28, a first sealing member 75A (e.g., an O-ring) is provided at the joint between the culture tank 20 and the cap 40.

[0024] The cap 40 includes an atmosphere replacement unit 42 for replacing the atmosphere in the culture space 28 and a culture medium replacement unit 50 for replacing the culture medium in the culture space 28. A user can replace the atmosphere in the culture space 28, replace the culture medium, or seed cells in the culture space 28 through a passage formed inside the cap 40. As shown in FIG. 1 , the upper peripheral surface of the cap 40 is formed with a gas inlet 44A for introducing gas into the culture space 28, a gas outlet 44B for discharging gas from the culture space 28, and a culture medium replacement port 52 for replacing the culture medium in the culture space 28.

[0025] As shown in Fig. 4, the atmosphere replacement unit 42 is composed of an atmosphere replacement port 44, a gas flow path 46, and a first closing member 48. The gas flow path 46 is formed inside the main body 10 (cap 40) and communicates with the culture space 28. Here, Figs. 4 and 5 are cross-sectional views taken along line IV-IV in Fig. 3, but for convenience, the gas inlet 44A, gas inlet path 46A, and gas inlet closing member 48A, which are not actually located on the cross section, are also shown. The gas outlet 44B, gas outflow path 46B, and gas outlet closing member 48B are not shown in Figs. 4 and 5, but are similarly arranged and configured to the gas inlet 44A, gas inlet path 46A, and gas inlet closing member 48A except for their positions in the y direction.

[0026] The atmosphere replacement port 44 includes a gas inlet 44A and a gas outlet 44B (see FIG. 1 ), the gas flow path 46 includes a gas inlet channel 46A and a gas outlet channel 46B, and the first closing member 48 includes a gas inlet closing member 48A and a gas outlet closing member 48B. As shown in FIG. 4 , the gas inlet 44A opens to the outside of the main body 10 on the upper peripheral surface of the cap 40 and communicates with the gas inlet channel 46A. The gas inlet channel 46A is a passage formed inside the cap 40, terminates at the lower surface of the cap 40, and communicates with the culture space 28. The gas inlet closing member 48A is a lid member that closes the gas inlet 44A when atmosphere replacement is not being performed. To ensure airtightness, a second sealing member 75B (e.g., an O-ring) is provided between the gas inlet 44A and the gas inlet closing member 48A.

[0027] The gas outlet 44B opens to the outside of the main body 10 on the upper peripheral surface of the cap 40 and communicates with the gas outflow path 46B. The gas outflow path 46B is a passage formed inside the cap 40, terminates at the lower surface of the cap 40, and communicates with the culture space 28. The gas outlet closing member 48B is a lid member that closes the gas outlet 44B when the atmosphere is not being replaced. To ensure airtightness, a sealing member (not shown) having a configuration similar to that of the second sealing member 75B is provided between the gas outlet 44B and the gas outlet closing member 48B.

[0028] When replacing the atmosphere, as shown in FIG. 5, a gas supply pipe 80 is attached to the gas inlet 44A. Anaerobic gas is introduced from the gas supply pipe 80 into the gas inlet 44A and supplied to the culture space 28 through the gas inlet path 46A. At the same time, a gas exhaust pipe (not shown) is attached to the gas outlet 44B, and gas in the culture space 28 is discharged through the gas outlet path 46B and from the gas outlet 44B to the gas exhaust pipe. In this manner, the atmosphere in the culture space 28 can be replaced. Once the atmosphere replacement is complete, the gas supply pipe 80 and the gas exhaust pipe are removed from the gas inlet 44A and the gas outlet 44B, and instead, a gas inlet closing member 48A and a gas outlet closing member 48B are attached to the gas inlet 44A and the gas outlet 44B, respectively.

[0029] As shown in FIG. 4, the culture medium exchange unit 50 is composed of a culture medium exchange port 52, a culture medium exchange passage 54, and a second closing member 56. The culture medium exchange port 52 opens to the outside of the main body 10 on the upper peripheral surface of the cap 40 and communicates with the culture medium exchange passage 54. The culture medium exchange passage 54 is formed inside the main body 10 (cap 40) and communicates with the culture space 28, and a culture medium supply pipe 94 (see FIG. 5) can be inserted from the culture medium exchange port 52. The culture medium exchange passage 54 terminates at the bottom surface of the cap 40 and is connected to the passage of the guide part 36 of the culture tank 20. The second closing member 56 is a lid member that closes the culture medium exchange port 52 when the culture medium is not being exchanged. To ensure airtightness, a third sealing member 75C (e.g., an O-ring) is provided between the culture medium exchange port 52 and the second closing member 56.

[0030] When replacing the culture medium, a syringe-like culture medium supply device 90 capable of injecting and aspirating a fluid can be used, as shown in FIG. 5 . The culture medium supply device 90 includes a syringe 92 that contains a fluid and can inject and aspirate the fluid using a piston mechanism, and a flexible culture medium supply tube 94 extending from the tip of the syringe 92. The culture medium supply device 90 is attached to the culture medium exchange port 52, and the culture medium supply tube 94 is introduced into the culture medium exchange passage 54. The culture medium supply tube 94 passes through the culture medium exchange passage 54 and the passage of the guide portion 36, and is guided by the guide portion 36 to the retraction portion 34, where it is directed away from the first cell culture body 100 formed on the perforated body 32. That is, the retraction portion 34 accommodates the tip 94A of the culture medium supply tube 94 so that the tip 94A does not face the perforated body 32 within the culture space 28. This reduces physical stimulation to the cultured cells when the culture medium is supplied from the tip 94A. By pushing and pulling the piston of the culture medium supplying device 90, the first culture medium 300 in the culture space 28 can be sucked out and supplied to the culture space 28 through the culture medium supplying pipe 94, thereby allowing the first culture medium 300 in the culture space 28 to be replaced. Cell seeding can also be performed by supplying a liquid containing cells into the culture space 28 using the culture medium supplying device 90. In this way, by providing the culture medium replacing unit 50, it is possible to perform cell seeding and culture medium replacement while maintaining the culture space 28 in an anaerobic state.

[0031] The cap 40 has a through-hole 58 for inserting the oxygen sensor 70. The through-hole 58 passes through the cap 40 in the z direction.

[0032] The cap 40, like the culture vessel 20, can be made of any material commonly used in cell culture applications, as long as it is gas impermeable.

[0033] (Well 60) The well 60 is a cylindrical member with a bottom placed under the main body 10, and supplies oxygen to the first cell culture 100 from the underside of the culture tank 20 through the perforated body 32. The well 60 has a storage space 68 that stores a culture solution (second medium 400) under aerobic conditions. The culture tank 20 of the main body 10 is inserted into the well 60 so that the perforated body 32 is exposed to the storage space 68.

[0034] The well 60 has a cylindrical well peripheral wall 62, and the flange 24 of the culture vessel 20 is placed on the upper end of the well peripheral wall 62. The inner diameter of the well peripheral wall 62 is larger than the outer diameter of the peripheral wall 22 of the culture vessel 20 so that the well 60 can receive the lower part of the culture vessel 20. A gas-permeable substrate 66 is provided in a lower opening 64 formed at the lower end of the well peripheral wall 62. The gas-permeable substrate 66 is configured to be permeable to gas but not to liquid so that oxygen can be supplied to the storage space 68 from outside the storage space 68. In other words, the gas-permeable substrate 66 allows oxygen-containing gas to pass from the lower side of the well 60 to the storage space 68. The gas-permeable substrate 66 may be made of a material such as, but not limited to, polydimethylsiloxane (PDMS). The storage space 68 is defined by the well peripheral wall 62 and the gas-permeable substrate 66. When the culture device 1 is used in the atmosphere, the gas-permeable substrate 66 allows oxygen to be continuously supplied from the outside to the culture space 28. Note that the method of supplying oxygen is not limited to the above example, and can be achieved by any means, such as providing oxygen supply holes in the well peripheral wall 62.

[0035] The storage space 68 stores the second culture medium 400 under aerobic conditions. As described above, oxygen is supplied to the second culture medium 400 from outside the storage space 68 through the gas-permeable substrate 66. The porous body 32 housed in the storage space 68 has holes that supply oxygen to the first cell culture 100 from outside the main body 10, so that oxygen in the second culture medium 400 is supplied to the first cell culture 100 through the porous body 32 and consumed by the first cell culture 100. In this way, the first cell culture 100 consumes oxygen from outside the main body 10, thereby maintaining the closed culture space 28 in an anaerobic state.

[0036] (Oxygen sensor 70) The oxygen sensor 70 is disposed so as to pass through the through-hole 58 of the cap 40, and measures the oxygen concentration in the culture space 28. As shown in FIG. 5 , the tip of the oxygen sensor 70 may be immersed in the first culture medium 300 in the culture space 28, and the amount of dissolved oxygen in the first culture medium 300 can be measured. To ensure airtightness, a fourth sealing member 75D (e.g., an O-ring) is provided between the oxygen sensor 70 and the upper part of the cap 40.

[0037] The above-described configuration provides a culture device 1 that can be used in the atmosphere, and oxygen can be supplied to the first cell culture medium 100 on the porous body 32 while maintaining the culture space 28 under anaerobic conditions. This allows for high versatility while improving the reproducibility of the in vivo environment. The culture device 1 does not need to be used in an anaerobic chamber, and can therefore be used in a normal CO2 incubator. Furthermore, because the culture device 1 can be used in the atmosphere, there is no need to worry about oxygen depletion in the aerobic region, and it can be easily incorporated into other culture systems that require oxygen supply. Furthermore, the culture device 1 is highly versatile in that well plates with a general configuration can be used as the wells 60.

[0038] The shape, structure, size, arrangement, material, etc. of each component of the culture device 1 are not limited to the above examples, and any configuration can be used as long as it is possible to maintain anaerobic conditions in the culture space 28.

[0039] [Cell culture] The first cell culture 100 is preferably a cell layer or tissue containing aerobic cells. Examples of cells constituting the first cell culture 100 include, but are not limited to, various epithelial cells such as intestinal epithelial cells, as well as Caco-2 cells, HT29 cells, and T84 cells. The cells constituting the first cell culture 100 may be cells derived from a living organism, or may be cells induced to differentiate from pluripotent stem cells such as ES cells and iPS cells, or somatic stem cells. For example, the cells constituting the first cell culture 100 may be various intestinal epithelial cells derived from stem cells, including induced pluripotent stem cells (iPS cells). Furthermore, the cells may be genetically modified or unmodified. The first cell culture 100 may contain any combination of the above cells.

[0040] The second cell culture 200 is preferably made of anaerobic bacteria. For example, the cells constituting the second cell culture 200 include, but are not limited to, anaerobic enterobacteria such as Bifidobacterium, Bacteroides, Eubacterium, Clostridium, Lactobacillus, Escherichia coli, and Enterococcus.

[0041] The first culture medium 300 and the second culture medium 400 can be selected appropriately depending on the cells to be cultured, etc. The second culture medium 400 may be the same as the first culture medium 300 or may be different.

[0042] [Culture production method] A method for producing a culture by culturing cells using the above-described culture device 1 will now be described. The culture medium manufacturing method of this embodiment is a culture medium manufacturing method for manufacturing a culture medium by co-culturing a first cell culture medium and a second cell culture medium using the above-mentioned culture device, and includes the steps of culturing the first cell culture medium on the porous body, closing the culture space from the outside of the main body and making the atmosphere in the culture space anaerobic, introducing the second cell culture medium into the culture space, and co-culturing the second cell culture medium with the first cell culture medium in the culture space.

[0043] First, with the cap 40 removed from the culture tank 20, cells of the first cell culture body 100 are seeded onto the surface of the porous body 32 facing the culture space 28, and the cells are cultured until the seeded cells form a cell layer or tissue that covers the surface of the porous body 32 facing the culture space 28.

[0044] Next, the cap 40 is attached to the top of the culture tank 20 to assemble the main body 10. The culture space 28 is closed by the cap 40. With the gas supply pipe 80 connected to the gas inlet 44A and the gas outlet 44B connected to a gas exhaust pipe and the medium exchange port 52 closed with the second closing member 56, the atmosphere in the culture space 28 is replaced with an anaerobic gas such as nitrogen. Because the perforated body 32 is covered with the first cell culture body 100, the culture space 28 becomes a closed space under anaerobic conditions.

[0045] While continuing to supply gas, the second closing member 56 is removed from the culture medium exchange port 52, and the culture medium supply pipe 94 of the culture medium supply device 90 is inserted into the culture medium exchange port 52. The first culture medium 300 and the cells of the second cell culture 200 are introduced from the syringe 92 through the culture medium supply pipe 94 into the culture space 28, thereby seeding the cells of the second cell culture 200 in the culture space 28 while maintaining the anaerobic conditions. After the seeding and culture medium supply are completed, the culture medium supply pipe 94 is pulled out, and the culture medium exchange port 52 is closed with the second closing member 56. Thereafter, the atmosphere replacement is completed, and the gas inlet 44A and gas outlet 44B are closed with the gas inlet closing member 48A and gas outlet closing member 48B, respectively.

[0046] The main body 10 is inserted into the well 60 with the second culture medium 400 stored in the storage space 68, and the first cell culture 100 and the second cell culture 200 are co-cultured. While the culture space 28 is maintained under anaerobic conditions, oxygen taken in from the gas-permeable substrate 66 can be supplied to the first cell culture 100 through the porous body 32. In this way, a culture can be produced by co-culturing the first cell culture 100 and the second cell culture 200.

[0047] As shown in FIG. 4, the tip of the gas flow path 46 is located above the first culture medium 300. Therefore, during atmosphere replacement, gas replacement occurs only in the gas phase portion of the culture space 28, and bubbling does not occur in the culture medium. This reduces physical irritation to the cultured cells. Furthermore, during culture medium replacement, inserting the culture medium supply pipe 94 while continuing gas replacement in the gas phase portion prevents oxygen from flowing into the culture space 28 through the culture medium replacement port 52. As described above, the tip 94A of the culture medium supply pipe 94 is guided to the retraction portion 34 by the guide portion 36, so that the tip 94A reaches the same position each time and does not point toward the perforated body 32. This reduces physical irritation to the cultured cells during culture medium replacement.

[0048] [Drug evaluation method] A method for evaluating a drug using the culture produced by the above culture production method will now be described. The drug evaluation method of this embodiment is a drug evaluation method using a culture produced by the above-mentioned culture production method, and includes the steps of contacting a drug with the culture, and measuring the response of the culture to stimulation caused by contact with the drug, or the permeability or permeability of the drug to the culture.

[0049] As used herein, the term "drug" includes not only drugs such as pharmaceuticals, but also cosmetics and quasi-drugs. For example, a drug can be brought into contact with the first cell culture 100 by applying the drug to the culture or by introducing the drug into the culture space 28 using the culture medium supply device 90. Furthermore, by evaluating, by any known method, changes in the first cell culture 100 after the drug is added, the state of diffusion and permeation of the drug into the first cell culture 100, and the state of diffusion and permeation of the drug through the first cell culture 100 and the porous body 32 from the culture space 28 (anaerobic side) to the storage space 68 (aerobic side), the response of the culture to stimulation caused by contact with the drug or the permeability or permeability of the drug into the culture can be measured.

[0050] According to this drug evaluation method, compared to conventional methods, various drugs can be evaluated in an environment closer to the actual intestinal tract of a living organism, where an anaerobic environment and oxygen supply to cells coexist. [Example]

[0051] [Experimental Example 1: Confirmation of oxygen permeability of porous materials] Two of the above culture devices were produced using a three-dimensional printer. Hereinafter, they will be referred to as Device 1 and Device 2. For Devices 1 and 2, oxygen supply from the wells through the perforated body of the culture device to the culture tank was confirmed as follows.

[0052] Without forming a cell layer on the porous membrane (i.e., without cell culture), the gas phase of the culture space was replaced with anaerobic gas (a 9:1 mixture of nitrogen and carbon dioxide) at a flow rate of 500 mL / min for 20 seconds, creating an anaerobic culture space. After the atmosphere replacement was completed, the culture device was sealed and left stationary. An oxygen sensor was used to monitor the time-dependent change in oxygen concentration in the culture space during and after this series of operations. D-MEM high glucose (10% FBS, 1% NEAA, 1% PSA) was used as the culture medium (as in the following experimental examples). In this experimental example, since no cell layer was formed on the porous membrane and no oxygen consumption occurred in the cell layer, it was expected that oxygen would flow into the culture space through the porous membrane, resulting in an increase in oxygen concentration over time.

[0053] Figure 6 is a graph showing the change in oxygen concentration over time for Devices 1 and 2 in Experimental Example 1. Initially, a decrease in oxygen concentration was observed due to the atmosphere replacement operation, but it was confirmed that the oxygen concentration then increased over time. In both Devices 1 and 2, the oxygen concentration exceeded 15% in about 4 hours. From these results, it was confirmed that when there is a difference in oxygen concentration between the top and bottom of the porous body, oxygen flows through the porous body from the side with the higher oxygen concentration (the well side) to the side with the lower oxygen concentration (the culture space side).

[0054] [Experimental Example 2: Confirmation of oxygen consumption capacity of cell layer without atmosphere replacement] In this experimental example, for Devices 1 and 2 manufactured in Experimental Example 1, a cell layer was formed on the porous media, and the time course of the oxygen concentration in the sealed culture space was examined without atmosphere replacement. The ratio of lactate production to glucose consumption and the TEER value were measured before and after sealing to evaluate the cell culture state. The lactate production and glucose consumption were calculated by comparing the lactate and glucose concentrations of sampled culture medium with those of fresh culture medium. The TEER value was calculated by measuring the electrical resistance by inserting electrodes on both sides of the porous media on which a cell layer had been formed, and subtracting the electrical resistance measured in the same way for the porous media on which no cell layer had been formed. Here, the cell layer was formed by seeding Caco-2 cells on the porous media at a density of 2 × 10 5 cells / cm 2 After seeding and culturing for several days, the TEER measurement value was 100 Ω cm 2 In this experiment, no atmospheric replacement was performed, so oxygen was present in the culture space at atmospheric composition. However, because a cell layer was formed on the porous body, oxygen consumption occurred in the cell layer, and it was expected that the oxygen concentration in the culture space would decrease over time.

[0055] 7 is a graph showing the change in oxygen concentration over time for Devices 1 and 2 in Experimental Example 2. It was confirmed that the oxygen concentration decreased over time, and the oxygen concentration for Devices 1 and 2 decreased to an average of about 3% in about 3 hours.

[0056] 8 is a graph showing the lactate production / glucose consumption ratio before and after sealing the culture device in Experimental Example 2. The values ​​shown are the average values ​​for Devices 1 and 2, and the error bars indicate the standard deviation. Comparing the values ​​before sealing and 24 hours after sealing, there was a slight increase in the value, but the effect of sealing was almost unnoticeable.

[0057] Figure 9 is a graph showing the TEER values ​​before and after sealing of the culture device in Experimental Example 2. The values ​​shown are the average values ​​for Devices 1 and 2, and the error bars indicate the standard deviation. No significant difference was observed between the values ​​before sealing and the values ​​24 hours after sealing. As shown in Figure 7, the culture space became anaerobic after sealing, but no effect on the barrier function of the cell layer was observed.

[0058] [Experimental Example 3: Confirmation of oxygen consumption capacity of cell layer when atmosphere replacement is performed] In this experimental example, for devices 1 and 2 manufactured in experimental example 1, a cell layer was formed on the porous body in the same manner as in experimental example 2, and the atmosphere was replaced in the same manner as in experimental example 1 to examine the change in oxygen concentration over time in the sealed culture space. In this experimental example, oxygen consumption by the cell layer occurred after atmosphere replacement, and it was predicted that the oxygen concentration in the culture space would decrease more quickly than in Example 2.

[0059] Figure 10 is a graph showing the change in oxygen concentration over time for Devices 1 and 2 in Experimental Example 3. The oxygen concentration decreased much more rapidly than in Experimental Example 2 (Figure 7), and the oxygen concentration decreased to about 1% on average for Devices 1 and 2 in a shorter time than in Experimental Example 2. Furthermore, since no increase in oxygen concentration was observed as in Experimental Example 1 (Figure 6), it is believed that the oxygen supplied from the outside through the porous body was consumed by the cell layer.

[0060] From the above, it was confirmed that the culture device having the above configuration can sufficiently reduce the oxygen concentration in the culture space. [Explanation of symbols]

[0061] 1...culture device, 10...main body, 20...culture tank, 28...culture space, 30...opening, 32...porous body, 34...retraction section, 36...guiding section, 40...cap, 42...atmosphere replacement section, 50...culture medium exchange section, 60...well, 66...gas-permeable substrate, 68...storage space, 70...oxygen sensor, 80...gas supply pipe, 90...culture medium supply device, 100...first cell culture, 200...second cell culture, 300...first culture medium, 400...second culture medium.

Claims

1. A culture device for co-culturing a first cell culture medium and a second cell culture medium, The present invention provides a method for culturing a microorganism comprising: a main body having a culture space formed therein that allows culture under anaerobic conditions; the main body having an opening that opens from the culture space to the outside of the main body; and a porous body provided at the opening; the first cell culture medium is held in the porous body so as to cover the culture space side of the porous body, and the second cell culture medium is cultured in the culture space; The main body is configured so that the culture space can be closed from the outside of the main body except for the opening, a well having a storage space for storing a culture solution under aerobic conditions, the main body being inserted into the well so that the porous body is exposed to the storage space; The well has a gas-permeable substrate that is impermeable to liquids but permeable to gases, and by having at least the gas-permeable substrate, oxygen is supplied to the storage space from outside the storage space.

2. 2. The culture device according to claim 1, wherein the porous body has holes that supply oxygen to the first cell culture from outside the body, and the first cell culture consumes oxygen from outside the body, thereby maintaining the closed culture space in an anaerobic state.

3. 3. The culture device according to claim 1, wherein the main body comprises a culture tank having the culture space, and a cap that is detachably attached to the culture tank and closes an upper side of the culture space, and the opening is formed on a lower side of the culture space.

4. The culture device according to any one of claims 1 to 3, further comprising an atmosphere replacement unit that replaces the atmosphere in the culture space, wherein the atmosphere replacement unit has a gas flow path formed inside the main body and communicating with the culture space.

5. The culture device according to any one of claims 1 to 4, further comprising a culture medium exchange unit that exchanges the culture medium in the culture space, the culture medium exchange unit having a culture medium exchange passage formed inside the main body, communicating with the culture space, and into which a culture medium supply pipe can be inserted.

6. The culture device according to claim 5 , wherein the main body has a retraction portion that accommodates the tip of the culture medium supply pipe so that the tip does not point toward the perforated body within the culture space.

7. The culture device according to any one of claims 1 to 6, further comprising an oxygen sensor that measures the oxygen concentration in the culture space.

8. The culture device according to any one of claims 1 to 7, wherein the first cell culture is a cell layer or tissue containing aerobic cells.

9. The culture device according to any one of claims 1 to 8, wherein the first cell culture is a cell layer of intestinal epithelial cells, and the second cell culture is intestinal bacteria.

10. A culture production method for producing a culture by co-culturing a first cell culture and a second cell culture using the culture device according to any one of claims 1 to 9, Cultivating the first cell culture on the porous body; closing the culture space from the outside of the main body and making the atmosphere in the culture space anaerobic; introducing the second cell culture construct into the culture space; co-culturing the second cell culture with the first cell culture in the culture space; A method for producing a culture medium, comprising:

11. A drug evaluation method using a culture produced by the culture production method according to claim 10, comprising: contacting the culture with an agent; measuring the response of the culture to stimulation by contact with the drug or the permeability or permeability of the drug through the culture; A drug evaluation method comprising:

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