Cell culture method

Abstract

A co-culture device (100) includes a container body (110) and a cell culture insert (120) in both of which a culture medium can be stored. The cell culture insert (120) includes a membrane (122) that separates the container body (110) and the cell culture insert (120) from each other and has oxygen permeability. The cell culture method comprises: (a) a step for culturing cells so as to cover the membrane (122) in the cell culture insert (120); (b) a step for introducing a first culture medium (161) having a first color into the container body (110); (c) a step for introducing a second culture medium (162) containing a bacterium and having a second color different from the first color into the cell culture insert (120); and (d) a step for co-culturing the cells and the bacterium in a state where the membrane (122) is disposed so as to be in contact with the first culture medium.

Description

Cell culture method

[0001] The present disclosure relates to cell culture methods.

[0002] Development of a cell culture system that simulates the intestinal environment of an organism is underway. International Publication No. 2022 / 009672 (Patent Document 1) discloses a system that uses a co-culture vessel in which an anaerobic medium and an aerobic medium are separated by a porous membrane, and co-cultures intestinal epithelial cells and bacteria contained in the medium in the anaerobic medium.

[0003] International Publication No. 2022 / 009672

[0004] In the cell culture system described above, after the culture process is completed, each medium is removed into a separate container such as an Eppendorf tube, and analysis of metabolites, etc. is performed in subsequent processing steps such as analysis. If the removed medium is mixed up, the analysis results will be incorrect, making accurate analysis and evaluation impossible.

[0005] The present disclosure has been made to solve such problems, and its purpose is to prevent the mixing of media after culture in a cell culture method in which cells and bacteria are co-cultured using two different media.

[0006] The present disclosure relates to a method for co-culturing a first biological component and a second biological component using a co-cultivation device. The co-cultivation device includes a first chamber and a second chamber capable of storing a culture medium therein. The second chamber separates the first and second chambers and includes an oxygen-permeable membrane. The method includes the steps of: (a) disposing the first biological component inside the second chamber so that the membrane is covered; (b) introducing a first culture medium having a first color into the first chamber; (c) introducing a second culture medium having a second color different from the first color and including a second biological component into the second chamber; and (d) culturing the first biological component and the second biological component with the membrane in contact with the first culture medium.

[0007] In the cell culture method according to the present disclosure, the two media used for co-culture are different in color from each other. This allows easy identification of the media when each medium is removed after the culture is completed. Therefore, in a cell culture method in which different biological elements are co-cultured using two different media, mixing up the media after culture can be prevented.

[0008] FIG. 6 is a configuration diagram of a cell culture system used in the cell culture method of embodiment 1. FIG. 7 is a flowchart showing the culture steps in the cell culture method. FIG. 8 is a diagram for explaining the state in which the culture medium is collected after completion of culture. FIG. 9 is a diagram showing an example of the state of the culture medium when cells are damaged during culture. FIG. 10 is a flowchart for explaining an example of a sample processing step after completion of culture. FIG. 11 is a configuration diagram of a cell culture system used in the cell culture method of embodiment 2. FIG. 12 is an exploded perspective view of a co-culture device used in the cell culture system of FIG.

[0009] Hereinafter, embodiments of the present disclosure will be described in detail with reference to the drawings. In the drawings, the same or corresponding parts are designated by the same reference numerals, and description thereof will not be repeated.

[0010] In this specification, biological cells and microorganisms are collectively referred to as biological elements. Examples of biological cells include organ-forming cells (specifically, organ-forming cells such as organoids). Examples of microorganisms include fungi or bacteria.

[0011] [Embodiment 1] (Configuration of cell culture system) Fig. 1 is a configuration diagram of a cell culture system 10 used in a cell culture method of embodiment 1. The cell culture system 10 includes a cell culture device 100, a pump 60, culture medium containers 70 and 75, and a measuring device 180. The cell culture system 10 is placed, for example, in an anaerobic chamber, and the surroundings are maintained in an anaerobic environment.

[0012] The cell culture device 100 includes a container body 110, a cell culture insert 120, cover members 130 and 140, and an electrode 150. The container body 110 is a generally cylindrical container having an upper wall 111, a bottom wall 112, and a side wall 113. The side wall 113 connects the upper wall 111 and the bottom wall 112. The container body 110 is formed, for example, from a resin material. A culture medium 161 (first culture medium) is stored inside the container body 110. Note that the interior of the container body 110 is maintained in an atmospheric environment (aerobic environment) by attaching a cell culture insert 120, in which cells (organ-forming cells) 170 are cultured in a sheet form, to the container body 110 in an atmospheric environment. Note that the "container body 110" in the embodiment corresponds to the "first chamber" in this disclosure, and the "cell culture insert 120" corresponds to the "second chamber" in this disclosure.

[0013] An opening 114 is formed in the top wall 111 of the container body 110, penetrating the top wall 111 in the thickness direction. As will be described later, a cell culture insert 120 is placed in this opening 114. Note that while FIG. 1 shows an example of a configuration in which the top wall 111 is formed integrally with the side wall 113, the top wall 111 may be formed as a separate member from the side wall 113.

[0014] An electrode 151 is embedded in the bottom wall 112 of the container body 110. The electrode 151 is exposed on the inner and outer surfaces of the bottom wall 112. Inside the container body 110, the electrode 151 is in contact with a culture medium 161, and the electrode 151 and the culture medium 161 can be electrically connected to each other.

[0015] The cell culture insert 120 is a generally cylindrical container having a cylindrical portion 121 forming the side wall of the container, a membrane 122 forming the bottom wall of the container, and a flange portion 123. The lower end of the cylindrical portion 121 is closed by the membrane 122. The membrane 122 is formed of an oxygen-permeable material such as a track-etched membrane made of polycarbonate. The membrane 122 may also be a porous membrane formed of other materials such as PET (polyethylene terephthalate) or a collagen vitrigel membrane.

[0016] The upper end of the cylindrical portion 121 is open and is sealed by a removable lid member 130. The upper end of the cylindrical portion 121 is provided with a flange portion 123 that protrudes in the outer circumferential direction.

[0017] The cell culture insert 120 is inserted into the opening 114 in the upper wall 111 of the container body 110. At this time, the flange portion 123 of the cell culture insert 120 is supported by the upper wall 111. The cell culture insert 120 is then fixed to the container body 110 by a removable lid member 140. When the cell culture insert 120 is fixed, the membrane 122 on the underside of the cell culture insert 120 is immersed in the culture medium 161.

[0018] A culture medium 162 is stored inside the cell culture insert 120. The dissolved oxygen concentration of the culture medium 162 is lower than the dissolved oxygen concentration of the culture medium 161 in the container body 110. In other words, the culture medium 161 is an aerobic culture medium, and the culture medium 162 is an anaerobic culture medium. The culture medium 162 contains a biological element. In the example of the first embodiment, the biological element contained in the culture medium 162 is an anaerobic bacterium. The anaerobic bacterium may be a facultative anaerobe such as lactic acid bacteria, or an obligate anaerobe such as bifidobacteria. The anaerobic bacterium corresponds to the "second biological element" in the present disclosure.

[0019] Pipes 50 and 55 pass through the cover member 130. A pump 60 disposed in the pipe 50 introduces a culture medium 162 (second culture medium) stored in a culture medium container 70 outside the device into the cell culture insert 120. The pipe 55 is a pipe for discharging the culture medium 162 from the cell culture insert 120. The pipe 55 continues to a culture medium container 75 for storing culture medium for disposal. The culture medium 162 that overflows from the cell culture insert 120 is discharged through the pipe 55 into the culture medium container 75.

[0020] Biological elements are cultured on the inner surface of the membrane 122. In the example of the first embodiment, the biological elements are cells (organ-constituting cells) 170. The cells 170 are, for example, intestinal epithelial cells that form tight junctions on the membrane 122. A specific example of the cells 170 is Caco-2 cells. As described above, the membrane 122 is oxygen permeable. Oxygen in the culture medium 161 is supplied to the cells 170 through the membrane 122. By co-culturing cells and bacteria in the anaerobic culture medium 162 in this manner, the intestinal environment in a living organism can be simulated. Note that the "cells 170" correspond to the "first biological element" in the present disclosure.

[0021] An electrode 150 is further disposed on the cover member 130. Although not shown in FIG. 1 , one end of the electrode 150 is in contact with a culture medium 162 stored in the cell culture insert 120 and is electrically connected to the culture medium 162.

[0022] The electrode 150 disposed on the lid member 130 of the cell culture insert 120 and the electrode 151 embedded in the bottom wall 112 of the container body 110 are connected to a measuring device 180 provided outside the device. The measuring device 180 measures the electrical resistance between the electrodes 150 and 151 by applying a voltage between the electrodes 150 and 151. The electrical resistance is measured by the measuring device 180, for example, by a four-terminal method.

[0023] The resistance value between the electrodes changes depending on whether or not tight junctions are formed in the cells 170 cultured on the membrane 122. Therefore, by monitoring the electrical resistance value during culture, it is possible to determine whether or not the tight junctions of the cells 170 are normally formed.

[0024] 1, an oxygen sensor may be provided to detect the dissolved oxygen concentration in the culture medium 161 and the culture medium 162. By detecting the dissolved oxygen concentration in each culture medium with the oxygen sensor, the growth status of the bacteria during co-culture can be monitored.

[0025] When the co-cultivation process of cells and bacteria using the cell culture system described above is completed, each medium is collected into another container such as an Eppendorf tube and stored in a freezer for a predetermined period of time or for subsequent analysis. If the media collected in the containers have similar appearances (colors), there is a risk that users will confuse the aerobic medium with the anaerobic medium when handling the containers.

[0026] Furthermore, during cell culture, the cells and / or membrane may be partially damaged, causing one medium to penetrate into the other, resulting in the mixing of the aerobic and anaerobic media. In other words, in such a case, the culture process itself may fail. However, if the colors of the culture media during culture are similar, the mixing of the media may not be detected, and the culture media may end up being analyzed as a failed culture.

[0027] If the analytical process is carried out while the medium is being mixed up after collection and / or the medium is being mixed during cultivation, erroneous results may be obtained, which may lead to incorrect judgment of the experimental results and evaluation.

[0028] Therefore, in the first embodiment, the aerobic side culture medium 161 and the anaerobic side culture medium 162 are made different colors that can be visually distinguished by the user. This makes it possible to easily distinguish whether the culture medium is an aerobic culture medium or an anaerobic culture medium after the culture medium is collected in a container such as an Eppendorf tube. Furthermore, by making the colors of the culture media different even during cultivation, it is possible to easily detect mixing of the culture media during cultivation.

[0029] It is sufficient that at least one of the two media is colored, and the other may be colorless. The medium can be colored using, for example, a coloring agent that has little effect on the culture treatment.

[0030] The color of the medium may be a fixed color that does not change before and after culture, or may change depending on the condition of the medium. For example, when an indicator whose color changes depending on pH, such as methyl orange, phenolphthalein (PP), or bromothymol blue (BTB), is added to the medium, the color of the medium may change depending on the pH of the medium as the culture progresses.

[0031] To prevent the mix-up of the media after collection, it is not essential that the two media have different colors from the beginning of the culture treatment, but it is sufficient that the colors of the two media differ at the completion of the culture due to the elapsed time or changes in the state of the media. On the other hand, to detect the mixed state of the media during culture, it is preferable that the colors of the media are different from the early stage of culture.

[0032] Fig. 2 is a flowchart showing an example of a culture process performed using the cell culture system 10 of Fig. 1. Each step in the flowchart of Fig. 2 may be performed manually by a user, or some or all of the steps may be performed automatically using a handling device (not shown) or the like.

[0033] 2, in step (hereinafter, step will be abbreviated as S) 100, cells are cultured over the entire surface of the membrane 122 in the cell culture insert 120. Then, in S110, the cell culture insert 120 is placed in the opening 114 of the container body 110.

[0034] In S120, a first color culture medium 161 is introduced into the container body 110. At this time, the culture medium 161 is introduced to a height such that the membrane 122 on the lower surface of the cell culture insert 120 is immersed in the culture medium 161. Then, in S130, a second color culture medium 162 containing bacteria is introduced into the cell culture insert 120. As described in FIG. 1 , fresh culture medium 162 is supplied from the culture medium container 70 to the cell culture insert 120 by the pump 60 even during cultivation.

[0035] The culture media 161, 162 used in S120, S130 may be commercially available and already colored, or the method may include a step of coloring a colorless culture medium using a coloring agent and / or an indicator, etc., prior to S120, S130.

[0036] When the culture medium is introduced into the container body 110 and the cell culture insert 120, in S140, the cells and bacteria are cultured for a predetermined time while the electrical resistance and dissolved oxygen concentration are monitored by the measuring device 180.

[0037] Fig. 3 is a diagram illustrating the state in which media 161 and 162 are collected after the end of culture. Fig. 3 shows the state in which media 161 and 162 are different colors at the stage when culture is completed. In the example of Fig. 3, medium 161 is shown in a light color, and medium 162 is shown in a dark color. Even when media 161 and 162 are collected in Eppendorf tubes 210 and 220, respectively, it is possible to distinguish between medium 161 and medium 162 based on the difference in their colors.

[0038] FIG. 4 shows an example of the state of the medium when the cells 170 and membrane 122 are damaged during culture. For example, if the pressure of the medium 162 is higher than the pressure of the medium 161, and the cells 170 and membrane 122 are damaged during culture, a portion of the medium 162 in the cell culture insert 120 will leak into the medium 161 from the damaged area, as shown in region RG in FIG. 4. In this case, if the colors of the medium 161 and the medium 162 are different, the medium 162 that has leaked into the medium 161 can be easily detected. Note that if the pressure of the medium 161 is higher than the pressure of the medium 162, and the cells 170 and membrane 122 are damaged during culture, the medium 161 will leak into the medium 162.

[0039] 5 is a flowchart illustrating an example of a sample processing step after the end of the culture. In the flowchart of FIG. 5, the processes of S200 to S240 are performed in an anaerobic environment, and the processes of S250 to S280 are performed in an aerobic environment.

[0040] 5, after the culture is completed, in S200, a specific amount of anaerobic medium 162 is collected into a container in an anaerobic environment. In addition, in S210, a specific amount of aerobic medium 161 is collected into another container. If necessary, each of the collected media is allowed to stand for a predetermined time in the anaerobic environment.

[0041] Thereafter, the medium 161 is inoculated onto an agar medium without dilution to confirm that no bacteria are present in the aerobic environment (S220). On the other hand, the medium 162 is a medium co-cultured with bacteria, and since it is necessary to count the bacteria, it is diluted and then inoculated onto an agar medium (S230). The dilution ratio differs depending on conditions such as the type of bacteria and / or the turbidity of the medium.

[0042] Once the inoculation of the agar medium is complete, the container containing the remaining medium is removed to an aerobic environment, and each medium is centrifuged using a centrifuge in S240. In S250, the supernatant of each medium after centrifugation is transferred to a separate container such as an Eppendorf tube and stored in a specified environment.

[0043] Thereafter, in S260, the remaining liquid of each medium is collected and the pH of each medium is measured. In addition, in S270, the pellet of the precipitated medium 162 is suspended in an appropriate amount of distilled water and the turbidity is measured.

[0044] If the two culture media are mixed up or mixed during the culture during the sample processing step shown in Figure 5, not only will the work up to that point be wasted, but the desired analysis results will not be obtained. As in the first embodiment, by making the two culture media different colors that can be visually distinguished by the user, the culture media can be easily distinguished after collection, thereby preventing the two culture media from being mixed up. Furthermore, even if the culture media leaks due to cell damage or the like during culture, the leakage state can be easily detected, allowing for early detection of experimental failures.

[0045] [Embodiment 2] Another example of the configuration of the cell culture device will be described in Embodiment 2. Fig. 6 is a configuration diagram of a cell culture system 10A used in the cell culture method of Embodiment 2.

[0046] 6, cell culture system 10A includes cell culture device 100A, pumps 60A and 60B, culture medium containers 70A, 70B, 75A, and 75B, measuring device 180, and sealed container 400. Cell culture system 10A is placed, for example, in an anaerobic chamber, and the surroundings are maintained in an anaerobic environment.

[0047] The cell culture device 100A includes a co-culture device 300. The co-culture device 300 has two adjacent flow paths 320 and 330 formed through a membrane 310. The flow path 320 is a path through which an aerobic medium (medium 161) flows, and the flow path 330 is a path through which an anaerobic medium (medium 162) flows.

[0048] Cells 170 are cultured on the surface of the membrane 310 on the side of the flow channel 320. Oxygen is supplied to the cells 170 from the culture medium 161 flowing through the flow channel 320 via the membrane 310.

[0049] The sealed container 400 is a container whose internal space can be sealed. The sealed container 400 includes a main body 410 and a lid member 420. The main body 410 has a cylindrical shape with one end closed by a bottom wall and the other end open. A removable lid member 420 is attached to the open end of the main body 410. The internal space of the sealed container 400 is maintained in an atmospheric environment, i.e., an aerobic environment.

[0050] A culture medium container 70A storing a culture medium 161, a pipe 50A, and a pump 60A attached to the pipe 50A are arranged inside the sealed container 400. The pipe 50A connects the culture medium container 70A to a flow path 320 of the co-culture device 300. By driving the pump 60A, the culture medium 161 in the culture medium container 70A is supplied to the flow path 320 through the pipe 50A. The culture medium 161 that has passed through the flow path 320 is discharged through the pipe 55A into a waste culture medium container 75A.

[0051] Culture medium 162 is stored in culture medium container 70B placed in an anaerobic environment. Culture medium container 70B is connected to flow path 330 of co-culture device 300 by piping 50B. By driving pump 60B provided on piping 50B, culture medium 162 in culture medium container 70B is supplied to flow path 330 through piping 50B. Culture medium 162 that has passed through flow path 330 is discharged through piping 55B to waste culture medium container 75B.

[0052] In the co-culture device 300, an electrode 350 is arranged so as to be exposed within the flow channel 320, and an electrode 355 is arranged so as to be exposed within the flow channel 330. The electrode 350 can be electrically connected to the culture medium 161 flowing within the flow channel 320, and the electrode 355 can be electrically connected to the culture medium 162 flowing within the flow channel 330. The electrodes 350 and 355 are connected to a measuring device 180. By applying a voltage to the electrodes 350 and 355 from the measuring device 180, the electrical resistance value between the electrodes 350 and 355 can be monitored.

[0053] As in the first embodiment, an oxygen sensor may be provided to detect the dissolved oxygen concentration in the culture media 161 and 162 flowing through each flow path.

[0054] Next, a detailed configuration of the co-culture device 300 in Fig. 6 will be described. Fig. 7 is an exploded perspective view of the co-culture device 300 used in the cell culture system 10A in Fig. 6.

[0055] 7 , the co-culture device 300 includes flat glass plates 301 and 302, and flat resin sheets 305 and 306 disposed between the glass plates 301 and 302. The glass plates 301 and 302 and the resin sheets 305 and 306 are stacked in the order of the glass plate 301, the resin sheet 305, the resin sheet 306, and the glass plate 302.

[0056] A specific example of the resin sheets 305 and 306 is silicone rubber. The glass plate and the resin sheet, and the resin sheets themselves, are bonded together by applying pressure to the bonding surfaces in a state where the bonding surfaces have been activated by oxygen plasma, for example.

[0057] A through-hole 361 is formed in the glass plate 301 so as to penetrate the glass plate 301 in the thickness direction. The pipe 50A is connected to the through-hole 361. That is, the through-hole 361 serves as an inlet of the flow path 320 through which the culture medium 161 flows.

[0058] A groove 340 is formed on the surface of the resin sheet 305 facing the glass plate 301. The flow path 320 in Fig. 6 is formed by this groove 340 and the surface of the glass plate 301. One end of the groove 340 communicates with a through-hole 361 in the glass plate 301. The other end of the groove 340 is formed with a through-hole 362 that penetrates the resin sheet 305 in the thickness direction.

[0059] Through holes 363 and 364 are formed in the resin sheet 306 and the glass plate 302, respectively, at positions corresponding to the through hole 362 in the resin sheet 305. The pipe 55A is connected to the through hole 364 in the glass plate 302. That is, the through hole 364 in the glass plate 302 serves as an outlet of the flow path 320 through which the culture medium 161 flows.

[0060] A groove 380 is formed on the surface of the resin sheet 306 that faces the glass plate 302. The groove 380 and the surface of the glass plate 302 form the flow path 330 shown in FIG.

[0061] In the glass plate 302, a through-hole 371 is formed at a position corresponding to one end of the groove portion 380, penetrating in the thickness direction, and a through-hole 372 is formed at a position corresponding to the other end of the groove portion 380, penetrating in the thickness direction. Pipe 50B is connected to through-hole 371 in the glass plate 302. Pipe 55B is connected to through-hole 372 in the glass plate 302. That is, through-holes 371 and 372 in the glass plate 302 serve as the inlet and outlet, respectively, of a flow path 330 through which the culture medium 162 flows.

[0062] When viewed from above in the normal direction of the resin sheet 305, a portion of the groove portion 340 formed in the resin sheet 305 overlaps in parallel with the groove portion 380 formed in the resin sheet 306. A slit 341 penetrating through the resin sheet 305 in the thickness direction is formed in the overlapping portion. Furthermore, a slit 381 is formed in the groove portion 380 of the resin sheet 306 at a position facing the slit 341. The membrane 310 is disposed between the slit 341 and the slit 381.

[0063] This configuration realizes a configuration in which two adjacent flow paths 320, 330 are connected via the membrane 310. That is, in the cell culture system 10A used in the cell culture method of the second embodiment, the flow path 320 in Fig. 6 corresponds to the container body 110 of the cell culture system 10 of the first embodiment, and the flow path 330 corresponds to the cell culture insert 120 of the cell culture system 10. That is, the "flow path 320" and the "flow path 330" in the second embodiment correspond to the "first chamber" and the "second chamber," respectively, in the present disclosure.

[0064] Furthermore, even when using the cell culture system 10A, by making the color of the aerobic culture medium different from the color of the anaerobic culture medium, it is possible to prevent the two culture media from being confused during processing after the end of the culture, and it is also possible to easily detect the mixing of the culture media during the culture.

[0065] [Notes] It will be understood by those skilled in the art that the exemplary embodiments described above are specific examples of the following aspects.

[0066] (Item 1) One aspect of the cell culture method relates to a method for co-culturing a first biological component and a second biological component using a co-culture device. The co-culture device includes a first chamber and a second chamber capable of storing a culture medium therein. The second chamber separates the first and second chambers and includes an oxygen-permeable membrane. The cell culture method includes the steps of (a) culturing the first biological component inside the second chamber so that the membrane is covered, (b) introducing a first culture medium having a first color into the first chamber, (c) introducing a second culture medium having a second color different from the first color into the second chamber, and (d) co-culturing the first biological component and the second biological component with the membrane in contact with the first culture medium.

[0067] According to the cell culture method of the first aspect, by performing co-culture in the co-culture device using two media (aerobic and anaerobic media) that have different colors, it is possible to prevent the two media from being mixed up when each media is collected into a separate container after the co-culture is completed. Furthermore, if the media become mixed up during the culture due to cell damage or the like, it becomes possible to easily detect the abnormal state.

[0068] (Item 2) In the cell culture method described in item 1, the second color is a color that can be visually distinguished from the first color by a user.

[0069] According to the cell culture method of the second aspect, the colors of the culture media are set to be visually distinguishable by the user, so that the two culture media can be easily distinguished from one another.

[0070] (Item 3) The cell culture method according to item 1 or 2 further includes a step of coloring the first culture medium a first color and a step of coloring the second culture medium a second color.

[0071] According to the method of the third aspect, when the original culture medium is colorless, the two culture media can be easily distinguished by coloring the culture medium with a coloring agent or the like.

[0072] (Item 4) In the cell culture method according to item 1 or 2, at least one of the first culture medium and the second culture medium contains an indicator whose color changes depending on the state of the medium.

[0073] According to the cell culture method of the fourth aspect, when the state of the culture medium (e.g., pH) changes during the culture process, an indicator that changes color depending on the state of the culture medium can be used to confirm the change in the state of the culture medium and to easily distinguish the culture medium after culture by the difference in color.

[0074] (Item 5) In the cell culture method according to any one of items 1 to 4, the dissolved oxygen concentration of the first culture medium is higher than the dissolved oxygen concentration of the second culture medium.

[0075] According to the cell culture method of the fifth aspect, the first medium can be used as an aerobic medium, and the second medium can be used as an anaerobic medium.

[0076] (Item 6) In the cell culture method described in any one of Items 1 to 5, the second chamber is a cell culture insert that further includes a cylindrical portion and a membrane arranged to close the lower end of the cylindrical portion.

[0077] According to the cell culture method of the fifth aspect, a cell culture insert that is widely used in general can be used as the second chamber.

[0078] (Item 7) In the cell culture method according to any one of Items 1 to 6, the first biological component is an organ-forming cell, and the second biological component is a microorganism.

[0079] According to the cell culture method of the seventh aspect, the cell culture method of the present disclosure can be applied to co-culture of organ-constituting cells and microorganisms.

[0080] The embodiments disclosed herein should be considered to be illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims, not by the description of the above embodiments, and is intended to include all modifications within the meaning and scope of the claims.

[0081] 10, 10A Cell culture system, 50, 50A, 50B, 55, 55A, 55B Piping, 60, 60A, 60B Pump, 70, 70A, 70B, 75, 75A, 75B Culture medium container, 100, 100A Cell culture device, 110 Container body, 111 Top wall, 112 Bottom wall, 113 Side wall, 114 Opening, 120 Cell culture insert, 121 Cylindrical portion, 122, 310 Membrane, 123 Flange portion, 130, 140, 420 Lid member, 150, 151, 350, 355 Electrode, 161, 162 Culture medium, 170 Cell, 180 Measuring device, 210, 220 Eppendorf tube, 300 Co-culture device, 301, 302 Glass plate, 305, 306 Resin sheet, 320, 330 Flow path, 340, 380 Groove portion, 341, 381 Slit, 361 to 364, 371, 372 Through hole, 400 Sealed container, 410 Main body portion, RG region.

Claims

1. 1. A cell culture method for co-culturing a first biological component and a second biological component using a co-culture device, comprising: The co-culture device includes a first chamber and a second chamber capable of storing a culture medium therein; the second chamber separating the first chamber from the second chamber and including an oxygen-permeable membrane; The cell culture method comprises: culturing the first biological component within the second chamber and overlying the membrane; introducing a first culture medium having a first color into the first chamber; introducing a second medium into the second chamber, the second medium including the second biological component and exhibiting a second color different from the first color; co-culturing the first biological component and the second biological component with the membrane disposed in contact with the first culture medium; A cell culture method, wherein the second color is a color that can be visually distinguished from the first color by a user.

2. coloring the first culture medium with the first color; The cell culture method according to claim 1 , further comprising the step of coloring the second culture medium with the second color.

3. The cell culture method according to claim 1 , wherein at least one of the first culture medium and the second culture medium contains an indicator whose color changes depending on the state of the culture medium.

4. The cell culture method according to claim 1 , wherein the dissolved oxygen concentration of the first culture medium is higher than the dissolved oxygen concentration of the second culture medium.

5. The cell culture method according to claim 1 , wherein the second chamber is a cell culture insert further including a cylindrical portion, the membrane being arranged to close the lower end of the cylindrical portion.

6. the first biological component is an organ-forming cell; The cell culture method of claim 1 , wherein the second biological component is a microorganism.

7. A cell culture method for co-culturing a first biological element and a second biological element using a co-culture device, comprising: The co-culture device includes a first chamber and a second chamber capable of storing a culture medium therein; the second chamber separating the first chamber from the second chamber and including an oxygen-permeable membrane; The cell culture method comprises: culturing the first biological component within the second chamber and overlying the membrane; introducing a first culture medium having a first color into the first chamber; introducing a second medium into the second chamber, the second medium including the second biological component and exhibiting a second color different from the first color; co-culturing the first biological component and the second biological component with the membrane disposed in contact with the first culture medium; coloring the first culture medium with the first color; and coloring the second medium the second color.