Culture device

Abstract

A culture device is provided in which the influence of the flow of culture medium on cells in a membrane is suppressed during the liquid supply. [Solution] The culture device comprises a well plate having one or more well portions with a first through-hole and a flow path for guiding culture medium to or from the well portion; a contact member having a second through-hole with a diameter larger than the diameter of the bottom side of the first through-hole and contacting the back surface of the well plate; a blocking member that blocks the second through-hole; an integrating member that integrates the well plate, the contact member, and the blocking member; a membrane that is placed within the first through-hole of the well portion and divides the first through-hole into a first space and a second space; and a culture vessel that fits into the well portion; when integrated by the integrating member, the culture medium flows between the flow path and the second space via the back surface portion of the well plate that is exposed from the second through-hole.

Description

[Technical Field]

[0001] The present invention relates to a culture device. [Background technology]

[0002] In recent years, advances in regenerative medicine and life science research have led to a growing demand for the development of various culture devices that accurately mimic the in vivo environment. One important example of such a culture device is a device for culturing cells that exist at the boundary between anaerobic (low-oxygen) and aerobic (aerobic) environments. For example, it has become known that the activity of intestinal bacteria influences health and even various diseases. The intestine is an anaerobic environment inside the intestine and an aerobic environment outside the intestine, with intestinal epithelial cells existing at the boundary between these two environments. Intestinal bacteria interact with intestinal epithelial cells in the anaerobic environment, and substances produced by intestinal bacteria can penetrate intestinal epithelial cells and act outside the intestine. Various devices, such as co-culture devices, that mimic the in vivo environment and simultaneously culture multiple types of cells or microorganisms in the same culture environment have been researched and are commercially available.

[0003] However, to realize an environment where anaerobic and aerobic atmospheres are adjacent to each other, it is necessary to isolate the two atmospheres. While the isolation is in place, it is necessary to replace the culture medium on both the anaerobic and aerobic sides, and preferably circulate it. It is difficult to precisely achieve the opposing environmental conditions of anaerobic and aerobic atmospheres simultaneously. Currently, co-culture devices being developed, manufactured, and sold are difficult to handle and do not have a stable supply, posing practical challenges. Currently, experiments are often conducted under limited conditions, such as only aerobic or only anaerobic environments. However, these experimental conditions pose a challenge, as the reactions of cells and bacteria differ from those in vivo.

[0004] Patent Document 1 discloses a co-culture device technology including a first sealed container, a co-culture device disposed outside the first sealed container, a first medium source disposed within the first sealed 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-culture device and the first medium source. The co-culture device has a membrane including a first main surface and a second main surface, a first flow path partially defined by the first main surface and arranged to allow the first medium to flow through it, and a second flow path partially defined by the second main surface and arranged to allow the second medium to flow through it. Cells are cultured on the membrane. The inlet of the first flow path is connected to the first conduit. The co-culture device is housed in an anaerobic chamber. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2022-130304 Summary of the Invention [Problem to be solved by the invention]

[0006] In conventional co-culture devices, the flow of culture medium can affect cells on the membrane. Furthermore, when completely discharging the medium from the co-culture device, there is also the problem that the membrane tends to dry out. There is still room for improvement in such culture devices, and a new culture device is desired.

[0007] The present invention has been made in consideration of the above circumstances, and aims to provide a culture device that has a simple configuration, is small, and can easily perform one culture or multiple cultures while isolating the upper and lower atmospheres, and in which the flow of culture medium is suppressed from affecting the cells on the membrane. [Means for solving the problem]

[0008] According to one aspect of the present invention, there is provided a well plate having one or more wells with a first through-hole and a flow path for guiding a culture medium to the wells or for guiding the culture medium from the wells, a plate-shaped contact member having a second through-hole corresponding to the wells and having a diameter larger than the diameter of the bottom side of the first through-hole, the contact member contacting the back surface of the well plate, a blocking member blocking the second through-hole of the contact member, an integration member integrating the well plate, the contact member, and the blocking member, and a blocking member disposed in the first through-hole of the well. a membrane that vertically divides the first through-hole of the well portion into a first space and a second space for culturing the culture medium, and a cylindrical culture vessel that fits into the well portion so as to form the first space inside, wherein when the well plate, the abutting member, and the closure member are integrated by the integrating member, the culture medium flows between the main flow groove and the second space via the back surface portion of the well plate that is exposed from the second through-hole of the abutting member. [Effects of the Invention]

[0009] A culture device according to one embodiment of the present invention has a simple configuration, is compact, and can perform one or more cultures while isolating the upper and lower atmospheres. During the transfer process, the flow direction of the culture medium from the channel for guiding the culture medium to the second space is changed so that it passes through the back surface of the well plate exposed through the second through-hole. The culture medium then flows into the second space after first flowing diagonally downward and then laterally. This reduces the force of the medium as it enters the space enclosed by the space of the second through-hole and the closure member. This reduces the impact on cells on the membrane compared to conventional culture devices that introduce culture medium vertically. Furthermore, the culture medium accumulates in the space enclosed by the space of the second through-hole and the closure member. Even when the culture medium is drained, the medium remains in the space enclosed by the thickness of the contact member, preventing excessive drying. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 2 is an exploded perspective view showing a state before a culture vessel and a piping port are fitted into a well portion of a well plate of the culture device according to the first embodiment of the present invention. [Figure 2] FIG. 2 is a perspective view showing the piping port, culture vessel, and membrane of the culture device according to the first embodiment of the present invention in an exploded state. [Figure 3] FIG. 1 is a perspective view showing a culture device according to a first embodiment of the present invention. [Figure 4] FIG. 4 is a cross-sectional view taken along line IV-IV in FIG. [Figure 5] FIG. 2 is a partially cutaway perspective view showing the well plate of the culture device according to the first embodiment of the present invention as viewed from the back side. [Figure 6] FIG. 2 is a partially cutaway perspective view showing a state in which an abutting member is abutted against a well plate of the culture device according to the first embodiment of the present invention from the back side. [Figure 7] 1 is a schematic diagram showing a culture system including a culture device according to a first embodiment of the present invention. [Figure 8] FIG. 1 is a schematic diagram showing the flow of a culture medium. [Figure 9] FIG. 10 is a schematic diagram showing the flow of culture medium in a well plate of a modified example according to the first embodiment of the present invention. [Figure 10] FIG. 10 is an exploded perspective view showing a state before the culture vessels and piping ports are fitted into wells of a well plate of a culture device according to a second embodiment of the present invention. [Figure 11] FIG. 10 is a perspective view showing a culture device according to a third embodiment of the present invention. [Figure 12] FIG. 10 is a rear view of a well plate according to a third embodiment of the present invention. [Figure 13] FIG. 10 is an exploded perspective view showing a state before the culture vessels and piping ports are fitted into the wells of the well plate of the culture device according to the third embodiment of the present invention. [Figure 14] FIG. 10 is a rear view of a modified example of the well plate according to the third embodiment of the present invention. [Figure 15] FIG. 10 is a schematic diagram showing a culture system including a culture device according to a fourth embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0011] Below, specific embodiments of the present invention will be described in detail, using as examples culture devices 100-103 and culture systems 200-201 as co-culture devices that realize a co-culture environment in which the upper side (e.g., first space) is an anaerobic atmosphere (anaerobic environment) and the lower side (e.g., second space) is an aerobic atmosphere (aerobic environment), and perform cell culture at the boundary portion.

[0012] The present invention is not limited to the following embodiments and may be modified as appropriate without departing from the spirit and scope of the present invention. Furthermore, the components including the culture devices 100-103, the culture systems 200-201, and their respective components may also include optional components not shown in the drawings. In the drawings, some or all of the components are depicted schematically and at a different scale, as appropriate. In this specification, identical components may be designated by the same reference numerals and their description may be omitted. The following description will refer to the XYZ Cartesian coordinate system shown in FIG. 1 and other figures as appropriate. In this XYZ Cartesian coordinate system, the X direction is the left-right direction, the Y direction is the front-back direction, and the Z direction is the up-down direction. For each of the X, Y, and Z directions, the direction indicated by the arrow will be referred to as the + direction (e.g., +X direction) or + side (e.g., +X side), and the direction opposite the arrow will be referred to as the - direction (e.g., -X direction) or (e.g., -X side). For example, the +X direction is referred to as the right direction, and the -X direction is referred to as the left direction. The +Y direction is referred to as the front direction, and the -Y direction is referred to as the rear direction. The +Z direction is referred to as the upward direction, and the -Z direction is referred to as the downward direction. A surface perpendicular to the Y direction is an XZ surface. A surface perpendicular to the Z direction is an XY surface. The XY surface is a surface that is parallel to the X direction (first direction) and perpendicular to the Z direction. For each surface, the XY surface on the +Z side will be referred to as the front surface (front surface), and the XY surface on the -Z side will be referred to as the back surface, with the +Z side referred to as the front side and the -Z side referred to as the back side, as appropriate.

[0013] [Embodiment 1] <Culture equipment> Fig. 1 is a perspective view showing a state before a culture vessel 7 and a piping port 8 are fitted into a well portion 1a of a well plate 1 of a culture device 100 according to embodiment 1. Fig. 2 is a perspective view showing a state in which the piping port 8, the culture vessel 7, and the membrane 6 of the culture device 100 are disassembled, and Fig. 3 is a perspective view showing a state in which the culture device 100 is in use. Fig. 4 is a cross-sectional view taken along line IV-IV in Fig. 3. Fig. 5 is a partially cutaway perspective view showing the well plate 1 as viewed from the backside. Fig. 6 is a partially cutaway perspective view showing a state in which an abutting member 2 is abutted against the well plate 1 from the backside.

[0014] As shown in FIGS. 1 to 4, the culture device 100 includes a well plate 1, a contact member 2, a closing member 3, an integrated member U, a membrane 6, a culture container 7, and a piping port 8.

[0015] The well plate 1 has a rectangular plate shape extending in the left-right direction (see FIG. 1). As shown in FIG. 1, the well plate 1 has a well portion 1a. The well plate 1 has one or more well portions 1a, and in embodiment 1, an example having two well portions 1a will be described. The well plate 1 has two well portions 1a, each having a first through-hole 1b, arranged side by side in the left-right direction.

[0016] The well plate 1 also has a flow path for guiding the culture medium to or from the well portion 1a. The "flow path for guiding the culture medium to the well portion 1a" refers to a flow path in which the ends of the tributary channels 1h do not reach the circumference of the inner circle (the circle at the bottom) of the well portion 1a (first through-hole 1b), as shown in Figures 5 and 6 of the first embodiment, and includes a case in which the culture medium flows obliquely downward along the back surface portion B of the well plate 1 exposed from the second through-hole 2a, between the ends of the tributary channels 1h and the circumference of the inner circle of the first through-hole 1b, so as to pass over a weir, and also includes a flow path in which the ends of the tributary channels 1h reach the circumference of the inner circle, as shown in Figure 9, which will be described later, and in which there is no weir and the culture medium flows obliquely downward along the back surface portion B. In the first embodiment, as shown in Fig. 5, the well plate 1 has main flow channels 1f, 1k provided on the back surface, and first flow channels 1e and second flow channels 1j that are medium flow channels and include connection channels 1g, 1m connected to the main flow channels 1f, 1k and whose ends open on the front surface (front face) or side face. In the first embodiment, an example will be described in which the ends of the first flow channel 1e and the second flow channel 1j open on the front surface, as shown in Fig. 4. The well plate 1 will be described in further detail later.

[0017] As shown in FIG. 1, the abutment member 2 is plate-shaped. The abutment member 2 has a contour shape in a plan view that is substantially the same as that of the well plate 1. The abutment member 2 is provided with second through-holes 2a that correspond to the well portions 1a and have a diameter larger than the diameter of the bottom side (-Z side) of the first through-hole 1b of the well plate 1 (see FIGS. 1 and 4). In the example shown in FIG. 1, the abutment member 2 has two second through-holes 2a that correspond to the two well portions 1a provided in the well plate 1. The abutment member 2 abuts against the back surface of the well plate 1 (see FIG. 4). The abutment member 2 abuts against the back surface of the well plate 1 with the second through-holes 2a of the abutment member 2 aligned with the first through-holes 1b of the well plate 1 (see FIG. 4). The second through-holes 2a of the contact member 2 correspond to the well portions 1a of the well plate 1, so that when the well plate 1 and the contact member 2 are in contact with each other, the central axes of the first through-holes 1b of the two well portions 1a provided in the well plate 1 and the central axes of the two second through-holes 2a provided in the contact member 2 are substantially coaxial. The contact member 2 will be described in more detail later.

[0018] The blocking member 3 is disposed below the contact member 2. The blocking member 3 blocks the second through hole 2a of the contact member 2. The blocking member 3 is, for example, a rectangular plate-like shape, and has substantially the same contour shape in a plan view as the contact member 2. The blocking member 3 also functions as a support member that supports the contact member 2 from the rear side (back surface side) of the contact member 2 while blocking the second through hole 2a.

[0019] The integrated member U is a member that integrates the well plate 1, the contact member 2, and the blocking member 3. In the first embodiment, as an example of the integrated member U, an example in which the integrated member U is composed of a cover 5, a housing 4, a hinge shaft 9, and a locking member 10 will be mainly described.

[0020] Fig. 1 shows the well plate 1, the contact member 2, and the blocking member 3 in a state before they are integrated (sometimes referred to as the "non-integrated state"). Figs. 2 to 4 show the well plate 1, the contact member 2, and the blocking member 3 in a state where they are integrated by an integration member U (sometimes referred to as the "integrated state").

[0021] 1 to 4, the housing 4 houses the well plate 1, the contact member 2, and the blocking member 3. The housing 4 may be configured to house at least a portion of the well plate 1, the contact member 2, and the blocking member 3 when they are in an integrated state, rather than the entire well plate 1, the contact member 2, and the blocking member 3. However, in the first embodiment, an example will be described in which the housing 4 houses substantially the entire well plate 1, the contact member 2, and the blocking member 3 when they are in an integrated state.

[0022] The housing 4 has a bottom surface slightly larger than the closure member 3. The cover 5 covers the well plate 1 housed in the housing 4 from the front side. The cover 5 has a surface that can be in surface contact with the upper surface of the well plate 1, and covers the upper surface of the well plate 1 in surface contact. The membrane 6 is disposed within the first through-hole of the well portion 1a and divides the first through-hole 1b of the well portion 1a into a first space A1 and a second space A2 (described below) for culturing the cultured organism (see Figure 4). The cultured organism includes various cells of animals, plants, and microorganisms, as well as transformants derived therefrom, hybridomas, tissues, organs, organoids, spheroids, viruses, and other organisms that are used for culturing. The culture vessel 7 is cylindrical so as to form the first space A1 inside, and is fitted into the well portion 1a (see Figures 2 to 4). The piping port 8 is configured so that piping for introducing or discharging a medium or gas into or from the culture vessel 7 can be attached / detached, and is fitted into the opening of the culture vessel 7 (see FIGS. 3 and 4).

[0023] The above culture device 100 is configured so that, as will be described later, when the well plate 1, the contact member 2, and the closing member 3 are integrated by the unifying member U, that is, when the cover 5 presses against the surface of the well plate 1 housed in the housing 4 to integrate the well plate 1, the contact member 2, and the closing member 3, the culture medium flows between the first flow path 1e or the second flow path 1j and the second space A2 via the back surface portion B of the well plate 1 exposed from the second through-hole 2a of the contact member 2. Each component of the culture device 100 will be described in detail below.

[0024] <Well plate> As shown in FIG. 4, first through-holes 1b of well portions 1a of well plate 1 penetrate well plate 1 in the vertical direction. Internal threads 1c are provided on the inner surface of the upper portions of first through-holes 1b, and an annular step 1d is formed at the bottom of first through-hole 1b. The step 1d has a smaller diameter than the upper portion of first through-hole 1b where internal threads 1c are provided. That is, the diameter of the lower portion of first through-hole 1b (the inner diameter of the bottom portion of step 1d) is smaller than the diameter of the upper portion. In culture device 100, membrane 6 and O-ring 11 serving as a sealing member (described later) can be placed on step 1d, allowing for easy installation of membrane 6 and O-ring 11. The ratio of the height of the upper surface of step 1d to the height of first through-hole 1b may be, for example, 0.4 to 0.8.

[0025] As shown in Figure 5, a first flow path 1e that supplies culture medium to the well portion 1a is formed on the front side (+Y side) of the well plate 1, and a second flow path 1j that discharges culture medium from the well portion 1a is formed on the rear side (-Y side).

[0026] The first flow path 1e has a main flow channel 1f, a connecting channel 1g, two branch flow channels 1h, and two reservoirs 1i. The first flow path 1e supplies the culture medium to the well portions 1a. The main flow channel 1f is a groove formed on the back surface of the well plate 1 and extends in the left-right direction in front of the two well portions 1a (+Y side), the same as the direction in which the two well portions 1a are lined up. The culture device 100 has two well portions 1a, so multiple cultures can be performed simultaneously. Since the main flow channel 1f extends in the direction in which the well portions 1a are lined up, the structure of the first flow path 1e connecting the main flow channel 1f and each well portion 1a can be made efficient. This allows the culture medium to be supplied in a single system (with a single pump) without supplying the culture medium to each well portion 1a individually, and allows the culture medium to flow more uniformly toward each well portion 1a.

[0027] The connecting channel 1g is provided at approximately the center of the main channel 1f in the left-right direction, i.e., it is connected to the main channel 1f and extends in the shape of a round hole in the vertical direction, with its upper end (end on the +Z side) opening on the surface of the well plate 1. The connecting channel 1g has a reservoir (pool) structure with a lower end (end on the -Z side) larger in diameter than the upper end, and is configured to store, for example, culture medium and make the flow from the connecting channel 1g in the direction branching at the main channel 1f (left-right direction) more uniform. The connection path 1g is not limited to being open on the surface of the well plate 1, but may be open on the side surface of the well plate 1, such as the upper part of the side surface.

[0028] The tributary channels 1h are provided so as to branch off from both ends of the main channel 1f, and extend from the front side to the rear side toward the well portion 1a so that their rear ends (the ends on the -Y side) (the ends facing the well portion 1a from the main channel 1f) are located on the back surface portion B of the well plate 1 exposed from the abutment member 2 (see FIGS. 5 and 6). The rear ends of the tributary channels 1h are located, for example, in front of the well portion 1a (the side away from the well portion 1a (the main channel 1f side in this example)), and are preferably located, for example, 0.5 mm to 3 mm in front of the well portion 1a (from the well portion 1a), and may be located, for example, 1 mm in front.

[0029] The width of tributary channel 1h is, for example, narrower than the width of main channel 1f, and the depth of tributary channel 1h is, for example, slightly shallower than the depth of main channel 1f. By making the width of tributary channel 1h narrower than the width of main channel 1f, it is possible to limit the flow rate from main channel 1f to tributary channel 1h while ensuring the flow rate of main channel 1f. This prevents too much medium from flowing into wells 1a near connecting channel 1g on the culture medium supply side, and allows the culture medium to flow more uniformly at the required flow rate in each well 1a.

[0030] The reservoir 1i is located where the tributary channel 1h branches off from the main channel 1f and stores the culture medium. The reservoir 1i is circular in plan view and slightly deeper than the main channel 1f. After the culture medium is stored in the reservoir 1i, it flows between the main channel 1f and the tributary channel 1h, so the reservoir 1i acts as a buffer, further stabilizing the flow rate between the main channel 1f and the tributary channel 1h. Furthermore, because the culture medium flows toward each well 1a with the flow rate stabilized, the culture medium can be set to flow more uniformly at the required flow rate.

[0031] The shape and size of each part of the first flow path 1e can be determined by preliminary experiments or simulations so that the culture medium flows uniformly into the two well portions 1a at the required flow rate. The width of the tributary channel 1h is preferably, for example, between 1 / 4 and 3 / 4 of the width of the main channel 1f. For example, the width of the tributary channel 1h may be 1 / 2 or less of the width of the main channel 1f. When the width of the tributary channel 1h is within the above range, the pipeline resistance is appropriate, the main channel 1f is filled before the medium flows into the tributary channel 1h, there is no need to increase the discharge pressure of the pump 16 described below, and the risk of culture medium leakage is reduced. The diameter of the reservoir 1i is preferably, for example, between 1.5 and 3 times the width of the main channel 1f. For example, the diameter of the reservoir 1i may be more than twice the width of the main channel 1f.

[0032] The second flow path 1j has a main flow channel 1k, a connecting channel 1m, two tributary flow channels 1n, and two reservoirs 1p. The second flow path 1j discharges the culture medium from the well portion 1a. The main flow channel 1k, the connecting channel 1m, the tributary flow channel 1n, and the reservoir 1p have the same shapes as the main flow channel 1f, the connecting channel 1g, the tributary flow channel 1h, and the reservoir 1i, respectively. The main flow channel 1k differs from the main flow channel 1f in that it extends behind the two well portions 1a in the left-right direction in which the two well portions 1a are lined up. The tributary flow channel 1n differs from the tributary flow channel 1h in that it extends from the rear side to the front side toward the well portion 1a.

[0033] 1 and 4, a joint 13 is attached to the upper end of a connection path 1g of the first flow path 1e. The joint 13 is configured so that a pipe 15 (described later) for supplying the culture medium can be attached, and a joint 14 is attached to the upper end of a connection path 1m of the second flow path 1j, and the joint 14 is configured so that a pipe 18 (described later) for discharging the culture medium can be attached. The ends of the joints 13 and 14 may be tapered and configured to allow the attachment of a stop member (blocking member), such as by fitting a cap to the end, or may have a valve that blocks the fluid (flow path). For example, a "stop fitting" type joint may be used as the joints 13 and 14. This allows the first flow path 1e and the second flow path 1j to be configured to be sealable, and allows the culture device 100 to be transported after the culture is interrupted. Instead of attaching the joints 13 and 14 directly to the well plate 1, such joints may be provided in the middle of the pipes 15 and 18.

[0034] As indicated by arrow F1 in FIG. 4, the culture medium is introduced through the joint 13 and flows through the connecting passage 1g of the first flow path 1e, the main flow channel 1f, the reservoir 1i, and the tributary flow channel 1h into the well portion 1a (second space A2). At this time, the culture medium flows between the tributary flow channel 1h (flow path) and the well portion 1a (second space A2) through the back surface portion B of the well plate 1 exposed through the second through-hole 2a. Here, "flowing through the back surface portion B" includes a case in which the end of the tributary flow channel 1h does not reach the inner circle of the well portion 1a (first through-hole 1b), and the culture medium flows obliquely downward along the back surface portion B, crossing a weir between the end of the tributary flow channel 1h and the circumference of the inner circle of the first through-hole 1b, as in the first embodiment, and a case in which the end of the tributary flow channel 1h reaches the circumference of the inner circle, there is no weir, and the culture medium flows obliquely downward along the back surface portion B, as in a modified example of the well plate 1 shown in FIG. 9 (described later). That is, the culture medium flows obliquely downward through region A4, which is a space (flow path) formed between the inner surface of the second through-hole 2a and the inner circle of the first through-hole 1b (rear surface portion B), the inner surface of the second through-hole 2a, and the upper surface of the blocking member 3. As shown in FIG. 8, the culture medium then flows gradually from the end of the tributary channel 1h along the rear surface portion B, i.e., along the shape of region A4. The culture medium initially flows downward and then flows laterally, losing momentum as it enters the culture medium storage space A3, which is the space surrounded by the space of the second through-hole 2a and the blocking member 3. Therefore, compared to conventional culture devices in which the culture medium flows vertically, the impact on the cells on the membrane 6 is reduced. Furthermore, the culture medium accumulates in the culture medium storage space A3. Even when the culture medium is drained, a portion of the culture medium remains, corresponding to the thickness of the contact member 2, preventing excessive drying.

[0035] 4, the culture medium is drained from the well portion 1a (second space A2) through the tributary channel 1n of the second flow path 1j, the reservoir 1p, the main channel 1k, the connecting channel 1m, and the joint 14. At this time, the culture medium flows between the well portion 1a (second space A2) and the tributary channel 1n through the back surface portion B of the well plate 1 exposed from the second through-hole 2a, the region A4 formed between the inner surface of the second through-hole 2a and the blocking member 3, and then flows into the tributary channel 1n through the back surface portion B of the contact member 2. At this time, the culture medium flows little by little from the second through-hole 2a of the contact member 2 to the tributary channel 1n along the shape of the region A4.

[0036] The well plate 1 can be made of a material that is chemically resistant and can be sterilized in an autoclave. Examples of such materials include synthetic resins such as PTFE (polytetrafluoroethylene) and polyacetal, with PTFE being preferred. When the well plate 1 is made of a material that can be sterilized in an autoclave, it can be made reusable.

[0037] <Contacting parts> The material of the abutting member 2 may be, for example, a material that is chemically resistant and can be sterilized by autoclave. The material of the abutting member 2 may also be a soft material. A preferred material for such a abutting member 2 is, for example, silicone resin. Forming the abutting member 2 from a soft material such as silicone can enhance adhesion between the well plate 1 and the closure member 3, enable sterilization by autoclave, and enable a reusable configuration. The thickness of the abutting member 2 and the difference between the radius of the second through-hole 2a of the abutting member 2 and the radius of the bottom of the first through-hole 1b of the well plate 1 can be determined by preliminary experiments or simulations so that the culture medium flows uniformly into the two well portions 1a at a required flow rate. The thickness of the abutting member 2 is preferably, for example, 0.5 mm or more and 2 mm or less. When the thickness of the abutting member 2 is within the above range, it has elasticity and good adhesion, and does not deform and intrude into the flow path. The difference between the radius of the second through-hole 2a and the radius of the bottom of the first through-hole 1b may be, for example, the length of the tributary channel 1h in the front-rear direction plus 1 to 2 mm. When the difference is within the above range, the culture medium flows in smoothly, the pressure difference when the culture medium is drained does not become large, and the culture medium can be drained smoothly. The remaining amount of culture medium can be adjusted by changing the thickness of the contact member 2. If it is desired to recover all of the enterobacteria that have permeated the membrane 6, a thickness of 0.5 mm, for example, will allow for almost complete drainage. If cells are also seeded on the blocking member 3 or the cover glass 43 in Figure 10 to examine their reaction with the enterobacteria 41 that have permeated the membrane 6, a thickness of 2 mm, for example, will prevent the culture medium from being completely drained, preventing the cells from drying out.

[0038] <Blocking member> Examples of the material for the blocking member 3 include glass and synthetic resin. Examples of the material for the blocking member 3 include materials that are chemically resistant and heat resistant and can be sterilized in an autoclave. Specific examples include borosilicate glass. If the blocking member 3 is made of glass, this is preferable because it can ensure flatness and improve adhesion with the contact member 2. Furthermore, if the blocking member 3 is made of a transparent material, such as glass, it becomes possible to observe the interior from the bottom of the culture device 100 using a microscope or the like. Furthermore, if the blocking member 3 is made of a material that can be sterilized in an autoclave, it can be configured to be reusable.

[0039] <Membrane> As described above, the membrane 6 is disposed at the bottom of the first through-hole 1b of the well portion 1a. The membrane 6 vertically divides the first through-hole 1b of the well portion 1a into a first space A1 and a second space A2 for culturing the culture medium. The membrane 6 is placed on the step 1d of the well plate 1. An O-ring 11 serving as a sealing member is abutted against the membrane 6. The membrane 6 is a membranous body, for example, a membranous body having pores. Commercially available membranes sold by various companies can be used as the membrane 6, and various culture conditions can be accommodated by selecting the material and permeable holes (pore size).

[0040] Furthermore, the membrane 6 is not limited to the above-described pore-containing membrane, but may be a so-called three-dimensional culture sheet, etc. For example, a "cell aggregate three-dimensional culture microwell plate TASCL" (manufactured by Sims Bio Co., Ltd.) may be used.

[0041] <Case> The housing 4 may be made of a material that can be sterilized by autoclave, for example. When the material can be sterilized by autoclave, the housing 4 can be reusable. A preferred material for the housing 4 is a metal such as aluminum. As shown in FIG. 4, a through-hole 4a having a rectangular shape in a plan view is provided on the bottom surface of the housing 4. As shown in FIG. 1, a protrusion 4b that protrudes toward the right is provided in the center of the right edge of the housing 4. A locking member 10 is attached to the protrusion 4b so as to be rotatable around the right edge as an axis (Y-axis). In this example, the locking member 10 is a hook. The locking member 10 locks the right edge of the cover 5 to the right edge of the housing 4. As shown in FIGS. 1 to 3, a hinge shaft 9 incorporating a hinge shaft is attached to the left edge of the housing 4. The hinge shaft 9 connects the left edge of the housing 4 to the corresponding left edge of the cover 5.

[0042] The locking member 10 is not limited to being attached to the protrusion 4b of the housing 4, but may be attached to a protrusion 5d of the cover 5, which will be described later.

[0043] <Cover> The cover 5 covers the top surface of the well plate 1 in surface contact. Examples of materials for the cover 5 include materials that can be sterilized by autoclave. Materials that can be sterilized by autoclave can be reusable. Furthermore, the cover 5 is preferably made of a material with high strength and rigidity, which reduces deflection during the integration and increases the contact surface area between the cover 5 and the well plate 1, resulting in a more secure integration. In particular, the greater the number of wells 1a provided on the well plate 1, the smaller the contact surface area between the cover 5 and the well plate 1. Therefore, the strength and rigidity of the material for the cover 5 are important. Examples of preferred materials for the cover 5 include metals such as stainless steel and aluminum, with stainless steel being preferred from the standpoints of strength and rigidity. As shown in FIGS. 2 and 4 , the cover 5 has through-holes 5a corresponding to the wells 1a and slightly larger in diameter than the upper portion of the first through-holes 1b of the wells 1a. The cover 5 covers the well plate 1 while exposing the wells 1a. The cover 5 is also provided with through-holes 5b so that the tips of the joints 13 and 14 protrude from the surface of the cover 5. As shown in FIG. 2 and other figures, the front long side and rear long side of the cover 5 are each provided with a side wall 5c that stands upright along the entire long side. The left end of each side wall 5c is connected to the hinge shaft 9, allowing the cover 5 to rotate relative to the housing 4. A protrusion 5d that protrudes toward the right is provided at the center of the right side of the cover 5. As shown in FIG. 1, the well plate 1, the abutting member 2, and the blocking member 3 are placed inside the housing 4, and as shown in FIG. 2, the cover 5 is rotated around the Y axis to overlap the protrusion 5d with the protrusion 4b. Then, the protrusion 5d and the right side of the protrusion 4b are engaged with the engaging member 10, thereby fixing the cover 5 to the housing 4. That is, the cover 5 is fixed to the housing 4 with the well plate 1, the contact member 2, and the closing member 3 sandwiched therebetween, thereby integrating the well plate 1, the contact member 2, and the closing member 3. As described above, the cover 5, the housing 4, the hinge shaft 9, and the hook 10 constitute the integrated member U.

[0044] The integration of the well plate 1, the abutment member 2, and the blocking member 3 is not limited to the above case, and the well plate 1, the abutment member 2, and the blocking member 3 may be integrated by clips, clamps, etc. without housing the well plate 1, the abutment member 2, and the blocking member 3 in the housing 4. In other words, the integration member U may be any member that integrates the well plate 1, the abutment member 2, and the blocking member 3, and may be, for example, a clip, a clamp, etc.

[0045] <Culture container> Examples of materials for the culture vessel 7 include synthetic resins such as PTFE and polyacetal, and among these, PTFE is preferred.

[0046] 4, the culture vessel 7 is cylindrical and has a flange 7a at its upper portion whose outer diameter is larger than that of its lower portion, and a male thread 7b that fits into the female thread 1c is provided on the outer surface of the lower portion of the flange 7a of the culture vessel 7, and is configured so that the male thread 7b is tightened onto the female thread 1c. With this configuration, the culture vessel 7 and the well portion 1a can be connected simply and effectively to form a more airtight connection, and as a result, the isolation between the first space A1 and the second space A2 can be improved.

[0047] With the well plate 1 fixed in the housing 4, the membrane 6 is placed on the upper surface of the step 1d of the well plate 1, an O-ring 11 with an outer diameter substantially equal to that of the membrane 6 is placed around the periphery of the membrane 6, and the male thread 7b of the culture vessel 7 is fastened to the female thread 1c of the well portion 1a. This allows the culture vessel 7 to fit airtightly into the well portion 1a.

[0048] <Piping port> Examples of the material for the piping port 8 include synthetic resins such as PTFE and polyacetal, and among these, PTFE is preferred.

[0049] As shown in FIG. 4, the piping port 8 is hollow and cylindrical, with a small-diameter cylindrical protrusion 8a at the bottom. The protrusion 8a has a diameter slightly smaller than that of the flange 7a of the culture vessel 7. The front and rear portions of the upper side of the piping port 8 are flat (see FIG. 3). A receiving groove 8b that opens to the outside is provided in the vertical center of the protrusion 8a. With an O-ring 12 accommodated in the receiving groove 8b of the piping port 8, the protrusion 8a is fitted into the flange 7a of the culture vessel 7.

[0050] A joint 21 is attached to the front portion of the piping port 8, and one end of a nozzle 25 is connected to the joint 21 inside the piping port 8, with the other end of the nozzle 25 protruding downward from the underside of the protrusion 8a. Culture medium is supplied from the other end of the nozzle 25.

[0051] A fitting 22 is attached to the right side of the fitting 21 in the front portion of the piping port 8 (see Figure 3), and one end of a nozzle 26 (described later) is connected to the fitting 22 inside the piping port 8, with the other end of the nozzle 26 protruding downward from the underside of the protrusion 8a. The culture medium is drained from the other end of the nozzle 26. The amount of culture medium drained is determined by the length of protrusion of the nozzle 26 from the underside of the protrusion 8a. Draining all of the culture medium may damage the cells, but leaving an appropriate amount of culture medium reduces the damage to the cells.

[0052] A joint 23 is attached to the rear portion of the piping port 8, and one end of a pipe 27 is connected to the joint 23 inside the piping port 8, with the other end of the pipe 27 protruding from the underside of the protrusion 8a. Low-oxygen gas is supplied from the other end of the pipe 27.

[0053] A fitting 24 is attached to the right of the fitting 23 in the rear portion of the piping port 8 (see FIG. 3), and one end of a pipe 28 (described later) is connected to the fitting 24 inside the piping port 8, with the other end of the pipe 28 protruding from the underside of the protrusion 8a. Low-oxygen gas is exhausted from the other end of the pipe 28.

[0054] For example, "stop fitting" type joints may be used as the joints 21, 22, 23, and 24. This allows the culture to be interrupted and the culture device 100 to be carried. Instead of a configuration in which the joints 21, 22, 23, and 24 are directly attached to the piping port 8, such joints may be provided in the middle of the piping 29, 32, 35, and 38. The piping port 8 of the culture device 100 can introduce or discharge the culture medium or gas into or from the first space A1 with a simple configuration.

[0055] <Culture system> 7 is a schematic diagram showing a culture system 200 in which a culture medium-filled container and the like are connected to the culture device 100. The culture system 200 includes one of the culture devices 100 to 103, a culture medium-filled container 17, a drainage container 20, a culture medium-filled container 31, a drainage container 34, and an anaerobic generating means 37.

[0056] One end of a pipe 15 is connected to a joint 13 attached to the upper end of the connection path 1g of the well plate 1, and the other end of the pipe 15 is connected to a culture medium filling container 17. The culture medium filling container 17 contains the culture medium to be supplied to the second space A2. A pump 16 is provided midway along the pipe 15.

[0057] One end of a pipe 18 is connected to a joint 14 attached to the upper end of the connection path 1m of the well plate 1, and the other end of the pipe 18 is connected to a drainage container 20. The drainage container 20 receives the drainage liquid discharged from the second space A2 from the pipe 18. A pump 19 is provided midway along the pipe 18.

[0058] One end of piping 29 is connected to fitting 21 attached to the front portion of piping port 8, and the other end of piping 29 is connected to culture medium filling container 31. Culture medium filling container 31 contains the culture medium to be supplied to first space A1. One end of piping 32 is connected to fitting 22 attached to the front portion of piping port 8, and the other end of piping 32 is connected to drainage container 34. Drainage discharged from first space A1 is sent from piping 32 and stored in drainage container 34. A pump 30 is provided midway through piping 29, and a pump 33 is provided midway through piping 32.

[0059] One end of pipe 35 is connected to fitting 23 attached to the rear portion of pipe port 8, and the other end of pipe 35 is connected to anaerobic generation means 37. One end of pipe 38 is connected to fitting 24 attached to the rear portion of pipe port 8, and the other end of pipe 38 is connected to anaerobic generation means 37. Pump 36 is provided in the middle of pipe 35, and pump 39 is provided in the middle of pipe 38.

[0060] <Culture method> An example of a cell culture method using the culture device 100 will be described below. As shown in FIG. 1, the blocking member 3, the contact member 2, and the well plate 1 are housed in the housing 4 in this order, and the cover 5 is closed and fixed with the locking member .

[0061] As shown in Figure 2, membrane 6 and O-ring 11 are placed on step 1d of well plate 1, and male thread 7b is tightened onto female thread 1c to fit culture vessel 7 into well portion 1a of well plate 1. With O-ring 12 fitted into receiving groove 8b of protrusion 8a of piping port 8, piping port 8 is fitted into flange 7a of culture vessel 7. At this time, the space sealed by membrane 6, well portion 1a, abutment member 2, and closure member 3 becomes second space A2 with an aerobic environment, and the space closed by culture vessel 7, membrane 6, and piping port 8 becomes first space A1 with an anaerobic environment. In this state, sterilization measures such as autoclaving are performed.

[0062] After sterilization, as shown in Figure 7, joint 13 of well plate 1 is connected to medium-filled container 17 via piping 15, and joint 14 is connected to drainage container 20 via piping 18. Next, joint 21 of piping port 8 is connected to medium-filled container 31 via piping 29, and joint 22 is connected to drainage container 34 via piping 32. Then, joints 23 and 24 are connected to anaerobic generating means 37 via piping 35 and 38, respectively.

[0063] Culture media from culture medium-filled containers 17 and 31 are sent to second space A2 and first space A1 through pipes 15 and 29, respectively. At this time, pump 30 is temporarily removed and a cell suspension is sent to first space A1 using a syringe or the like, or piping port 8 is removed and cells 40 (culture medium), such as intestinal epithelial cells, are directly seeded on membrane 6, and piping port 8 is then reattached. The joints 13 and 14 are sealed off from the outside air by the caps or valves described above.

[0064] An anaerobic atmosphere is sent from the anaerobic generating means 37 to the first space A1 by a pump 36 via a pipe 35 connected to the joint 23, and is then returned to the anaerobic generating means 37 by a pump 39 via a pipe 38 connected to the joint 24, and circulated. The oxygen concentration in the anaerobic generating means 37 can be maintained at a predetermined reduced level by continuously filling the anaerobic generating means 37 with nitrogen gas to expel oxygen from the anaerobic generating means 37, by filling a separate cylinder with anaerobic gas and continuously sending it to the anaerobic generating means 37, or by generating low-oxygen gas using an oxygen scavenger to adsorb the oxygen in the anaerobic generating means 37.

[0065] After confirming operation, the piping and container are removed, and only the housing 4 sealed with the cover 5 is placed in a general-purpose CO2 incubator to control the temperature. The joint 13 is connected to the culture medium filling container 17 via piping 15, and the culture medium is sent to the second space A2, and after a predetermined period of time, the culture medium is discharged into the drainage container 20 via piping 18 connected to the joint 14.

[0066] Instead of placing the housing 4 in a CO2 incubator, a heat source (heating device) may be provided in the housing 4 to keep it warm.

[0067] The joint 21 is connected to a culture medium-filled container 31 via piping 29, and sends the culture medium to the first space A1, and after a predetermined period of time, the culture medium is discharged via piping 32 connected to the joint 22 into a drainage container 34. For example, the pump 30 can be removed at a predetermined timing to introduce enterobacteria 41 as anaerobic culture medium, thereby reproducing a reaction in an aerobic co-culture environment, or it can also be used as a means for discharging overgrown enterobacteria 41.

[0068] As shown in Figures 2 and 5, the well portion 1a and tributary channel 1h provided in the well plate 1 are not directly connected. Instead, as shown in Figure 6, the end of tributary channel 1h is exposed on the annular back surface portion B of the well plate 1, which is exposed from the second through-hole 2a of the abutting member 2. The culture medium first flows from the end of tributary channel 1h to the well portion 1a via the back surface portion B. In other words, the culture medium flows between the main flow channel 1f and the second space A2 via the back surface portion B, which is exposed from the second through-hole 2a of the abutting member 2. Since the culture medium does not flow abruptly into the well portion 1a but flows over a weir, this has the effect of making the flow more uniform.

[0069] FIG. 8 is a schematic diagram showing the flow of culture medium, showing a portion of the right side of FIG. 6. As indicated by the arrows in FIG. 8, the culture medium flows gradually from the end of the tributary channel 1h through region A4 along the back surface portion B. That is, the culture medium first flows slightly circumferentially from the end of the tributary channel 1h along the underside of region A4, then enters the well portion 1a diagonally downward. Then, it gradually flows circumferentially along the entire length before entering the well portion 1a and flowing along the back surface portion B. As shown in FIG. 8, the culture medium does not flow directly to the center of the well portion 1a, so even if a culture medium, such as enterobacteria 41 that have penetrated the membrane 6, is present in the center, damage to the culture medium is reduced. In this way, the flow direction of the culture medium from the main channel 1f to the second space A2 changes as it passes through the back surface portion B, and then the culture medium flows into the second space A2. Instead of flowing directly into the second space A2, the culture medium reduces the impact on the cells, such as detachment of cells growing on the membrane 6, during the liquid transfer. Even when cells are seeded on the blocking member 3 or the cover glass 43 in FIG. 10 described below and a reaction with the enterobacteria 41 that has permeated the membrane 6 is examined, physical damage such as cell detachment can be reduced.

[0070] 4, the culture device 100 has a culture medium storage space (culture medium storage portion) A3 in the integrated state. The culture medium storage space A3 includes the space of the second through-hole 2a of the contact member 2 and the space surrounded by the blocking member 3. The culture medium storage space A3 is a space where the culture medium remains when, for example, draining the liquid from the connection path 1m of the second flow path 1j. When the culture medium storage space A3 is provided, the culture medium remains in the culture medium storage space A3 by the thickness of the contact member 2 when draining the liquid, making it possible to prevent the membrane 6 from drying out excessively.

[0071] After a predetermined period of time has passed, all operations are stopped, the housing 4 is removed from the CO2 incubator, and the piping port 8 is disconnected, allowing the enterobacteria 41 or other suspended matter in the culture medium in the first space A1 to be collected. Next, the culture vessel 7 is removed, and the cells grown on the membrane 6 can be collected. If it is desired to collect substances or cells that have permeated the membrane 6, they can be collected after cutting and removing the membrane 6 with scissors or the like without removing the hook 10, for example. Because the well plate 1, abutment member 2, and closure member 3 are integrated, the culture medium does not leak even when the culture vessel 7 is removed, so enterobacteria that have permeated the membrane 6 can be collected from the remaining culture medium.

[0072] In the culture device 100, the well plate 1, the contact member 2, and the closure member 3 are tightly attached and integrated. A first flow path 1e and a second flow path 1j are formed in the well plate 1, and by attaching joints 13 and 14 to two locations on the surface of the well plate 1, the culture medium is supplied to the well portion 1a and discharged from the well portion 1a. In other words, it is sufficient to connect piping at two locations on the top of the well plate 1, and there is no need to connect piping to each well portion of the well plate 1, which simplifies the piping connection operation and also simplifies the piping connection configuration, thereby reducing the installation space for the piping, the culture medium filling container, and the discharge container.

[0073] In some conventional products, the medium flow path is formed by bonding multiple components together with adhesives or the like, making it impossible to disassemble, and when dust accumulates in the medium flow path, it is difficult to remove, so the product is intended to be disposable.In the culture device 100, the first flow path 1e and the second flow path 1j are formed by through-holes and grooves formed on the back surface, making maintenance such as cleaning easy and also enabling reuse.

[0074] In the culture device 100, the well plate 1, contact member 2, and closure member 3 are easily integrated by closing the cover 5 on the housing 4 and locking it with the locking member 10. In addition, the cover 5 presses the upper surface of the well plate 1 by surface contact, allowing for more stable and strong integration.

[0075] In the culture device 100, the supply and discharge of culture medium can be performed using only two systems: a first flow path 1e that supplies the culture medium to the well portion 1a, and a second flow path 1j that discharges the culture medium from the well portion 1a. That is, the first flow path 1e has a main flow channel 1f, a connecting channel 1g, and a tributary flow channel 1h, and can be controlled to supply the culture medium uniformly to each well portion 1a with a single pump 16. Conventionally, each well portion has been individually controlled by switching using a solenoid valve or the like, that is, controlled using multiple systems, but this increases costs and becomes complicated.

[0076] The second flow path 1j also has a main flow channel 1k, a connecting channel 1m, and a tributary flow channel 1n, allowing a single pump 19 to uniformly control the amount of culture medium drained from each well 1a. If, for some reason, the culture medium cannot be uniformly drained, air will be drawn in from the wells that emptied first, lowering the drainage pressure and resulting in the intake of only air. This can lead to the wells with remaining culture medium not being able to draw in culture medium, so it is desirable to ensure that the drainage, like the liquid supply, flows uniformly. Ideally and preferably, the widths and lengths of the flow path grooves and holes in the first flow path 1e and the second flow path 1j are identical, but configurations in which they are approximately identical are not excluded from the technical scope of the present invention. That is, ideally and preferably, the widths and lengths of the tributary flow channels 1h on the connected liquid supply side are identical in each well 1a, and ideally and preferably, the widths and lengths of the tributary flow channels 1n on the connected liquid discharge side are identical. Note that "approximately identical" means that they are 95% or more identical.

[0077] Furthermore, since the first flow path 1e and the second flow path 1j have the same shape, the same function can be achieved regardless of which one is connected to the culture medium filling container 17. In other words, in this configuration, both the first flow path 1e and the second flow path 1j can be used as a flow path for supplying and draining the culture medium.

[0078] The culture device 100 can be applied to various researches on intestinal bacteria, infectious diseases, and patient-derived cancer cells. The culture device 100 can also be adapted to various MPS (mimetic biological environments) by replacing the well plate 1 or membrane 6.

[0079] According to the culture system 200, fittings 13, 14 are attached to the ends of the connection paths 1g, 1m, which open on the surface or side of the well plate 1 of the culture device 100, and piping 15, 18 are used to connect the fittings 13, 14 to the medium-filling container 17 or the drainage container 20, thereby allowing culture medium to be supplied to or discharged from the well portion 1a. In other words, the piping can be connected at the surface or side of the well plate 1 where the fittings 13, 14 are attached, eliminating the need to connect piping to each well portion 1a of the well plate 1. This simplifies the piping connection process and reduces the installation space required for the piping, medium-filling container, and drainage container. Since the piping, medium-filling container, and drainage container can be arranged above or to the side of the culture device 100, the flexibility of the installation location of the culture device 100 is improved. For example, the culture device can be configured to be placed directly on a laboratory table, microscope stage, etc. The culture system according to this configuration has a simple configuration and can be miniaturized. In this embodiment, the case where culture medium is supplied to the first space A1 and the second space A2 from different culture medium filling containers 17, 31 is described, but this is not limited to this, and culture medium may be supplied from the same culture medium filling container depending on the culture conditions.

[0080] <Modification> Figure 9 is a schematic diagram showing the flow of culture medium in a modified well plate 1. The same parts as in Figure 8 are given the same reference numerals and detailed explanations are omitted. The ends of the tributary channels 1h in the modified well plate 1 are not located in front of the well portion 1a in the back surface portion B, but are located on the circumferential portion of the well portion 1a (first through-hole 1b).

[0081] FIG. 9(A) is a schematic diagram showing the state in which the end of the tributary channel 1h extends rearward and reaches the circumference of the first through-hole 1b of the well portion 1a. In the case of FIG. 9(A), the culture medium first flows downward as shown in the cross-sectional view of FIG. 9(D), and then flows in the Y-axis direction, thereby entering the culture medium storage space A3 with reduced momentum. Therefore, compared to conventional culture devices in which the culture medium flows in the Z-axis direction, the impact on the cells on the membrane 6 is reduced. Furthermore, the culture medium accumulates in the culture medium storage space A3, and even when the culture medium is drained, the culture medium remains by the thickness of the abutment member 52, making it possible to prevent excessive drying.

[0082] 9(B) is a schematic diagram showing a state in which the reservoir 1i is slightly shifted to the right with respect to the center of the well 1a, and the end of the tributary channel 1h extends rearward and reaches the circumference of the first through-hole 1b of the well 1a. The circumferential length of the portion of the tributary channel 1h in FIG. 9(B) where it contacts the first through-hole 1b is longer than the circumferential length of the portion of the tributary channel 1h in FIG. 9(A) where it contacts the first through-hole 1b. In other words, the cross-sectional area (the area of ​​the surface through which the culture medium flows into the well 1a) is larger. As indicated by the arrows, the culture medium flows diagonally from right to left, not directly toward the center of the well 1a. The increased cross-sectional area reduces the flow rate, which reduces damage to the culture medium, such as enterobacteria 41 that have penetrated the membrane 6 and are present in the center of the well 1a. This also reduces damage to the closure member 3 when cells are seeded therein (see also FIG. 9(D)).

[0083] 9(C) is a schematic diagram showing a state in which tributary groove 1h extends approximately ¼ of the circumference of first through-hole 1b along the circumferential direction of back surface portion B, thereby increasing the cross-sectional area. Even in the case of FIG. 9(C), the culture medium flows as shown by the arrows, not directly toward the center of well portion 1a, and the flow rate decreases due to the increased cross-sectional area. This reduces damage to cultured organisms, such as enterobacteria 41 that have permeated membrane 6 and are present in the center of well portion 1a, and also reduces damage to cells seeded on closure member 3.

[0084] As described above, by increasing the cross-sectional area and designing the shape of the end of the tributary groove 1h, it is possible to prevent the culture medium from flowing forcefully toward the center of the well portion 1a, as shown in the cross-sectional view of Figure 9(D), thereby reducing damage to the cells.

[0085] [Embodiment 2] 10 is a perspective view showing an exploded state of members excluding the piping ports, culture vessels, and membranes of the culture device 101 according to embodiment 2. In the figure, the same parts are given the same reference numerals and detailed description thereof will be omitted.

[0086] In the culture device 101, instead of providing a rectangular through-hole 4a as in the culture device 100, a through-hole 4c that corresponds to the well portion 1a and is circular in plan view is provided in the bottom surface of the housing 4. For example, an aluminum support member 42 has a shape slightly smaller than the bottom surface of the housing 4, is provided with a through-hole 42a that is larger than the through-hole 4c, and is placed on the bottom surface of the housing 4 with the through-hole 42a aligned with the through-hole 4c. A cover glass 43 is placed on the bottom portion of the housing 4 that is exposed from the through-hole 42a. The cover glass 43 corresponds to a blocking member. The diameter of the cover glass 43 is smaller than the diameter of the through-hole 42a but larger than the diameter of the through-hole 4c. The surface of the cover glass 43 is coated with collagen or the like. In the culture device 101, the cover glass 43 that has been treated with a required coating or the like can be replaced according to the culture conditions.

[0087] In addition, in the culture device 101, cells can also be seeded on the cover glass 43. In this case, as explained in Figure 8 above, the culture medium does not flow directly into the well portion 1a, but flows along the back surface portion B of the ring in area A4, thereby reducing damage such as peeling to the cells on the cover glass 43 in the center of the well portion 1a.

[0088] [Embodiment 3] Figure 11 is an oblique view showing a culture device 102 relating to embodiment 3, Figure 12 is a back view of the well plate 51 of the culture device 102, and Figure 13 is an oblique view of the culture device 101 before the culture container 57 and piping port 58 are fitted into the well portion 51a of the well plate 51.

[0089] The culture device 102 includes a well plate 51 , a contact member 52 , a blocking member 53 , a housing 54 , a cover 55 , a membrane (not shown), a culture vessel 57 , and a piping port 58 .

[0090] <Well plate> As shown in Figures 11 to 13, unlike well plate 1 which has two well portions 1a arranged side by side in the left-right direction, well plate 51 has three well portions 51a arranged side by side in the row direction (left-right direction) and two well portions 51a arranged side by side in the column direction (front-back direction), for a total of six well portions 51a.

[0091] 13, first through-holes 51b of well portions 1a of well plate 51 penetrate well plate 51 in the vertical direction. An internal thread 51c is provided on the inner surface of the upper portion of first through-hole 51b, and an annular step 51d having a smaller diameter in plan view than the upper portion of first through-hole 51b where internal thread 51c is provided is formed on the bottom portion of first through-hole 51b. That is, the diameter of the lower portion of first through-hole 51b (the inner diameter of the bottom side of step 51d) is smaller than the diameter of the upper portion.

[0092] As shown in Figure 12, the well plate 51 has a first flow path 51e formed along the front, left, and rear edges to supply culture medium to the well portion 51a, and a second flow path 51j formed in the center of the front-to-rear direction, extending in the left-to-right direction, to discharge the culture medium from the well portion 51a.

[0093] The first flow path 51e has a main flow channel 51f, a connection channel 51g, six tributary flow channels 51h, and six storage portions 51i. The main flow channel 51f is a groove formed on the back surface of the well plate 51, and extends along the front, left, and rear sides of the well plate 51. The connection channel 51g is provided in approximately the center of the left side of the main flow channel 51f, extends in the vertical direction like a round hole, and has an upper end that opens at the surface of the well plate 51. The lower end of the connection channel 51g has a larger diameter than the upper end, and is configured to store the culture medium.

[0094] In the portion of the main flow channel 51f along the front edge of the well plate 51, three tributary channels 51h are provided branching out from three left-right locations corresponding to the three wells 51a. These tributary channels 51h extend toward the wells 51a so that their rear ends are located on the back surface of the well plate 51 exposed from the abutting member 52. In the portion of the main flow channel 51f along the rear edge of the well plate 51, three tributary channels 51h are provided branching out from three left-right locations. These tributary channels 51h extend toward the wells 51a so that their leading ends are located on the back surface of the well plate 51 exposed from the abutting member 52. The ends of the tributary channels 51h are preferably located 0.5 mm to 3 mm or less in front of the wells 51a, for example, and may be located 1 mm in front. The width of tributary groove 51h is narrower than that of main groove 51f, and the depth is slightly shallower than that of main groove 51f. Reservoir 51i is provided at the point where tributary groove 51h branches off from main groove 51f, has a circular shape in a plan view, and is slightly deeper than main groove 51f.

[0095] The shape and size of each part of the first flow path 51e can be determined by preliminary experiments or simulations so that the culture medium flows uniformly into the six well portions 51a at a required flow rate. For example, the width of the tributary channel 51h is preferably between 1 / 4 and 3 / 4 of the width of the main channel 51f. For example, the width of the tributary channel 51h may be 1 / 2 or less of the width of the main channel 51f. When the width of the tributary channel 51h is within the above range, the conduit resistance is appropriate, the main channel 51f is filled before the culture medium flows into the tributary channel 51h, there is no need to increase the discharge pressure of the pump 16, and the risk of culture medium leakage is reduced. For example, the diameter of the reservoir 51i is preferably between 1.5 and 3 times the width of the main channel 51f. For example, the diameter of the reservoir 51i may be at least twice the width of the main channel 51f.

[0096] The second flow path 51j has a main flow channel 51k, a connection channel 51m, six tributary flow channels 51n, and three reservoirs 51p. The main flow channel 51k is a channel formed in the center of the back surface of the well plate 51 in the front-to-back direction and extends in the left-to-right direction. The connection channel 51m is provided to the right of the connection channel 51g at the end of the main flow channel 51k, extends in the vertical direction like a round hole, and its upper end opens on the surface of the well plate 51. At three left-to-right locations on the main flow channel 51k corresponding to the well portions 51a, one tributary flow channel 51n is provided branching out to the front and ... back surface of the well plate 51 so that its end is located in the back surface portion of the well plate 51 exposed from the abutting member 52.

[0097] As shown in Figures 12 and 13, a fitting 59 is attached to the upper end of the connecting passage 51g of the first flow path 51e, and is configured so that a pipe for supplying the culture medium can be attached to the fitting 59, and a fitting 60 is attached to the upper end of the connecting passage 51m of the second flow path 51j, and is configured so that a pipe for discharging the culture medium can be attached to the fitting 60.

[0098] <Cover> As shown in FIG. 13 , the cover 55 is provided with a through-hole 55a corresponding to the well portion 51a and having a diameter slightly larger than the diameter of the upper portion of the first through-hole 51b of the well portion 51a, and through-holes (not shown) are provided so that the tips of the joints 59, 60 protrude from the surface of the cover 55. Side wall portions 55b are provided on the front and rear long sides of the cover 55, respectively, standing along the entire long sides. The left end of each side wall portion 55b is connected to the hinge shaft portion 9, allowing the cover 55 to rotate with respect to the housing 54. A protrusion 55c protruding toward the right is provided at the center of the right side of the cover 55. A protrusion 55d protruding in the front-to-rear direction is provided at the center of each side wall portion 55b of the cover 55.

[0099] <Case> A cantilever member 56 is attached to the housing 54. The cantilever member 56 has two support portions 56a provided on the front and rear sides of the housing 54 closer to the hinge shaft 9 than the two protrusions 55d, two lever portions 56b rotatably connected to the two support portions 56a, and a shaft portion 56c connecting the two lever portions 56b.

[0100] When protrusion 55c of cover 55 is aligned with protrusion 54b of housing 54, locking member 10 is locked with protrusion 55c and protrusion 54b, and the right edge of cover 55 is locked with the right edge of housing 54, lever 56b presses protrusion 55d, and cover 55 is fixed to housing 54. Furthermore, if protrusion 55d of cover 55 is made of a magnetic material and lever 56b is made of a material (magnetic body) that is attracted by the magnetism of protrusion 55d, when lever 56b is opened, the magnetism of protrusion 55d attracts lever 56b, so that cover 55 opens following the movement of lever 56b. Protrusion 55d is, for example, a magnet (e.g., permanent magnet, electromagnet).

[0101] <Culture container> The culture vessel 57 has the same configuration as the culture vessel 7 .

[0102] <Piping port> The piping port 58 has a configuration similar to that of the piping port 8 . 11 , a fitting 61 is attached to the front portion of the piping port 58, and one end of a nozzle 65 is connected to the fitting 61 inside the piping port 58, with the culture medium being supplied from the other end of the nozzle 65. A fitting 62 is attached to the right side of the fitting 61 in the front portion of the piping port 58, and one end of a nozzle 66 is connected to the fitting 62 inside the piping port 58, with the culture medium being drained from the other end of the nozzle 66.

[0103] A joint 63 is attached to the rear portion of the piping port 58, and one end of a pipe 67 is connected to the joint 63 inside the piping port 58, and low-oxygen gas is supplied from the other end of the pipe 67. A fitting 64 is attached to the right of the fitting 63 at the rear portion of the piping port 58, and one end of a pipe 68 is connected to the fitting 64 inside the piping port 58, and low-oxygen gas is exhausted from the other end of the pipe 68.

[0104] When the number of well portions 51a is increased, in an integrated structure of only hinge shafts 9 and locking members 10 as in culture device 100, adhesion between well portions 51a in the left-right center of housing 54, which are located away from hinge shafts 9 and locking members 10, and closing member 53 may decrease. According to culture device 102, lever portion 56b of cantilever member 56 presses protrusion 55d of cover 5, pressing not only the short sides but also the long sides of housing 54; in other words, four sides are fastened by cantilever member 56, which further improves adhesion and enables more stable and strong integration.

[0105] In the culture device 102, the first flow path 51e has a main channel 51f, a connecting channel 51g, and a branch channel 51h. Even if the number of wells 51a is increased, a single pump system can uniformly control the amount of medium supplied to each well 51a. As described above, the medium from the main channel 51f does not flow directly into the membrane 6 located at the bottom of the well 51a, reducing the risk of cells being detached during feeding. Furthermore, when the medium is drained, the medium remains by the thickness of the contact member 52, preventing excessive drying.

[0106] The second flow path 51j also has a main flow channel 51k, a connection channel 51m, and a branch flow channel 51n, and the amount of culture medium discharged from each well portion 51a can be uniformly controlled with one pump system.

[0107] In the above embodiment, the well plate 51 has six well portions 51a, but is not limited to this. The well plate of this embodiment can have any number of well portions, such as four or eight. In this case, the configuration corresponding to the well portions 51a (contact members, shape of each flow path, etc.) can be the same as that of the above embodiment.

[0108] <Modification> 14 is a rear view of a modified example of the well plate according to the third embodiment of the present invention. In the figure, the same parts as those in FIG. 12 are given the same reference numerals and detailed description thereof will be omitted. The well plate 51 of the modified example has six wells 51a arranged in the row direction (left and right direction) and four wells arranged in the column direction (front and back direction), for a total of 24 wells 51a. The first flow path 51e has a main flow channel 51f extending in the direction in which the well portions 51a (step portions 51d) are arranged, tributary grooves 51h which branch off from the main flow channel 51f and extend toward the well portions 51a so that their ends are located on the back surface portion B of the well plate 1 and which are narrower than the main flow channel 51f, a storage portion 51i which is provided at the portion where the tributary grooves 51h branch off from the main flow channel 51f and which stores the culture medium, and a connection path 51g which is connected to the main flow channel 51f and has an end which opens on the front or side surface of the well plate 51. In the first flow path 51e, one end of a connection path 51r which extends in the front-rear direction is connected to the storage portion 51i, and the other end of the connection path 51r is provided with the four tributary grooves 51h which respectively extend toward the four step portions 51d. In the second flow path 51j, a U-shaped connecting passage 51s is connected to the storage section 51p, and the connecting passage 51s has four tributary grooves 51n that branch off from the connecting passage 51s and extend toward the four step sections 51d, respectively. The contact member 52 is plate-shaped and is provided with a second through-hole 52a having a diameter larger than the diameter of the bottom side of the first through-hole 51b, corresponding to the well portion 51a, and is in contact with the rear surface of the well plate 51. Even if the number of wells 51a increases to 24, the amount of culture medium supplied to each well 51a can be uniformly controlled with one pump system, and the amount of culture medium discharged from the wells 51a can be uniformly controlled.

[0109] [Embodiment 4] <Culture system> 15 is a schematic diagram showing a culture system 201 including a culture device 103 according to embodiment 4 of the present invention. In the figure, the same parts as those in FIG. 7 are denoted by the same reference numerals and detailed description thereof will be omitted.

[0110] The culture device 103 of the culture system 201 is provided with a piping port 71 instead of the piping port 8 of the culture device 100. The piping port 71 is made of synthetic resin and is cylindrical with a through-hole 71a that penetrates vertically in the center in the front-to-rear direction, and has a protrusion 71b at the bottom whose outer diameter is smaller than that of the upper part. A storage groove 71c that opens to the outside is provided at the center in the vertical direction of the protrusion 71b. A lid may be provided on the upper opening surface of the piping port 71 so that it can be closed as necessary. With the O-ring 12 accommodated in the accommodation groove 71c of the piping port 71, the protrusion 71b is fitted into the culture vessel 7.

[0111] In the culture device 103 , the closure member 3 , the contact member 2 , and the well plate 1 are housed in the housing 4 , and the cover 5 is closed and fixed to the housing 4 by the locking member 10 . Next, the culture vessel 7 is screwed to the well portion 1a of the well plate 1. The aerobic culture medium is passed through the first flow path 1e from the joint 13 and sent to the second space A2. The culture medium on the anaerobic side is passed through the nozzle 25 from the joint 21 and sent to the first space A1.

[0112] Then, with the joints 13 and 14 closed with caps or valves, the culture device 103 is housed in the anaerobic container 70. The anaerobic container 70 may be any airtight container capable of maintaining the oxygen concentration in the system at a predetermined concentration between 3 and 15%, and an anaerobic jar, anaerobic bag, or the like may be used. By housing the culture device 103 in the anaerobic container 70, the first space A1 can be exposed to the anaerobic atmosphere of the anaerobic culture container 70.

[0113] Instead of using the anaerobic vessel 70, the sealed chamber described in Japanese Patent No. 7705637 can also be used. The sealed chamber includes a housing, a lid, and a cantilever member for simultaneously sealing and opening and closing the four sides of the lid. Two joints are attached to the sides of the housing to connect two tubes, allowing low-oxygen gas to be introduced through one joint and discharged through the other joint. After maintaining a predetermined oxygen concentration within this sealed chamber, the culture device 103 is placed inside, allowing the first space A1 to be exposed to the anaerobic atmosphere of the anaerobic vessel 70 in a well-sealed state.

[0114] The culture system 201 can also be used as the culture chamber in the cell culture device of Japanese Patent No. 7691707. The cell culture device includes a culture chamber, a reservoir tank containing an oxygen scavenger, and an O sensor. The reservoir tank has two on-off valves at its inlet and outlet, and the two on-off valves are connected to the culture chamber by tubes. The on-off valves can be used to completely close the inlet and outlet of the reservoir tank, thereby reducing the oxygen concentration in the entire device in a short time and accurately maintaining the oxygen concentration at a desired value. Furthermore, the sealed chamber housing the culture device 103 can be used as the culture system 201 and as the culture chamber of the cell culture device.

[0115] The culture system 201 of the fourth embodiment, in which the culture device 103 is housed in the anaerobic container 70, is compact and can be directly connected to a measuring instrument or the like.

[0116] [summary] A culture device according to one aspect of the present disclosure includes a well plate having one or more wells with a first through-hole and a flow path for guiding a culture medium to or from the well, a plate-shaped contact member having a second through-hole corresponding to the well and having a diameter larger than the diameter of the bottom side of the first through-hole, the contact member being in contact with the back surface of the well plate, a blocking member closing the second through-hole of the contact member, an integration member integrating the well plate, the contact member, and the blocking member, and a blocking member for blocking the first through-hole of the well. The device comprises a membrane that is disposed within the through-hole and that divides the first through-hole of the well portion into a first space and a second space above and below for culturing the cultured organism, and a cylindrical culture vessel that is fitted into the well portion to form the first space inside, and is configured so that when the well plate, the abutting member, and the blocking member are integrated by the integrating member, the culture medium flows between the flow path and the second space via the back surface portion of the well plate that is exposed from the second through-hole of the abutting member.

[0117] According to the above configuration, the well plate, abutment member, and closure member are integrated by an integrating member, which allows for easy assembly and disassembly of the well plate, abutment member, and closure member. In conventional products, the medium flow path is formed by bonding multiple members together with adhesives or the like, making it impossible to disassemble. However, in such products, if dust accumulates in the medium flow path, it is difficult to remove, and the product is intended to be disposable. In the culture device of this aspect, the well plate has a medium flow path, which makes maintenance such as cleaning easy and allows for reuse. In this embodiment of the culture device, the medium flows between the flow channel and the second space via the back surface of the well plate exposed through the second through-hole in the contact member to guide the medium to the well portion. This configuration changes the flow direction of the medium from the flow channel to the second space as it passes through the back surface of the well plate. As shown in the cross-sectional views of Figures 4 and 9(D), the medium initially flows downward and then laterally. This reduces the force of the medium as it enters the medium storage space, which is the space surrounded by the second through-hole and the blocking member. This reduces the impact on cells on the membrane compared to conventional culture devices that allow the medium to flow vertically. Furthermore, when the medium is drained from the medium storage space, the medium remains in the thickness of the contact member, preventing excessive drying.

[0118] In one aspect of the culture device of the present disclosure, the well plate has a plurality of well portions, and the flow path has a first flow path that supplies the culture medium to the well portions and a second flow path that discharges the culture medium from the well portions, and the first flow path and the second flow path each have a main flow channel that is provided on the back surface of the well plate and extends in the direction in which the well portions are arranged, a tributary channel that branches off from the main flow channel and extends toward the well portions so that its end is located within the back surface of the well plate and is narrower than the main flow channel, a storage section that is provided at the point where the tributary channel branches off from the main flow channel and stores the culture medium, and a connecting channel that is connected to the main flow channel and has an end that opens on the surface or side of the well plate.

[0119] In the above-described embodiment, when the flow path has a branch channel branching from the main channel and extending toward the well portion so that its end is located within the back surface of the well plate, the medium can be ejected from the branch channel with a simple configuration and flow into the second space via the back surface. Because the end of the branch channel is located "within" the back surface, the medium flows over a weir between the end of the branch channel and the inner circle of the lower end of the well portion, reducing its momentum and bypassing it to enter the second space. This prevents the liquid flow from affecting cells on the membrane, penetrating the membrane and affecting cells such as enterobacteria present in the center of the second space, or damaging cells seeded on the closure member. Furthermore, when a well plate has multiple well portions, a single system with a single pump can be controlled to uniformly deliver medium to each well portion, thereby simplifying the overall configuration.

[0120] The width of the tributary channel may be narrower than the width of the main channel. With this configuration, the flow rate from the main channel to the tributary channel can be restricted while ensuring the flow rate in the main channel. For example, when there are multiple wells, it is possible to prevent a large amount of medium from flowing into a well that is closer to the connection path on the culture medium supply side, and to set the culture medium to flow more uniformly at a required flow rate into each well.

[0121] The flow path may also be configured to include a reservoir for storing the culture medium at the point where the tributary channel branches off from the main channel. With this configuration, the culture medium is stored in the reservoir before flowing between the main channel and the tributary channel, and the reservoir acts as a buffer, further stabilizing the flow rate between the main channel and the tributary channel. Furthermore, since the culture medium flows toward each well with a stabilized flow rate, the flow rate to each well can be controlled to a constant level, allowing the culture medium to flow more uniformly at the required flow rate.

[0122] The well plate may have a plurality of wells, and the main flow channel may extend in the direction in which the wells are arranged. With this configuration, multiple cultures can be cultured simultaneously, and because the main flow channel extends in the direction in which the wells are arranged, the structure of the flow path connecting the main flow channel to each well can be made more efficient, and the medium can be supplied in a single system (with a single pump) without supplying the medium to each well, allowing the medium to flow more uniformly toward each well.

[0123] The flow path may be configured to include a first flow path that supplies the culture medium to the well portion and a second flow path that discharges the culture medium from the well portion. With this configuration, the supply and discharge of the culture medium can be performed using only two systems: the first flow path that supplies the culture medium to the well portion and the second flow path that discharges the culture medium from the well portion. Conventionally, the supply and discharge of the culture medium have been controlled individually for each well portion by switching using solenoid valves or the like, i.e., by using multiple systems, but this has resulted in increased costs and complicated operations. Furthermore, if the first flow path and the second flow path have the same configuration, the same function can be achieved regardless of which one the culture medium-filled container is connected to.

[0124] In conventional culture devices, when feeding or draining culture medium to or from the culture device, a pump is placed for each well, or when using only one pump, a solenoid valve is placed for each well to individually control the flow rate. This is one of the reasons why culture systems, in which the culture device is connected to a culture medium-filled container and a drainage container by piping, become large-scale. In this embodiment, by finely setting the flow path as described above, it is possible to control the flow rate to a constant level with just one pump, without the need for a solenoid valve.

[0125] A culture device according to one aspect of the present disclosure has a fitting attached to the end of the connection path at one end and a pipe at the other end, and the fitting may be configured to be able to block fluid.

[0126] According to the above configuration, by attaching a fitting to the end of the connection path of the well plate and connecting piping, culture medium can be supplied to the well portion or discharged from the well portion. In other words, the piping can be connected at the point where the fitting is attached to the well plate, and there is no need to connect piping to each well portion of the well plate, which simplifies the piping connection operation, simplifies the piping connection configuration, and reduces the installation space for the piping, culture medium filling container, and discharge container. By configuring the fitting to block fluid, the flow path and the second space can be easily sealed. An example of a fitting that can block fluid is a "stop fitting" type fitting. When such a fitting that can block fluid is used, it opens only when the piping is connected and closes when the piping is removed, so the second space can be made into an airtight space, and the culture can be suspended and the device can be transported. Note that such a fitting may be provided midway through the piping rather than being directly attached to the well plate.

[0127] a housing having a first side portion corresponding to the first side portion of the cover and a second side portion opposite the first side portion, the housing containing the well plate, the abutting member, and the closing member; a hinge shaft portion connecting the first side portion of the cover to the first side portion of the housing; and a locking portion locking the second side portion of the cover to the second side portion of the housing, the housing being configured such that the cover can be locked to the well plate when the locking portion is engaged with the closing member; The housing may be configured so that the abutting member and the blocking member press against the upper surface of the well plate and integrate the well plate, the abutting member, and the blocking member contained in the housing, the first through-hole of the well plate having a female thread at the top of its inner surface and a step portion below the female thread that is annular in diameter in plan view and smaller than the female thread portion of the first through-hole, the membrane is placed on the step portion and has a sealing member that abuts against the upper surface of the membrane placed on the step portion, the culture vessel having a male thread on its outer surface that engages with the female thread, and the lower end of the culture vessel presses against the sealing member by tightening the male thread onto the female thread, thereby connecting the lower end of the culture vessel, the sealing member, and the membrane.

[0128] According to the above configuration, the membrane and the seal member can be placed on the step portion, which allows for easy installation of the membrane and the seal member. Furthermore, in the case of a configuration in which the connection is made by tightening the male thread of the culture vessel to the female thread of the well portion, the connection between the culture vessel and the well portion can be made simply and effectively with a higher airtightness, thereby improving the isolation between the first space and the second space.

[0129] Furthermore, with a simple configuration, the culture medium or gas can be introduced into or discharged from the first space. For example, if a pipe for introducing a gas such as an anaerobic (low oxygen) gas into the culture medium in the first space within the culture vessel and a pipe for discharging the gas from the culture vessel are attached to the piping port, and if anaerobic gas is refluxed as the gas, the refluxed anaerobic gas will create an anaerobic environment in the culture medium, and the operation of making the culture medium anaerobic by degassing the refluxed culture medium will be unnecessary.

[0130] With the above-described structure including the integrating member, the well plate, the contact member, and the closure member are easily integrated by closing the cover on the housing and locking it with the locking portion. Furthermore, the cover presses against the top surface of the well plate through surface contact, so the above-described integration can be achieved more stably and strongly.

[0131] In one embodiment of the culture device of the present disclosure, the integrated member further includes a cantilever member having protrusions provided at the centers of two sides adjacent to the first side of the cover, two support portions provided on the two sides corresponding to the two sides of the housing closer to the hinge shaft than the two protrusions, two lever portions connected to the two support portions, and a shaft portion connecting the two lever portions, and when the second side of the cover is engaged with the second side of the housing, the lever portions press against the protrusions, and the cover is fixed to the housing.

[0132] With this configuration, the cantilever members simultaneously press and fasten the four sides, including the short sides, improving adhesion and enabling a more stable and strong integration. Even if the number of wells is increased, the long sides are not tightly pressed down, preventing leakage of culture medium.

[0133] A culture system according to one aspect of the present disclosure may include a culture device according to any of the above aspects, a culture medium filling container filled with culture medium to be supplied to the culture device, a drainage container for containing culture medium discharged from the culture device, and anaerobic generation means for generating low-oxygen gas.

[0134] According to the above configuration, a fitting is attached to the end of the connecting passage opening on the surface or side of the well plate of the culture device, and the fitting is connected to the medium-filling container or the drainage container via piping, allowing culture medium to be supplied to or drained from the well. In other words, the piping can be connected at the surface or side of the well plate where the fitting is attached, eliminating the need to connect piping to each well of the well plate. This simplifies the piping connection operation and reduces the installation space required for the piping, medium-filling container, and drainage container. Since the piping, medium-filling container, and drainage container can be placed above or to the side of the culture device, the flexibility of the installation location of the culture device can be improved. For example, the culture device can be configured to be placed directly on a laboratory table, microscope stage, etc. The culture system according to this configuration has a simple configuration and can be made compact.

[0135] A culture system according to one aspect of the present disclosure includes a culture device according to any of the above aspects, a sealed container that houses the culture device, and an anaerobic generation means that is connected to the sealed container, can be closed, and generates low-oxygen gas.

[0136] According to the above configuration, by making the anaerobic generation means closable, the oxygen concentration in the entire device can be reduced in a short period of time, and the oxygen concentration can be maintained at any desired value with high accuracy.

[0137] A well plate according to one embodiment of the present disclosure may have one or more well portions each having a first through-hole, a main flow groove provided on the back surface, and a culture medium flow path including a connecting path connected to the main flow groove and having an end opening on the front surface or side surface, and may be configured such that an abutting member having a second through-hole with a diameter larger than the diameter of the bottom side of the first through-hole is abutted against the back surface of the well plate, and when the second through-hole is blocked by a blocking member that blocks the second through-hole, the culture medium flows between the main flow groove and the well portion via the back surface portion of the well plate that is exposed from the second through-hole in the abutting member.

[0138] According to the above configuration, the flow path includes a groove formed on the back surface of the well plate and a connecting channel with an open end, which facilitates maintenance such as cleaning and enables reuse. Furthermore, the culture medium is configured to flow between the main flow groove and the well portion via the back surface of the well plate exposed through the second through-hole of the abutting member, and does not flow directly to the well portion, reducing the impact of the liquid flow on the cells on the membrane. Furthermore, when draining the liquid, the culture medium remains in an amount equal to the thickness of the abutting member, making it possible to prevent the membrane from drying out excessively.

[0139] The technical scope of the present invention is not limited to the aspects described in the above-mentioned embodiments, etc. One or more of the requirements described in the above-mentioned embodiments, etc. may be omitted. Furthermore, the requirements described in the above-mentioned embodiments, etc. may be combined as appropriate.

[0140] In the above embodiment, the culture device of the present invention is described as being used as a co-culture device, but is not limited thereto. For example, it may be used as a culture device that uses a three-dimensional culture sheet instead of a membrane and supplies different culture media (or atmospheres) to the upper first space A1 and the lower second space A2. That is, the membrane includes a three-dimensional culture sheet. Also, the culture media may be the same in the upper first space A1 and the lower second space A2.

[0141] Furthermore, the culture vessel is not limited to being screwed to the well portion, as long as the culture vessel is fitted airtightly to the well portion.

[0142] In addition, the cover may be fixed to the housing, and in addition to the well plate, abutment member, and support member being integrated, a second cover may be provided, and the second cover may be fixed to the first cover, thereby fixing the piping port and culture vessel to the well portion.

[0143] In the above description, the culture devices 100-103 and the culture systems 200-201 have been described as having an anaerobic atmosphere (anaerobic environment) on the upper side (first space A1) and an aerobic atmosphere (aerobic environment) on the lower side (second space A2). However, the present invention is not limited to this example and can be used for other purposes. For example, the environments of the first space A1 and the second space A2 are both arbitrary. For example, the environments of the first space A1 and the second space A2 may both be aerobic environments. In this case, the culture devices 100-103 and the culture systems 200-201 can also be used for various experiments, such as cell migration, movement, and infiltration from the first space A1 to the second space A2.

[0144] In addition, the length and width of the bottom surface of the housings 4, 54 of the culture devices 100 to 103 may be 85 mm x 127 mm, the same as a commercially available general well plate (or microplate), which allows them to be mounted on a microscope plate holder or various test devices, making observation and analysis easier. The culture device of the present invention may also be configured to measure transepithelial electrical resistance (TEER). For example, the first space A1 and the second space A2 may each be provided with electrodes used to measure transepithelial electrical resistance (TEER), and the electrical resistance flowing between the electrodes may be measured by an external device or an internal device of the culture device. [Explanation of symbols]

[0145] 1. 51-well plate 1a, 51a well section 1b, 51b 1st through hole 1c, 51c female thread 1d, 51d stepped section 1e, 51e First flow path 1f, 1k, 51f, 51k Main ditch 1g, 1m, 51g, 51m connecting route 1h, 1n, 51h, 51n tributary groove 1i, 1p, 51i, 51p Reservoir 1j, 51j Second flow path 2, 52 abutting member 3, 53 Closure member 4, 54 cabinet 5, 55 cover 6 Membranes 7, 57 Culture vessel 7b Male thread 8, 58, 71 Piping ports 9 Hinge shaft 10 Hooks 11, 12 O-ring 13, 14, 21, 22, 23, 24, 59, 60, 61, 62, 63, 64 Fittings 15, 18, 27, 28, 29, 32, 35, 38, 67, 68 Piping 16, 19, 30, 33, 36, 39 Pumps 17, 31 Medium-filled container 20, 34 Drainage container 25, 26, 65, 66 nozzles 40 Intestinal epithelial cells 41 Intestinal bacteria 70 Anaerobic chamber A1 1st space A2 2nd space A3 Medium storage space A4: The lower area of ​​the back surface of the well plate exposed through the second through-hole B. The back surface of the well plate exposed through the second through-hole U Integrated member 100, 101, 102, 103 Culture equipment 200, 201 Culture system

Claims

1. A well plate having one or more well portions with a first through-hole penetrating in the vertical direction, and a flow path for guiding a culture medium to the well portion or for guiding the culture medium from the well portion; a plate-shaped contact member that corresponds to the well portion, has a second through-hole with a diameter larger than the diameter of the bottom side of the first through-hole, and is in contact with the back surface of the well plate; a blocking member that blocks the second through hole of the contact member; an integration member that integrates the well plate, the abutment member, and the closing member; a membrane disposed within the first through-hole of the well portion so as to vertically divide the first through-hole into an upper first space for culturing a cultured organism and a lower second space; a cylindrical culture vessel that forms the first space therein and is fitted into the well portion; Equipped with When the well plate, the abutting member, and the closing member are integrated by the integrating member, a central axis of the second through hole of the abutting member is substantially coaxial with a central axis of the first through hole of the well plate, and a back surface portion of the well plate is exposed from the second through hole; an end of the flow path for guiding the culture medium to the well portion or for guiding the culture medium from the well portion is located at a position within the back surface portion of the well plate exposed from the second through-hole, and does not reach the first through-hole; A culture device configured so that the culture medium guided to the well portion enters the second through-hole located below the end of the flow path that guides the culture medium to the well portion, and flows into the second space via the back surface portion of the well plate exposed from the second through-hole.

2. the well plate has a plurality of well portions, The flow path is a first flow path that supplies the culture medium to the well portion and a second flow path that discharges the culture medium from the well portion; The first flow path and the second flow path each include: a main flow groove provided on the rear surface of the well plate and extending in the direction in which the well portions are arranged; a branch channel that branches off from the main channel, extends toward the well portion, and has an end that is located at a position that does not reach the first through-hole within the back surface portion of the well plate exposed from the second through-hole, and has a width narrower than that of the main channel; a storage section provided at a portion where the tributary channel branches from the main channel and in which the culture medium is stored; a connecting channel connected to the main channel and having an end opening on the surface or side of the well plate; The culture device according to claim 1 ,

3. A piping port is provided, which is detachable from the opening of the culture vessel and which introduces or discharges a medium or a gas into or from the culture vessel, The integrated member is a cover having a first side portion and a second side portion opposite to the first side portion, the cover being in surface contact with the top surface of the well plate to cover the well plate while the well portion is exposed; a housing having a first side portion corresponding to the first side portion of the cover and a second side portion opposing the first side portion, the housing accommodating the well plate, the contact member, and the closing member; a hinge shaft portion connecting the first side portion of the cover to the first side portion of the housing; a locking portion that locks the second side portion of the cover to the second side portion of the housing; and When the cover is locked by the locking portion, the cover presses the upper surface of the well plate to integrate the well plate, the contact member, and the closing member housed in the housing, The first through-hole of the well plate is There is a female thread on the top of the inner surface, A step portion having an annular shape and a smaller diameter than the portion where the female thread is provided in a plan view is provided below the portion where the female thread is provided, The membrane is placed on the step, a seal member that is in contact with an upper surface of the membrane that is placed on the step portion; The culture vessel has an outer surface provided with a male screw that mates with the female screw, The culture device according to claim 1 or 2, wherein the culture vessel is configured such that, by tightening the male screw onto the female screw, the lower end of the culture vessel presses against the sealing member, thereby connecting the lower end of the culture vessel, the sealing member, and the membrane.

4. The integrated member is protrusions provided at the centers of two sides of the cover adjacent to the first side; a cantilever member including two support portions provided on two sides corresponding to the two sides of the housing closer to the hinge shaft portion than the two protrusion portions, two lever portions connected to the two support portions, and a shaft portion connecting the two lever portions; and The culture device according to claim 3 , wherein when the second side of the cover is engaged with the second side of the housing, the lever presses the protrusion, and the cover is fixed to the housing.

Images