JPWO2020017067A1 - Attachment for perfusion culture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an attachment for mounting on an existing open-type culture vessel and performing perfusion culture with the culture vessel sealed. SOLUTION: The attachment for perfusion culture of the present invention is attached to a culture vessel 10A having an opening 103 at the upper end and having at least one bottomed tubular container portion 100 extending in the vertical direction, and the opening 103 is provided. Attachment 11A for closing and sealing the inner space of the container portion 100 to perform perfusion culture, which is flexible and is separate from the seal member 13 and the seal member 13 which come into contact with the container portion 100. A base member 12 made of a hard material, provided with a base member 12 in contact with the sealing member 13, a bottom wall 121 that fits inside the container 100 and protrudes downward, and one or both of the sealing member 13 and the base member 12. It is formed and has a plurality of attachment inner flow paths 15 and 16 having one end communicating with the outside and the other end communicating with the inner space of the container portion 100.

Description

The present invention relates to an attachment for perfusion culture in an open culture vessel, which is placed in a closed state and closed.

Although the evolution of cell culture technology is required for the practical application of regenerative medicine, existing research products (well plates, dishes) are currently used as culture containers at research and development sites for many cell products. ing. The background to this is that containers with a proven track record in their performance in conventional research processes are preferred. However, these culture containers are designed for culturing in an aerated state, and even if they have a lid, they cannot be sealed only by placing them. These conventional open-type culture vessels are highly versatile for cell culture applications for research purposes, but for cell culture applications for medical purposes, work is performed while opening and closing the lid manually. Therefore, there was a risk of contamination. There are also problems in ensuring the quality of cells and human labor and cost due to the need for specialized skills in handling.

Recently, there are automatic culture devices that eliminate the trouble of manual cell seeding, culture medium exchange, and recovery, and automatic perfusion culture devices that supply the culture solution at a constant rate and at the same time extract the same amount of culture solution. Various have been developed (see, for example, Patent Documents 1 and 2). For example, it is easy for an AI robot device that has mastered the handiwork of a researcher to operate an existing culture vessel, but a device that reproduces human operations is inevitably large-scale, so in a general small or medium-sized laboratory. If so, there is an issue of introduction cost. On the other hand, with a simple device, there is a problem that an existing culture container cannot be used because a dedicated container suitable for the device is required.

International Publication WO2016 / 170623 JP-A-2017-79633

The present invention has been conceived under such circumstances, and includes various culture solution exchanges and perfusion cultures by mounting the culture vessel in an existing open culture vessel and keeping the culture vessel in a sealed state. The main task is to provide an attachment suitable for the incubator.

In order to solve the above problems, the following technical means are adopted in the present invention.

The perfusion culture attachment provided by the present invention is attached to a culture vessel having an opening at one end in the first direction and having at least one bottomed tubular container portion extending in the first direction. , An attachment for performing perfusion culture by closing the opening and sealing the inner space of the container portion, which is flexible and is separate from the sealing member which comes into contact with the container portion and the sealing member. A base member made of a hard material of the body, provided with a base member in contact with the seal member, a protruding portion that fits inside the container portion and protrudes toward the other side in the first direction, and the seal member and the base member. It is formed on either one or both, and has a plurality of internal flow paths in the attachment, one end of which leads to the outside and the other end of which leads to the inner space of the container portion.

In a preferred embodiment, the base member comprises a bottom wall constituting the protrusion, a tubular side wall rising from the bottom wall, and an annular shape extending radially outward from the end of the side wall. The sealing member has a flange and a plate portion that is interposed between the flange and the peripheral edge portion of the opening in the first direction and can be in contact with all the peripheral edges of the opening.

In a preferred embodiment, the sealing member has a tubular portion extending from the plate portion to the other side in the first direction, and the tubular portion is press-fitted between the container portion and the side wall. Will be done.

In a preferred embodiment, the base member has a bottom wall forming the protrusion and a tubular side wall that rises from the bottom wall, and the seal member has the container part and the side wall. It is press-fitted between.

In a preferred embodiment, the seal member has a seal member bottom wall portion constituting the protrusion, and the seal member bottom wall portion has a thickness relatively smaller than that of other portions. It has a transparent thin-walled portion, and a through hole is formed in the bottom wall of the base member so as to overlap the thin-walled portion in the first direction and penetrates in the first direction.

In a preferred embodiment, the seal member includes a seal member bottom wall portion constituting the projecting portion, a tubular portion rising from the seal member bottom wall portion, and a radial portion outside the end portion of the tubular portion. The seal member bottom wall portion has an annular plate portion extending in the direction, and the seal member bottom wall portion has a transparent thin-walled portion having a thickness relatively smaller than that of other portions. , Has an annular flange that can come into contact with all the upper surfaces of the plate portion.

In a preferred embodiment, the surface of the bottom wall facing the other side in the first direction is hydrophilized or hydrophobized.

In a preferred embodiment, the base member is transparent.

In a preferred embodiment, one end of each of the attachment inner channels points in a direction that is substantially perpendicular to the first direction.

In a preferred embodiment, the plurality of attachment inner flow paths include a fluid introduction flow path for introducing a fluid from the outside into the inner space of the container portion, and a fluid introduction flow path for introducing the fluid from the inner space of the container portion to the outside. Includes a fluid derivation channel for derivation.

In a preferred embodiment, the culture vessel comprises a plurality of vessel portions that are spaced apart from each other in the in-plane direction perpendicular to the first direction, and the base member is the container portion of the container portion. A bottom wall that fits inside and constitutes the protrusion, and a plurality of first bottomed cylinders, each of which has a tubular side wall that rises from the bottom wall, and an integral body that extends outward from the end of each side wall. The seal member is interposed between the pressing portion and the peripheral edge portion of each opening in the first direction, and is interposed with all the peripheral edges of the respective openings. It has a plate part that can come into contact with it.

In a preferred embodiment, each of the sealing members has a plurality of tubular portions each extending from the plate portion to the other side in the first direction and into which the first bottomed tubular portion can be inserted. The tubular portion is press-fitted between the container portion and the side wall.

In a preferred embodiment, the base member is transparent.

In a preferred embodiment, the culture vessel comprises a plurality of vessel portions spaced apart from each other in the in-plane direction perpendicular to the first direction, and the base member is a plate-shaped pressing portion. The seal member has a plate portion that is interposed between the pressing portion and the peripheral edge portion of each of the openings in the first direction, and is capable of contacting all the peripheral edges of the respective openings, and the plate portion. It has a tubular portion extending from the first direction to the other side, and a plurality of second bottomed tubular portions each having a bottom wall portion of a seal member constituting the protruding portion.

In a preferred embodiment, each of the bottom wall portions of the sealing member has a transparent thin-walled portion having a thickness relatively smaller than that of the other portions, and each of the pressing portions has the first one. A plurality of through holes are formed so as to overlap with any of the thin-walled portions in a directional view and penetrate in the first direction.

In a preferred embodiment, one end of each of the attachment inner channels points in a direction that is substantially perpendicular to the first direction.

In a preferred embodiment, each of the attachment inner flow paths is formed in the seal member, and one end thereof is formed in the seal member inner flow path facing one side in the first direction and the holding portion. It includes a flow path in the base member that communicates with the one end of the flow path in the seal member and penetrates in the first direction.

In a preferred embodiment, the plurality of attachment inner flow paths include a plurality of fluid introduction flow paths for introducing a fluid into the inner space of each of the plurality of container portions from the outside, and the plurality of container portions. Includes a plurality of fluid lead-out channels for deriving fluid from each of the inner spaces to the outside.

In a preferred embodiment, the sealing member has at least one communication flow path that allows the inner spaces of the container to communicate with each other.

In a preferred embodiment, the plurality of attachment inner flow paths are a fluid introduction flow path for introducing a fluid from the outside into the inner space of the first container portion among the plurality of container portions, and the above. Among the plurality of container portions, the connecting flow path includes a fluid lead-out flow path for guiding a fluid from the inner space of the second container portion to the outside, and the connecting flow path is the above-mentioned above of the first container portion. It is provided so as to communicate from the inner space through the inner space of the adjacent container portion and sequentially to the inner space of the second container portion.

In a preferred embodiment, the connecting flow path includes a groove formed on a surface of the plate portion facing the one side in the first direction, and the groove is formed in the plate portion. By overlapping the pressing portions, the one-sided end in the first direction is closed to form a part of the connecting flow path.

Other features and advantages of the present invention will become more apparent with the detailed description given below with reference to the accompanying drawings.

It is a schematic block diagram which shows an example of the perfusion culture system which was configured with the attachment for perfusion culture which concerns on this invention. It is an exploded view of the perfusion culture system shown in FIG. It is a top view which shows the 1st Embodiment of the attachment for perfusion culture which concerns on this invention, and the culture container unit provided with this. It is sectional drawing which follows the IV-IV line of FIG. It is sectional drawing which follows the VV line of FIG. It is an exploded perspective view of the culture vessel unit shown in FIG. It is the same cross-sectional view as FIG. 5, and shows the state which contained the contents in the culture vessel unit. It is a top view which shows the modification of the culture vessel unit and the attachment for perfusion culture shown in FIG. It is sectional drawing which follows the IX-IX line of FIG. It is an exploded perspective view of the culture vessel unit shown in FIG. It is the same cross-sectional view as FIG. 4 which shows the modification of the culture vessel unit and the attachment for perfusion culture shown in FIG. It is the same cross-sectional view as FIG. 4 which shows the modification of the culture vessel unit and the attachment for perfusion culture shown in FIG. It is the same cross-sectional view as FIG. 4 which shows the modification of the culture vessel unit and the attachment for perfusion culture shown in FIG. It is a top view which shows the modification of the culture vessel unit and the attachment for perfusion culture shown in FIG. It is sectional drawing which follows the XV-XV line of FIG. It is sectional drawing which follows the XVI-XVI line of FIG. It is an exploded perspective view of the culture vessel unit shown in FIG. It is a top view which shows the 2nd Embodiment of the attachment for perfusion culture which concerns on this invention, and the culture container unit provided with this. It is a perspective view of the culture vessel unit shown in FIG. It is a perspective view of the state in which the attachment of the culture vessel unit shown in FIG. 18 is appropriately disassembled. It is sectional drawing which follows the XXI-XXI line of FIG. It is a top view which shows the 3rd Embodiment of the attachment for perfusion culture which concerns on this invention, and the culture container unit provided with this. It is a perspective view of the culture vessel unit shown in FIG. 22. It is an exploded perspective view of the culture vessel unit shown in FIG. 22. FIG. 2 is a cross-sectional view taken along the line XXV-XXV of FIG. It is a top view which shows the 4th Embodiment of the attachment for perfusion culture which concerns on this invention, and the culture container unit provided with this. It is a perspective view of the culture vessel unit shown in FIG. 26. FIG. 6 is an exploded perspective view of the culture vessel unit shown in FIG. 26. FIG. 6 is a cross-sectional view taken along the line XXIX-XXIX of FIG. 26. FIG. 6 is a cross-sectional view taken along the line XXX-XXX of FIG. 26. It is a top view which shows the 5th Embodiment of the attachment for perfusion culture which concerns on this invention, and the culture container unit provided with this. FIG. 3 is a cross-sectional view taken along the line XXXII-XXXII of FIG. FIG. 3 is a cross-sectional view taken along the line XXXIII-XXXIII of FIG. It is an exploded perspective view of the culture vessel unit shown in FIG. 31.

Hereinafter, preferred embodiments of the present invention will be specifically described with reference to the drawings.

1 and 2 show an example of a perfusion culture system configured with the attachment for perfusion culture according to the present invention. The perfusion culture system A10 of the present embodiment includes a culture vessel unit 1A, a culture solution container 2, a vacuum vessel unit 3, a first flow path 41, a second flow path 42, a first connection means 51, and the like. A second connecting means 52 and a narrowed portion 6 are provided. Although the details will be described later, the perfusion culture system A10 supplies the new culture solution contained in the culture solution container 2 to the culture container unit 1A, and collects the old culture solution in the culture container unit 1A into the vacuum container unit 3. It is for doing.

As shown in FIGS. 1 and 4 to 6, in the present embodiment (first embodiment), the culture vessel unit 1A includes a culture vessel 10A and an attachment 11A. As the culture container 10A, an existing open culture container such as a dish (Petri dish) or a well plate is used, and in the present embodiment, the case where the culture container 10A is a dish is shown. The culture container 10A includes one container portion 100, and the container portion 100 has a bottom plate 101 and a cylindrical side plate that stands up from the peripheral edge of the bottom plate 101 and has an opening 103 at the upper end (one side end in the first direction). It is composed of 102 and.

The upper surface of the bottom plate 101 is a flat cell culture surface for culturing cells. The cell culture surface (upper surface of the bottom plate 101) is appropriately subjected to surface treatment for improving cell adhesion, if necessary. Examples of the surface treatment include corona discharge treatment and plasma treatment (hydrophilization treatment).

The attachment 11A is attached to the culture container 10A and is for closing the opening 103 of the culture container 10A (container portion 100) to seal the inner space of the container portion 100. The attachment 11A is an example of the attachment for perfusion culture according to the present invention, and includes the base member 12 and the seal member 13 in the present embodiment.

The base member 12 has a bottom wall 121, a cylindrical side wall 122 extending upright from the peripheral edge of the bottom wall 121, and a flange 123. The bottom wall 121 fits inside the side plate 102 in the culture vessel 10A at a distance from the side plate 102, and protrudes downward (the other side in the first direction). The bottom wall 121 constitutes the protruding portion referred to in the present invention. The flange 123 extends radially outward from the upper end of the side wall 122 and has an annular shape. The base member 12 having such a configuration overlaps all the openings 103 of the culture vessel 10A in a plan view.

The culture vessel 10A and the base member 12 are made of, for example, a translucent or transparent hard plastic material. As the plastic material, for example, polystyrene, methylpentene, polycarbonate, cycloolefin polymer, cycloolefin copolymer, and other materials having transparency are preferably used, but the plastic material is not limited thereto.

In the present embodiment, at least the bottom plate 101 of the culture vessel 10A and the bottom wall 121 of the base member 12 are transparent. As a result, the bottom plate 101 and the bottom wall 121 are sites where the inner space of the culture vessel 10A can be visually recognized from the outside.

The seal member 13 is, for example, a rubber molded product, and has a cylindrical portion 131 extending vertically, a plate portion 132, and an annular protrusion 133 as shown in FIGS. 4 and 5. The seal member 13 is interposed between the side plate 102 of the culture container 10A (container portion 100) and the side wall 122 or the flange 123 of the base member 12. The cylindrical portion 131 extends downward (the other side in the first direction) from the inner peripheral portion of the plate portion 132, and is fitted onto the side wall 122 of the base member 12. The natural inner diameter of the cylindrical portion 131 is slightly smaller than the outer diameter of the side wall 122, whereby the cylindrical portion 131 (seal member 13) is pressed and held by the side wall 122 (base member 12). The annular protrusion 133 projects radially outward from the outer circumference of the cylindrical portion 131, and is in pressure contact with the inner peripheral surface of the side plate 102 when the attachment 11A is attached to the culture vessel 10A. As a result, when the attachment 11A is attached, the cylindrical portion 131 is press-fitted between the side plate 102 (container portion 100) and the side wall 122 (base member 12). The plate portion 132 extends radially outward from the upper end of the cylindrical portion 131 and has an annular shape. The plate portion 132 overlaps with the flange 123 of the base member 12 in a plan view. Further, when the attachment 11A is attached to the culture vessel 10A, the plate portion 132 comes into contact with all the peripheral edges of the opening 103 in the culture vessel 10A (container portion 100). With such a configuration, the base member 12 and the seal member 13 (attachment 11A) seal the inner space of the culture container 10A (container portion 100).

The seal member 13 has a fluid introduction flow path 15 and a fluid lead-out flow path 16. The fluid introduction flow path 15 is a flow path for introducing a fluid such as a culture solution from the outside of the container portion 100 into the inner space of the container portion 100. In the present embodiment, the fluid introduction flow path 15 is composed of holes and grooves formed in the seal member 13. More specifically, the fluid introduction flow path 15 has a configuration in which a hole formed in the plate portion 132 and a groove formed in the inner surface of the cylindrical portion 131 are connected to each other. One end of the fluid introduction flow path 15 is a fluid inflow port 151 leading to the outside, and the other end of the fluid introduction flow path 15 is connected to the inner space of the closed culture container 10A (container portion 100). The fluid inlet 151 faces a direction substantially perpendicular to the vertical direction (first direction).

The fluid lead-out flow path 16 is a flow path for leading out the fluid from the inner space of the container portion 100 to the outside of the container portion 100. In the present embodiment, the fluid lead-out flow path 16 is provided on the opposite side of the fluid introduction flow path 15 with the center of the seal member 13 interposed therebetween in a plan view. In the present embodiment, the fluid lead-out flow path 16 is configured to connect a hole formed in the plate portion 132 and a groove formed in the inner surface of the cylindrical portion 131. One end of the fluid lead-out flow path 16 is a fluid outlet 161 that leads to the outside, and the other end of the fluid lead-out flow path 16 leads to the inner space of the closed culture container 10A (container portion 100). The fluid outlet 161 faces a direction substantially perpendicular to the vertical direction (first direction). The fluid inlet 151 and the fluid outlet 161 face opposite to each other. The fluid introduction flow path 15 and the fluid lead-out flow path 16 constitute the attachment inner flow path referred to in the present invention.

The sealing member 13 is made of a flexible and elastic material. Examples of the material constituting the seal member 13 include silicone rubber, natural rubber, urethane rubber, and elastomer resin. Although the details will be described later, considering the contact between the sealing member 13 and the contents (for example, a culture solution), the material of the sealing member 13 is more preferably a medical grade silicone rubber having no cytotoxicity and biocompatibility. .. Regarding the hardness of the seal member 13, for example, the rubber hardness is preferably about 20 to 40 degrees.

The culture solution container 2 shown in FIG. 1 is a closed container body for accommodating a replacement culture solution. The culture solution container 2 is provided with a lead-out port 21 for leading out the contents. In the present embodiment, the culture solution container 2 is preferably configured to be flexible and changeable in volume. Examples of such a culture solution container 2 include a bellows-shaped container, a film-shaped container, a syringe, a laminated peeling bottle, and the like. In this embodiment, the case where the culture solution container 2 is a bellows-shaped container is shown.

The inside of the vacuum vessel unit 3 is adjusted to a negative pressure state, and the culture solution or the like inside the culture vessel unit 1A is recovered. In the present embodiment, the vacuum container unit 3 includes a pipe body 31 and a plug 32. The tubular body 31 has a bottomed cylindrical shape having an open end 311 at the tip. The plug 32 closes the opening end 311, and the inside of the sealed tube 31 is adjusted to a negative pressure state. A part of the stopper 32 is made of a rubber material capable of puncturing, for example, a puncture needle. As the vacuum container unit 3 having such a configuration, for example, a general-purpose blood collection tube can be used.

The first flow path 41 is a fluid flow path that connects the culture container unit 1A and the culture solution container 2, and is composed of, for example, a flexible tube. One end of the first flow path 41 can communicate with the inside of the culture vessel unit 1A via the fluid inflow port 151. The other end of the first flow path 41 can communicate with the inside of the culture solution container 2 via the outlet 21.

The first connection means 51 shown in FIG. 1 is provided in the first flow path 41, and connects the culture container unit 1A and the culture solution container 2 in a closed state. Further, the first connecting means 51 can separate the culture container unit 1A and the culture solution container 2 in a sealed state (see FIG. 2). The first connecting means 51 having such a configuration is realized by, for example, a luer lock connection structure having a female luer connector 511 and a male luer connector 512. In the configuration shown in FIGS. 1 and 2, the first flow path 41 is a partial flow path 411 connected to the culture vessel unit 1A (fluid inflow port 151) and a partial flow path connected to the culture solution container 2 (outlet port 21). It is composed of a road 412. Then, for example, a female luer connector 511 is provided at the tip of the partial flow path 411, and a male luer connector 512 is provided at the tip of the partial flow path 412.

The second flow path 42 is a fluid flow path connecting between the culture container unit 1A and the vacuum container unit 3, and is composed of, for example, a flexible tube. One end of the second flow path 42 can communicate with the inside of the culture vessel unit 1A via the fluid outlet 161. A puncture needle 43 capable of puncturing the stopper 32 of the vacuum container unit 3 is attached to the other end of the second flow path 42. The other end of the second flow path 42 can communicate with the inside of the vacuum container unit 3 (tube 31) via the puncture needle 43.

The second connecting means 52 shown in FIG. 1 is provided in the second flow path 42, and connects the culture vessel unit 1A and the vacuum vessel unit 3 in a closed state. Further, the second connecting means 52 can separate the culture container unit 1A and the vacuum container unit 3 in a sealed state (see FIG. 2). The second connecting means 52 having such a configuration is realized by, for example, a luer lock connection structure having a female luer connector 521 and a male luer connector 522. In the configuration shown in FIGS. 1 and 2, the second flow path 42 is composed of a partial flow path 421 connected to the culture vessel unit 1A (fluid outlet 161) and a partial flow path 422 connected to the vacuum vessel unit 3. To. Then, for example, a female luer connector 521 is provided at the tip of the partial flow path 421, and a male luer connector 522 is provided at the tip of the partial flow path 422.

The puncture needle 43 described above is attached to the end of the partial flow path 422. Further, the puncture needle 43 is surrounded by a tubular holder 44 to prevent danger. The inner diameter of the holder 44 is larger than the outer diameter of the pipe body 31, and as can be understood from FIGS. 1 and 2, the pipe body 31 (vacuum container unit 3) is inserted into the holder 44 to insert the plug 32. It is possible to safely puncture the puncture needle 43.

The narrowed portion 6 is provided in at least one of the first flow path 41 and the second flow path 42. The narrowed portion 6 has a portion where the cross section of the flow path is narrowed, and the flow rate of the fluid passing through the narrowed portion 6 is adjusted to be desired.

In the present embodiment, the narrowed portion 6 is composed of a columnar body having micropores penetrating in the longitudinal direction. A fluid (for example, about 0.02 to 0.05 mm) that passes through the narrowed portion 6 by appropriately selecting the pore diameter (for example, about 0.02 to 0.05 mm) and the length of the micropore (dimension in the longitudinal direction: about 5 to 20 mm). The flow rate of the culture solution, etc.) can be adjusted. As shown in FIGS. 1 and 2, in the present embodiment, the narrowed portion 6 is provided in the partial flow path 422 of the second flow path 42.

Next, the operation of this embodiment will be described.

The attachment 11A of the present embodiment accommodates cultured cells and a culture solution in a closed culture container unit 1A configured by mounting on an open culture container 10A, and the culture solution is maintained in a cell culture state. Used to make a replacement. The cultured cells and the culture solution contained in the culture container 10A (container portion 100) are not particularly limited.

In cell culture using the culture vessel unit 1A, first, the cells to be cultured are seeded in the culture vessel 10A (container portion 100), and the inside of the container portion 100 is filled with the culture solution. The seeding of cells may be carried out by removing the attachment 11A from the culture vessel 10A and directly injecting the culture solution containing the cells into the culture vessel 10A, but it can also be carried out by using the perfusion culture system A10. ..

Although detailed illustration will be omitted, when cells are seeded using the perfusion culture system A10, a culture medium container 2 containing a culture medium containing the cells to be cultured is prepared, and the culture medium container 2 is used. Connect to the culture vessel unit 1A. Further, a syringe is prepared in place of the vacuum container unit 3, and the syringe is connected to the culture container unit 1A. Then, by pulling the piston to make the inside of the syringe negative pressure, the culture solution (including cells) in the culture solution container 2 is guided into the culture vessel unit 1A, and the inside of the culture vessel 10A, the culture vessel unit 1A, and the syringe are introduced. Fill the flow path between and the culture medium. FIG. 7 shows a state in which the inside of the culture container 10A (container portion 100) is filled with the culture solution M1. After filling the inside of the culture container unit 1A with the culture solution, the connection between the culture container unit 1A and the culture solution container 2 and the connection between the culture container unit 1A and the syringe are separated from each other.

The cells in the culture vessel unit 1A (culture vessel 10A) adhere to the cell culture surface (upper surface of the bottom plate 101) over time, and proliferation begins. When the upper surface of the bottom plate 101 is subjected to the above-mentioned surface treatment for improving cell adhesion, the cells adhere to the cell culture surface more reliably. Therefore, such a configuration is preferable for the proper progress of cell culture in the culture vessel unit 1A.

With respect to the culture solution in the culture vessel unit 1A, nutrients decrease as the culture cells proliferate, and waste products excreted from the culture cells increase. Therefore, it is necessary to replace the culture solution in the culture vessel unit 1A as appropriate. When exchanging the culture solution in the culture container unit 1A during cell culture, a partial flow having a culture solution container 2 containing the replacement culture solution, a vacuum container unit 3, a constriction portion 6, and a puncture needle 43. Road 422 and is prepared (see FIG. 2). Then, the first connecting means 51 between the culture vessel unit 1A and the culture solution container 2 and the second connecting means 52 between the culture vessel unit 1A and the vacuum vessel unit 3 are separated from each other (FIG. FIG. 2) to the connected state as shown in FIG.

Here, the culture container unit 1A and the culture solution container 2 communicate with each other via the first flow path 41, and the culture container unit 1A and the vacuum container unit 3 communicate with each other via the second flow path 42. Then, the old culture solution in the culture container unit 1A moves into the vacuum container unit 3 via the second flow path 42 by the suction force of the vacuum container unit 3 whose inside is in a negative pressure state, and the culture solution container 2 The new culture solution in the culture medium moves into the culture vessel unit 1A via the first flow path 41. When the exchange of the culture solution in the culture vessel unit 1A is completed, the first connecting means 51 and the second connecting means 52 are returned from the connected state to the separated state.

In the culture vessel unit 1A, cells can be cultured in a closed state by attaching the attachment 11A to the existing open culture vessel 10A. The attachment 11A (base member 12 and seal member 13) closes the opening 103 of the culture container 10A (container portion 100), and the flexible seal member 13 plays a role of sealing the inner space of the container portion 100. .. Further, the seal member 13 is formed with a fluid introduction flow path 15 and a fluid lead-out flow path 16 (flow path in the attachment), and the culture container unit is formed by using the fluid introduction flow path 15 and the fluid lead-out flow path 16. By connecting the culture fluid container 2 and the vacuum vessel unit 3 to 1A, perfusion culture in a closed state is possible.

The culture solution in the culture vessel unit 1A can be exchanged by a one-touch operation such as changing the first connecting means 51 and the second connecting means 52 from the separated state to the connected state as described above. In addition, the culture solution exchange work can be performed while the culture container unit 1A is closed, and the risk of contamination can be avoided. Therefore, according to the culture container unit 1A to which the attachment 11A is attached, it is possible to reduce the operational burden on the operator and ensure the quality of the cells when exchanging the culture solution. Further, in the perfusion culture system A10 configured to include the culture vessel unit 1A, it is possible to exchange the culture solution in the culture vessel unit 1A without using an electric device such as a controller or a power source. Therefore, the perfusion culture system A10 can be miniaturized without worrying about failure.

As described above, if the attachment 11A is used, the open culture vessel 10A can be used as the closed culture vessel unit 1A. Therefore, even if the culture vessel unit 1A is tilted, inconveniences such as liquid leakage do not occur. Therefore, if the tubes (partial flow path 411 and partial flow path 421) are closed by a heat seal or the like with the culture solution container 2 and the vacuum container unit 3 removed, the culture container unit 1A maintains the cell culture state. It is possible to transport while.

The attachment 11A has a bottom wall 121 (protruding portion), and the bottom wall 121 projects downward (the other side in the first direction) inside the culture container 10A (container portion 100). According to such a configuration, the storage space of the culture solution in the culture container 10A (container portion 100) is reduced, and the amount of the culture solution used during cell culture can be reduced.

The attachment 11A includes a potential sealing member 13 and a base member 12 made of a hard material separate from the sealing member 13. Then, in the state where the attachment 11A is attached to the culture container 10A, the plate portion 132 of the seal member 13 is interposed between the flange 123 of the base member 12 and the peripheral edge of the opening 103 of the culture container 10A (container portion 100). , It comes into contact with all the peripheral edges of the opening 103. According to such a configuration, the sealing member 13 can seal the culture container 10A (container portion 100) while being supported by the base member 12. Further, when the attachment 11A is attached, the cylindrical portion 131 of the seal member 13 is press-fitted between the side plate 102 of the culture container 10A and the side wall 122 of the base member 12. As a result, the culture container 10A can be appropriately sealed by simply attaching the attachment 11A to the culture container 10A.

In the present embodiment, the bottom plate 101 of the culture vessel 10A and the bottom wall 121 of the base member 12 are transparent. Thereby, it is possible to observe the situation inside the culture vessel unit 1A from either the upper portion or the lower portion of the culture vessel unit 1A through the transparent bottom wall 121 or the bottom plate 101. With such a configuration, irradiation light from upper and lower light sources, lasers, echoes, etc. can be transmitted, and cells can be transmitted using an optical microscope, a phase contrast microscope, a laser microscope, an ultrasonic microscope, and various other observation devices. Monitoring and shooting are possible.

In the present embodiment, the bottom wall 121 of the base member 12 projects downward. By arranging the bottom wall 121 at a low position in this way, the culture solution in the culture vessel unit 1A comes into contact with the surface of the bottom wall 121. As a result, the liquid surface of the culture solution becomes flat due to the effect of underwater eyeglasses, diffuse reflection of irradiation light is suppressed, and the effect of facilitating focusing of the microscope can be expected.

In the fluid introduction flow path 15 and the fluid lead-out flow path 16 provided in the seal member 13, the fluid inlet 151 and the fluid outlet 161 leading to the outside are substantially perpendicular to the vertical direction (first direction). Looking in the direction. According to such a configuration, the size of the culture vessel unit 1A to which the attachment 11A is attached can be reduced in the vertical direction (first direction), and the culture vessel unit 1A can be miniaturized.

8 to 17 show modified examples of the above-mentioned culture vessel unit 1A and attachment 11A. In these figures, only the configuration of the attachment (attachment for perfusion culture) is different from the attachment 11A described above. In the drawings after FIG. 8, the same or similar elements as those in the above embodiment are designated by the same reference numerals, and the description thereof will be omitted as appropriate.

In the attachment 11B shown in FIGS. 8 to 10, the arrangement of the fluid introduction flow path 15 and the fluid lead-out flow path 16 provided in the seal member 13 is different from that of the above attachment 11A, and the plate portion 132 (seal) is accompanied by this. The shapes of the member 13) and the flange 123 (base member 12) are different from those of the above embodiment.

In the above attachment 11A, the fluid lead-out flow path 16 is provided on the opposite side of the fluid introduction flow path 15 with the center of the seal member 13 interposed therebetween in a plan view. The fluid lead-out flow path 16 is provided relatively close to each other. The fluid inlet 151 and the fluid outlet 161 leading to the outside in the fluid introduction flow path 15 and the fluid outlet flow path 16 are all oriented in a direction substantially perpendicular to the vertical direction (first direction). , Face each other in the same direction.

The attachment 11B of this modified example accommodates cultured cells and a culture solution inside a closed culture container unit 1B configured by being attached to an open culture container 10A, and the culture solution is maintained in a cell culture state. Used to make a replacement.

In the culture vessel unit 1B, cells can be cultured in a closed state by attaching the attachment 11B to the existing open culture vessel 10A. The attachment 11B (base member 12 and seal member 13) closes the opening 103 of the culture container 10A (container portion 100), and the flexible seal member 13 plays a role of sealing the inner space of the container portion 100. .. Further, the seal member 13 is formed with a fluid introduction flow path 15 and a fluid lead-out flow path 16 (flow path in the attachment), and the culture container unit is formed by using the fluid introduction flow path 15 and the fluid lead-out flow path 16. By connecting the culture fluid container 2 and the vacuum vessel unit 3 to 1B, perfusion culture in a closed state is possible.

In addition, the attachment 11B and the culture vessel unit 1B of the present modification have the same effects as those of the attachment 11A and the culture vessel unit 1A described above.

In the attachment 11C shown in FIG. 11, the sealing member 13 is not provided with the cylindrical portion 131, and the sealing member 13 is not press-fitted between the culture container 10A and the base member 12. By pressing the base member 12 downward, the culture container 10A (container portion 100) can be sealed.

In the attachment 11C of this modified example, the cultured cells and the culture solution are housed inside the closed culture container unit 1C configured by mounting the culture container 10A in an open system, and the culture solution is maintained while maintaining the cell culture state. Used to make a replacement.

In the culture vessel unit 1C, cells can be cultured in a closed state by attaching the attachment 11C to the existing open culture vessel 10A. The attachment 11C (base member 12 and seal member 13) closes the opening 103 of the culture container 10A (container portion 100), and the flexible seal member 13 plays a role of sealing the inner space of the container portion 100. .. Further, the seal member 13 is formed with a fluid introduction flow path 15 and a fluid lead-out flow path 16 (flow path in the attachment), and the culture container unit is formed by using the fluid introduction flow path 15 and the fluid lead-out flow path 16. By connecting the culture fluid container 2 and the vacuum vessel unit 3 to 1C, perfusion culture in a closed state is possible.

In addition, the attachment 11C and the culture vessel unit 1C of the present modification have the same effects as those of the attachment 11A and the culture vessel unit 1A described above.

In the attachment 11D shown in FIG. 12, the seal member 13 has a bottom wall portion 134. The bottom wall portion 134 is connected to the lower end of the cylindrical portion 131 and closes the inside of the cylindrical portion 131. The central portion of the bottom wall portion 134 is a thin-walled portion 135 having a relatively small thickness, and the thin-walled portion 135 is transparent. On the other hand, the cylindrical portion 131 is not provided with the annular protrusion 133, and there is a gap between the cylindrical portion 131 and the side plate 102 of the culture vessel 10A, and the cylindrical portion 131 is not press-fitted. The base member 12 does not include a bottom wall 121 and a side wall 122, but includes only an annular flange 123. As a result, the seal member 13 is not press-fitted between the culture container 10A and the base member 12. In the attachment 11D having such a configuration, the culture container 10A (container portion 100) can be sealed by pressing the base member 12 downward.

In the attachment 11D of this modified example, the cultured cells and the culture solution are housed inside the closed culture container unit 1D configured by mounting the culture container 10A in an open system, and the culture solution is maintained while maintaining the cell culture state. Used to make a replacement.

In the culture vessel unit 1D, cells can be cultured in a closed state by attaching the attachment 11D to the existing open culture vessel 10A. The attachment 11D (base member 12 and seal member 13) closes the opening 103 of the culture container 10A (container portion 100), and the flexible seal member 13 plays a role of sealing the inner space of the container portion 100. .. Further, the seal member 13 is formed with a fluid introduction flow path 15 and a fluid lead-out flow path 16 (flow path in the attachment), and the culture container unit is formed by using the fluid introduction flow path 15 and the fluid lead-out flow path 16. By connecting the culture fluid container 2 and the vacuum vessel unit 3 to 1D, perfusion culture in a closed state is possible.

In addition, the attachment 11D and the culture vessel unit 1D of the present modification have the same effects as those of the attachment 11A and the culture vessel unit 1A described above.

In the attachment 11E shown in FIG. 13, the seal member 13 is not provided with the plate portion 132, and the seal member 13 includes only the cylindrical portion 131. The cylindrical portion 131 is fitted onto the side wall 122 of the base member 12, and the inner diameter dimension of the cylindrical portion 131 in the natural state is slightly smaller than the outer diameter dimension of the side wall 122. As a result, the cylindrical portion 131 (seal member 13) is pressed and held by the side wall 122 (base member 12). In this modification, the upper end of the cylindrical portion 131 is in contact with the flange 123 of the base member 12, and this configuration allows the cylindrical portion 131 (seal member 13) to be positioned in the vertical direction with respect to the base member 12.

Further, the outer diameter dimension of the cylindrical portion 131 in the natural state is slightly larger than the inner diameter dimension of the side plate 102 in the culture vessel 10A (container portion 100). As a result, when the attachment 11E is attached, the cylindrical portion 131 is press-fitted between the side plate 102 (container portion 100) and the side wall 122 (base member 12).

In the attachment 11E of this modified example, the cultured cells and the culture solution are housed inside the closed culture container unit 1E configured by mounting the culture container 10A in an open system, and the culture solution is maintained while maintaining the cell culture state. Used to make a replacement.

In the culture vessel unit 1E, cells can be cultured in a closed state by attaching the attachment 11E to the existing open culture vessel 10A. The attachment 11E (base member 12 and seal member 13) closes the opening 103 of the culture container 10A (container portion 100), and the flexible seal member 13 serves to seal the inner space of the container portion 100. Carry. Further, the seal member 13 is formed with a fluid introduction flow path 15 and a fluid lead-out flow path 16 (flow path in the attachment), and the culture container unit is formed by using the fluid introduction flow path 15 and the fluid lead-out flow path 16. By connecting the culture fluid container 2 and the vacuum vessel unit 3 to 1E, perfusion culture in a closed state is possible.

In addition, the attachment 11E and the culture vessel unit 1E of the present modification have the same effects as those of the attachment 11A and the culture vessel unit 1A described above.

In the attachment 11F shown in FIGS. 14 to 17, the seal member 13 has a bottom wall portion 134. The bottom wall portion 134 is connected to the lower end of the cylindrical portion 131 and closes the inside of the cylindrical portion 131. The central portion of the bottom wall portion 134 is a thin-walled portion 135 having a relatively small thickness, and the thin-walled portion 135 is transparent. On the other hand, the cylindrical portion 131 is not provided with the annular protrusion 133, and there is a gap between the cylindrical portion 131 and the side plate 102 of the culture vessel 10A, and the cylindrical portion 131 is not press-fitted. The base member 12 includes a bottom wall 121, a side wall 122, and a flange 123, but the configuration of the bottom wall 121 is different from that of the above embodiment. In this modification, the bottom wall 121 is formed with a through hole 121a penetrating in the thickness direction. The through hole 121a has a substantially circular shape and is formed in the center of the bottom wall 121. The through hole 121a is located so as to overlap the thin portion 135 of the seal member 13 in the vertical view (first direction view). Due to the above configuration, the seal member 13 is not press-fitted between the culture container 10A and the base member 12. In the attachment 11F having such a configuration, the culture container 10A (container portion 100) can be sealed by pressing the base member 12 downward.

The attachment 11F of this modified example accommodates cultured cells and a culture solution inside a closed culture container unit 1F configured by mounting on an open culture container 10A, and the culture solution is maintained in a cell culture state. Used to make a replacement.

In the culture vessel unit 1F, cells can be cultured in a closed state by attaching the attachment 11F to the existing open culture vessel 10A. The attachment 11F (base member 12 and seal member 13) closes the opening 103 of the culture container 10A (container portion 100), and the flexible seal member 13 plays a role of sealing the inner space of the container portion 100. .. Further, the seal member 13 is formed with a fluid introduction flow path 15 and a fluid lead-out flow path 16 (flow path in the attachment), and the culture container unit is formed by using the fluid introduction flow path 15 and the fluid lead-out flow path 16. By connecting the culture fluid container 2 and the vacuum vessel unit 3 to the 1st floor, perfusion culture in a closed state is possible.

The attachment 11F has a bottom wall portion 134 (protruding portion), and the bottom wall portion 134 projects downward (the other side in the first direction) inside the culture vessel 10A (container portion 100). There is. According to such a configuration, the storage space of the culture solution in the culture container 10A (container portion 100) is reduced, and the amount of the culture solution used during cell culture can be reduced.

In the culture container unit 1F, the thin portion 135 of the seal member 13 is transparent. Further, the through hole 121a formed in the bottom wall 121 of the base member 12 overlaps the thin portion 135 in the vertical view. Since the bottom plate 101 of the culture container 10A and the thin-walled portion 135 of the seal member 13 are both transparent, the culture container is passed through the transparent thin-walled portion 135 or the bottom plate 101 from either the upper part or the lower part of the culture container unit 1F. It is possible to observe the situation inside the unit 1F. With such a configuration, irradiation light from upper and lower light sources, lasers, echoes, etc. can be transmitted, and cells can be transmitted using an optical microscope, a phase contrast microscope, a laser microscope, an ultrasonic microscope, and various other observation devices. Monitoring and shooting are possible.

In addition, the attachment 11F and the culture vessel unit 1F of this modification have the same effects as those of the attachment 11A and the culture vessel unit 1A described above.

18 to 21 show another embodiment (second embodiment) of the attachment for perfusion culture according to the present invention and the culture vessel unit provided with the attachment. The culture container unit 1G of the present embodiment includes a culture container 10B and a plurality of attachments 11G. 18 and 19 show a state in which the attachment 11G is attached to the culture vessel 10B. FIG. 20 is a perspective view of the attachment 11G in an appropriately disassembled state.

The culture container 10B corresponds to a so-called well plate, and has a plurality of (six in this embodiment) wells as container portions 100. Each of the plurality of container portions 100 is composed of a common elongated bottom plate 101 and a cylindrical side plate 102 that stands up from an appropriate position of the bottom plate 101 and has an opening 103 at the upper end (one side end in the first direction). ing. These container portions 100 are arranged apart from each other in the in-plane direction which is perpendicular to the vertical direction (first direction).

The upper surface portion of the bottom plate 101 corresponding to each container portion 100 is a flat cell culture surface for culturing cells. The cell culture surface (upper surface of the bottom plate 101) is appropriately subjected to surface treatment for improving cell adhesion, if necessary. Examples of the surface treatment include corona discharge treatment and plasma treatment (hydrophilization treatment).

The attachment 11G is attached to each container portion 100 of the culture container 10B, and is for closing the opening 103 of the container portion 100 and sealing the inner space of the container portion 100. The attachment 11G includes a base member 12 and a seal member 13, and the specific configuration thereof is similar to the attachment 11B described above with reference to FIGS. 8 to 10.

The base member 12 has a bottom wall 121, a cylindrical side wall 122 extending upright from the peripheral edge of the bottom wall 121, and a flange 123. The bottom wall 121 fits inside the side plate 102 in the container portion 100 at a distance from the side plate 102, and protrudes downward (the other side in the first direction). The bottom wall 121 constitutes the protruding portion referred to in the present invention. The flange 123 extends radially outward from the upper end of the side wall 122 and has an annular shape. The base member 12 having such a configuration overlaps all the openings 103 of the container portion 100 in a plan view.

In the present embodiment, the base member 12 further includes a holding cylinder portion 124 and an extension piece 125. The holding cylinder portion 124 extends vertically from the outer peripheral edge of the flange 123, and is a portion for an operator to pick and hold by hand. The extension piece 125 is a portion extending radially outward from the outer circumference of the upper end portion. In the present embodiment, a pair of extension pieces 125 are provided so as to sandwich the axial center of the holding cylinder portion 124.

The culture vessel 10B and the base member 12 are made of, for example, a translucent or transparent hard plastic material. As the plastic material, for example, polystyrene, methylpentene, polycarbonate, cycloolefin polymer, cycloolefin copolymer, and other materials having transparency are preferably used, but the plastic material is not limited thereto.

In the present embodiment, at least the bottom plate 101 of the culture vessel 10B and the bottom wall 121 of the base member 12 are transparent. As a result, the bottom plate 101 and the bottom wall 121 are portions where the inner space of each container portion 100 can be visually recognized from the outside.

The seal member 13 is, for example, a rubber molded product, and has a cylindrical portion 131 extending vertically and a plate portion 132 as shown in FIG. 21. The seal member 13 is interposed between the side plate 102 of the culture container 10B (container portion 100) and the side wall 122 or the flange 123 of the base member 12. The cylindrical portion 131 extends downward (the other side in the first direction) from the inner peripheral portion of the plate portion 132, and is fitted onto the side wall 122 of the base member 12. The natural inner diameter of the cylindrical portion 131 is slightly smaller than the outer diameter of the side wall 122, whereby the cylindrical portion 131 (seal member 13) is pressed and held by the side wall 122 (base member 12). Further, the outer diameter dimension of the cylindrical portion 131 in the natural state is slightly larger than the inner diameter dimension of the side plate 102 in the container portion 100. As a result, when the attachment 11G is attached, the cylindrical portion 131 is press-fitted between the side plate 102 (container portion 100) and the side wall 122 (base member 12). The plate portion 132 extends radially outward from the upper end of the cylindrical portion 131 and has an annular shape. The plate portion 132 overlaps with the flange 123 of the base member 12 in a plan view. Further, when the attachment 11G is attached to the culture container 10B (container portion 100), the plate portion 132 comes into contact with all the peripheral edges of the opening 103 in the container portion 100. With such a configuration, the base member 12 and the seal member 13 (attachment 11G) seal the inner space of the container portion 100.

The seal member 13 has a fluid introduction flow path 15 and a fluid lead-out flow path 16. In the present embodiment, the fluid introduction flow path 15 is composed of holes and grooves formed in the seal member 13. More specifically, the fluid introduction flow path 15 has a configuration in which a hole formed in the plate portion 132 and a groove formed in the inner surface of the cylindrical portion 131 are connected to each other. One end of the fluid introduction flow path 15 is a fluid inflow port 151 leading to the outside, and the other end of the fluid introduction flow path 15 is connected to the inner space of the sealed container portion 100. The fluid inlet 151 faces a direction substantially perpendicular to the vertical direction (first direction).

In the present embodiment, the fluid lead-out flow path 16 is provided relatively close to the fluid introduction flow path 15, and the hole formed in the plate portion 132 and the groove formed in the inner surface of the cylindrical portion 131 are connected to each other. It is composed. One end of the fluid lead-out flow path 16 is a fluid outlet 161 that leads to the outside, and the other end of the fluid lead-out flow path 16 leads to the inner space of the sealed container portion 100. The fluid outlet 161 faces a direction substantially perpendicular to the vertical direction (first direction). The fluid inlet 151 and the fluid outlet 161 face each other in the same direction. The fluid introduction flow path 15 and the fluid lead-out flow path 16 constitute the attachment inner flow path referred to in the present invention.

The sealing member 13 is made of a flexible and elastic material. Examples of the material constituting the seal member 13 include silicone rubber, natural rubber, urethane rubber, and elastomer resin. Considering the contact between the sealing member 13 and the contents (for example, a culture solution), the material of the sealing member 13 is more preferably a medical grade silicone rubber having no cytotoxicity and biocompatibility. Regarding the hardness of the seal member 13, for example, the rubber hardness is preferably about 20 to 40 degrees.

The attachment 11G of the present embodiment accommodates cultured cells and a culture solution in a closed culture container unit 1G configured by mounting on an open culture container 10B, and the culture solution is maintained in a cell culture state. Used to make a replacement.

In the culture container unit 1G, cells can be cultured in a closed state by attaching the attachment 11G to the existing open culture container 10B (container portion 100). The attachment 11G (base member 12 and seal member 13) closes the opening 103 of the container portion 100, and the flexible seal member 13 plays a role of sealing the inner space of the container portion 100. Further, the seal member 13 is formed with a fluid introduction flow path 15 and a fluid lead-out flow path 16 (flow path in the attachment), and the culture container unit is formed by using the fluid introduction flow path 15 and the fluid lead-out flow path 16. By connecting the culture fluid container 2 and the vacuum vessel unit 3 to 1G, perfusion culture in a closed state is possible.

Further, in the present embodiment, the culture container 10B has a plurality of container portions 100, and a plurality of attachments 11G can be separately attached to each of the container portions 100. According to such a configuration, for example, perfusion culture under different culture conditions can be performed in parallel by appropriately differentiating the culture solution container 2 and the vacuum container unit 3 to be connected for each attachment 11G attached to the container portion 100. It is possible to do.

In addition, the attachment 11G and the culture vessel unit 1G of the present embodiment have the same effects as those of the attachment 11A and the culture vessel unit 1A described above.

22 to 25 show another embodiment (third embodiment) of the attachment for perfusion culture according to the present invention and the culture vessel unit provided with the attachment. The culture container unit 1H of the present embodiment includes a culture container 10B and an attachment 11H. 22 and 23 show a state in which the attachment 11H is attached to the culture vessel 10B. FIG. 24 is an exploded perspective view of the culture vessel unit 1H. The culture vessel 10B in the present embodiment corresponds to a so-called well plate and has a plurality of container portions 100, but is the same as the culture vessel 10B in the second embodiment described with reference to FIGS. 18 to 21. Since it is the configuration of, the description thereof will be omitted as appropriate.

The attachment 11H is attached to a plurality of container portions 100 of the culture container 10B, and is for closing the opening 103 of each of the container portions 100 to seal the inner space of the container portion 100. The attachment 11H includes a base member 12 and a seal member 13.

The attachment 11H of the present embodiment is configured to be able to collectively close each opening 103 of the plurality of container portions 100, and is significantly different from the attachment 11G of the above embodiment in this respect.

Specifically, the base member 12 has a pressing portion 120A and a plurality of first bottomed cylinder portions 120B. The pressing portion 120A has a long rectangular flat plate shape, and has a size corresponding to the plan view size of the culture vessel 10B. The plurality of first bottomed cylinder portions 120B are arranged so as to correspond to each of the plurality of container portions 100, and each of them can enter the inside of the container portion 100. The first bottomed tubular portion 120B has a bottom wall 121 and a tubular side wall 122 extending upright from the peripheral edge of the bottom wall 121. Each bottom wall 121 fits inside the side plate 102 in the container portion 100 at a distance from the side plate 102, and projects downward (the other side in the first direction). The bottom wall 121 constitutes the protruding portion referred to in the present invention. The upper end of each side wall 122 is connected to the holding portion 120A. As can be understood from FIGS. 24 and 25, the pressing portion 120A connected to each side wall 122 extends outward from the upper end portion of each side wall 122 and is integrally connected.

The culture vessel 10B and the base member 12 are made of, for example, a translucent or transparent hard plastic material. As the plastic material, for example, polystyrene, methylpentene, polycarbonate, cycloolefin polymer, cycloolefin copolymer, and other materials having transparency are preferably used, but the plastic material is not limited thereto.

In the present embodiment, at least the bottom plate 101 of the culture vessel 10B and the bottom wall 121 of the base member 12 are transparent. As a result, the bottom plate 101 and the bottom wall 121 are portions where the inner space of each container portion 100 can be visually recognized from the outside.

The seal member 13 is, for example, a rubber molded product, and has a plate portion 130A and a plurality of cylindrical portions 131 as shown in FIGS. 24 and 25. The plate portion 130A has a long rectangular flat plate shape, and has a size corresponding to the plan view size of the culture vessel 10B. The plurality of cylindrical portions 131 are arranged so as to correspond to each of the plurality of container portions 100, and each of them is on the lower side (the other side in the first direction) from the inner peripheral portion of the through opening provided at an appropriate position of the plate portion 130A. Extends to. The first bottomed tubular portion 120B of the base member 12 can be inserted inside the cylindrical portion 131, and the inner diameter dimension of the cylindrical portion 131 in the natural state is slightly smaller than the outer diameter dimension of the side wall 122. As a result, the cylindrical portion 131 (seal member 13) is pressure-welded and held by the side wall 122 (base member 12). Each cylindrical portion 131 can be inserted inside the container portion 100, and the natural outer diameter dimension of the cylindrical portion 131 is slightly larger than the inner diameter dimension of the side plate 102 in the container portion 100. As a result, when the attachment 11H is attached, each cylindrical portion 131 is press-fitted between the side plate 102 (container portion 100) and the side wall 122 (base member 12). The plate portion 130A overlaps the pressing portion 120A of the base member 12 in a plan view. Further, when the attachment 11H is attached to the culture container 10B (container portion 100), the plate portion 130A is interposed between the pressing portion 120A and the peripheral edge portion of the opening 103 in each container portion 100, and the peripheral portion of each opening 103. Contact with everything. With such a configuration, the base member 12 and the seal member 13 (attachment 11H) seal the inner space of each of the plurality of container portions 100.

The seal member 13 has a fluid introduction flow path 15 and a fluid lead-out flow path 16. In the present embodiment, the seal member 13 has a plurality of fluid introduction flow paths 15 and a fluid discharge flow path 16 corresponding to the plurality of container portions 100. A pair of fluid introduction flow paths 15 and a fluid lead-out flow path 16 are provided corresponding to each container portion 100. In the present embodiment, the fluid introduction flow path 15 is composed of holes and grooves formed in the seal member 13. More specifically, the fluid introduction flow path 15 is configured to connect a hole formed in the plate portion 130A and a groove formed on the inner surface of the cylindrical portion 131. One end of the fluid introduction flow path 15 is a fluid inflow port 151 leading to the outside, and the other end of the fluid introduction flow path 15 is connected to the inner space of the sealed container portion 100. The fluid inlet 151 faces a direction substantially perpendicular to the vertical direction (first direction).

In the present embodiment, the fluid lead-out flow path 16 is provided relatively close to the fluid introduction flow path 15, and the hole formed in the plate portion 130A and the groove formed in the inner surface of the cylindrical portion 131 are connected to each other. It is composed. One end of the fluid lead-out flow path 16 is a fluid outlet 161 that leads to the outside, and the other end of the fluid lead-out flow path 16 leads to the inner space of the sealed container portion 100. The fluid outlet 161 faces a direction substantially perpendicular to the vertical direction (first direction). In the paired fluid introduction flow path 15 and fluid lead-out flow path 16, the fluid inlet 151 and the fluid outlet 161 face each other in the same direction. The fluid introduction flow path 15 and the fluid lead-out flow path 16 constitute the attachment inner flow path referred to in the present invention.

The sealing member 13 is made of a flexible and elastic material. Examples of the material constituting the seal member 13 include silicone rubber, natural rubber, urethane rubber, and elastomer resin. Considering the contact between the sealing member 13 and the contents (for example, a culture solution), the material of the sealing member 13 is more preferably a medical grade silicone rubber having no cytotoxicity and biocompatibility. Regarding the hardness of the seal member 13, for example, the rubber hardness is preferably about 20 to 40 degrees.

The attachment 11H of the present embodiment accommodates cultured cells and a culture solution in a closed culture container unit 1H configured by being attached to an open culture container 10B, and the culture solution is maintained in a cell culture state. Used to make a replacement.

In the culture vessel unit 1H, cells can be cultured in a closed state by attaching the attachment 11H to the existing open culture vessel 10B (container portion 100). The attachment 11H (base member 12 and seal member 13) closes the opening 103 of the container portion 100, and the flexible seal member 13 plays a role of sealing the inner space of the container portion 100. Further, the seal member 13 is formed with a fluid introduction flow path 15 and a fluid lead-out flow path 16 (flow path in the attachment), and the culture container unit is formed by using the fluid introduction flow path 15 and the fluid lead-out flow path 16. By connecting the culture fluid container 2 and the vacuum vessel unit 3 to 1H, perfusion culture in a closed state is possible.

Further, in the present embodiment, the culture container 10B has a plurality of container parts 100, and by attaching a single attachment 11H to the culture container 10B, the plurality of container parts 100 can be hermetically sealed at once. According to such a configuration, with respect to the culture container 10B including the plurality of container parts 100, all the container parts 100 can be sealed by a simple operation. Further, according to the present embodiment, for example, the culture solution container 2 and the vacuum container unit 3 to be connected are appropriately different for each of the fluid introduction flow path 15 and the fluid out-flow flow path 16 corresponding to the container portion 100. Perfusion culture under culture conditions can be performed in parallel.

In addition, the attachment 11H and the culture vessel unit 1H of the present embodiment have the same effects as those of the attachment 11A and the culture vessel unit 1A described above.

26 to 30 show another embodiment (fourth embodiment) of the attachment for perfusion culture according to the present invention and the culture vessel unit provided with the attachment. The culture container unit 1J of the present embodiment includes a culture container 10B and an attachment 11J. 26 and 27 show a state in which the attachment 11J is attached to the culture vessel 10B. FIG. 28 is an exploded perspective view of the culture vessel unit 1J. The culture vessel 10B in the present embodiment corresponds to a so-called well plate and has a plurality of container portions 100, but is the same as the culture vessel 10B in the second embodiment described with reference to FIGS. 18 to 21. Since it is the configuration of, the description thereof will be omitted as appropriate.

The attachment 11J is attached to a plurality of container portions 100 of the culture container 10B, and is for closing the opening 103 of each of the container portions 100 to seal the inner space of the container portion 100. The attachment 11J includes a base member 12 and a seal member 13.

The attachment 11J of the present embodiment is configured to be able to collectively close each opening 103 of the plurality of container portions 100, similarly to the attachment 11H described above. On the other hand, the specific configurations of the base member 12 and the seal member 13 are significantly different from those of the attachment 11H, and the path through which the culture solution flows when the culture solution is exchanged is different from that of the attachment 11H.

In the present embodiment, the base member 12 substantially includes only the pressing portion 120A. The pressing portion 120A has a long rectangular flat plate shape, and has a size corresponding to the plan view size of the culture vessel 10B. A plurality of through holes 126 penetrating in the thickness direction are formed in the pressing portion 120A. The plurality of through holes 126 are formed at positions corresponding to each of the plurality of container portions 100. Further, in the present embodiment, a plurality of (two in the present embodiment) inner flow paths 15B and 16B in the base member are provided at appropriate positions of the pressing portion 120A so as to penetrate in the thickness direction (first direction). The significance of the through hole 126 and the flow paths 15B and 16B in the base member will be described later.

The culture vessel 10B and the base member 12 are made of, for example, a translucent or transparent hard plastic material. As the plastic material, for example, polystyrene, methylpentene, polycarbonate, cycloolefin polymer, cycloolefin copolymer, and other materials having transparency are preferably used, but the plastic material is not limited thereto. In the present embodiment, at least the bottom plate 101 of the culture vessel 10B is transparent. As a result, the bottom plate 101 is a portion where the inner space of each container portion 100 can be visually recognized from the outside.

The seal member 13 is, for example, a rubber molded product, and has a plate portion 130A and a plurality of second bottomed cylinder portions 130B as shown in FIGS. 28 and 29. The plate portion 130A has a long rectangular flat plate shape, and has a size corresponding to the plan view size of the culture vessel 10B. The plurality of second bottomed cylinder portions 130B are arranged so as to correspond to each of the plurality of container portions 100, and each of them can enter the inside of the container portion 100. The second bottomed tubular portion 130B has a cylindrical portion 131 and a bottom wall portion 134. The cylindrical portion 131 extends downward (the other side in the first direction) from the inner peripheral portion of the through opening provided at an appropriate position in the plate portion 130A. The bottom wall portion 134 is connected to the lower end of the cylindrical portion 131 and closes the inside of the cylindrical portion 131. The central portion of the bottom wall portion 134 is a thin-walled portion 135 having a relatively small thickness, and the thin-walled portion 135 is transparent.

The plate portion 130A overlaps the pressing portion 120A of the base member 12 in a plan view. Further, when the attachment 11J is attached to the culture container 10B (container portion 100), the plate portion 130A is interposed between the pressing portion 120A and the peripheral edge portion of the opening 103 in each container portion 100, and the peripheral portion of each opening 103. Contact with everything. On the other hand, in the present embodiment, there is a gap between the cylindrical portion 131 and the side plate 102 in the container portion 100, and the cylindrical portion 131 is not press-fitted. In the attachment 11J having such a configuration, the plurality of container portions 100 can be sealed by pressing the base member 12 downward.

When the attachment 11J is attached, in a plan view, the thin portion 135 of each second bottomed tubular portion 130B of the seal member 13 overlaps with any of the plurality of through holes 126 formed in the pressing portion 120A. Thereby, it is possible to observe the internal condition of each container portion 100 from above (outside) of the attachment 11J through the through hole 126 and the transparent thin-walled portion 135.

In this embodiment, the seal member 13 further includes an extension piece 128 and a protrusion 129. The extension piece 128 extends outward from each of the four outer peripheral edges of the holding portion 120A, and is a portion for the operator to pick by hand. The protrusions 129 project upward from the upper surface of each extension piece 128, and come into contact with or approach the four outer peripheral edges of the holding portion 120A of the base member 12 when the attachment 11J is attached. The configuration having such protrusions 129 plays a role of positioning the base member 12 and the seal member 13 with each other when the attachment 11J is attached.

The seal member 13 has flow paths 15A and 16A in the seal member. In the present embodiment, the seal member 13 is provided in one seal member inner flow path 15A corresponding to one container portion 100 (first container portion) and another container portion 100 (second container portion). It has one corresponding inner flow path 16A in the seal member. The flow paths 15A and 16A in the seal member are formed by holes penetrating the plate portion 130A in the thickness direction (vertical direction, that is, the first direction).

The flow path 15A in the seal member is provided in the vicinity of one second bottomed cylinder portion 130B. One end (upper end) of the flow path 15A in the seal member faces upward (one side in the first direction). The other end (lower end) of the flow path 15A in the seal member leads to the inner space of the sealed container portion 100 (first container portion). The flow path 16A in the seal member is provided in the vicinity of the other second bottomed tubular portion 130B. One end (upper end) of the flow path 16A in the seal member faces upward (one side in the first direction). The other end (lower end) of the flow path 16A in the seal member leads to the inner space of the sealed container portion 100 (second container portion).

When the attachment 11J is attached, the seal member inner flow paths 15A and 16A overlap with the base member inner flow paths 15B and 16B formed on the base member 12 (holding portion 120A), respectively, in a plan view. As a result, the inner flow paths 15A and 16A of the seal member communicate with the inner flow paths 15B and 16B of the base member, respectively, and communicate with the outside through the inner flow paths 15B and 16B of the base member, respectively. The seal member inner flow path 15A and the base member inner flow path 15B form a fluid introduction flow path 15. Further, the seal member inner flow path 16A and the base member inner flow path 16B form a fluid lead-out flow path 16. The fluid introduction flow path 15 and the fluid lead-out flow path 16 constitute a flow path in the attachment referred to in the present invention.

In the present embodiment, the seal member 13 has a plurality of communication flow paths 17. The communication flow path 17 is a flow path that allows the inner spaces of the pair of adjacent container portions 100 to communicate with each other. In the present embodiment, the connecting flow path 17 is composed of holes and grooves formed in the seal member 13. More specifically, the connecting flow path 17 is composed of a pair of holes 171 formed in the plate portion 130A and a groove 172. The pair of holes 171 extend in the thickness direction of the plate portion 130A, respectively. The lower ends of the pair of holes 171 communicate with the inner space of the pair of adjacent container portions 100. The groove 172 is formed on the upper surface (the surface facing one side in the first direction) of the plate portion 130A. Both ends of the groove 172 are connected to the upper ends of the pair of holes 171. By superimposing the base member 12 (holding portion 120A) on the plate portion 130A, the upper end of the groove 172 is closed, and the communication flow path 17 communicates the inner spaces of the adjacent container portions 100 with each other.

As can be understood from FIGS. 28 to 30, the plurality of connecting flow paths 17 are adjacent to each other from the inner space of the container portion 100 (first container portion) through which the lower end of the fluid introduction flow path 15 communicates. It is provided so as to communicate with the inner space of the container portion 100 (second container portion) in which the lower end of the fluid lead-out flow path 16 communicates through the inner space of 100 in sequence.

The sealing member 13 is made of a flexible and elastic material. Examples of the material constituting the seal member 13 include silicone rubber, natural rubber, urethane rubber, and elastomer resin. Considering the contact between the sealing member 13 and the contents (for example, a culture solution), the material of the sealing member 13 is more preferably a medical grade silicone rubber having no cytotoxicity and biocompatibility. Regarding the hardness of the seal member 13, for example, the rubber hardness is preferably about 20 to 40 degrees.

The attachment 11J of the present embodiment accommodates cultured cells and a culture solution in a closed culture container unit 1J configured by mounting on an open culture container 10B, and prepares the culture solution while maintaining the cell culture state. Used to make a replacement.

In the culture vessel unit 1J, cells can be cultured in a closed state by attaching the attachment 11J to the existing open culture vessel 10B (container portion 100). The attachment 11J (base member 12 and seal member 13) closes the opening 103 of the container portion 100, and the flexible seal member 13 plays a role of sealing the inner space of the container portion 100. Further, the base member 12 and the seal member 13 are formed with a fluid introduction flow path 15 and a fluid lead-out flow path 16 (flow path in the attachment), and the fluid introduction flow path 15 and the fluid lead-out flow path 16 are used. By connecting the culture fluid container 2 and the vacuum vessel unit 3 to the culture vessel unit 1J, perfusion culture can be performed in a closed state.

In the present embodiment, the culture container 10B has a plurality of container parts 100, and by attaching a single attachment 11J to the culture container 10B, the plurality of container parts 100 can be hermetically sealed at once. According to such a configuration, with respect to the culture container 10B including the plurality of container parts 100, all the container parts 100 can be sealed by a simple operation.

Further, in the present embodiment, the plate portion 130A (seal member 13) is provided with a plurality of communication flow paths 17. The lower end of the fluid outflow flow path 16 sequentially passes through the inner space of the adjacent container portion 100 from the inner space of the container portion 100 (first container portion) through which the lower end of the fluid introduction flow path 15 communicates with the connecting flow path 17. It communicates with the inner space of the container portion 100 (second container portion) with which the fluid communicates. According to such a configuration, the culture solution container 2 and the vacuum container unit 3 are connected to the culture container unit 1J by using the fluid introduction flow path 15 and the fluid out-flow flow path 16, thereby connecting the culture solution container 2 and the vacuum container unit 3 to the plurality of container units 100. New culture solutions can be fed in sequence. Therefore, according to the present embodiment, the culture broth can be exchanged collectively for the plurality of container portions 100, and it is suitable for performing perfusion culture under the same culture conditions in the plurality of container portions 100 in parallel.

In addition, the attachment 11J and the culture vessel unit 1J of the present embodiment have the same effects as those of the attachment 11A and the culture vessel unit 1A described above.

31 to 34 show another embodiment (fifth embodiment) of the attachment for perfusion culture according to the present invention and the culture vessel unit provided with the attachment. The culture container unit 1L of the present embodiment includes a culture container 10A and an attachment 11L. FIG. 31 shows a state in which the attachment 11L is attached to the culture vessel 10A. FIG. 34 is an exploded perspective view of the culture vessel unit 1L. The attachment 11L includes a base member 12 and a seal member 13. In the present embodiment, the configurations of the fluid introduction flow path 15 and the fluid lead-out flow path 16 are different from those in the above embodiment, and various changes have been made accordingly.

In the attachment 11L of the present embodiment, the fluid introduction flow path 15 and the fluid lead-out flow path 16 are provided on the base member 12. The base member 12 has a bottom wall 121, a side wall 122, a flange 123, and a pair of bulges 127. The seal member 13 has a cylindrical portion 131 and a plate portion 132.

The bulging portion 127 of the base member 12 is a portion for forming the fluid introduction flow path 15 and the fluid lead-out flow path 16. The bulging portion 127 projects from the upper surface of the flange 123 and the inner surface of the side wall 122, and has a substantially L-shape. The fluid introduction flow path 15 and the fluid lead-out flow path 16 are each configured to connect a hole formed in the bulging portion 127 and a hole formed in the bottom wall 121. One end of the fluid introduction flow path 15 is a fluid inflow port 151 leading to the outside, and the other end of the fluid introduction flow path 15 is connected to the inner space of the closed culture container 10A (container portion 100). The fluid inlet 151 faces a direction substantially perpendicular to the vertical direction (first direction). One end of the fluid lead-out flow path 16 is a fluid outlet 161 that leads to the outside, and the other end of the fluid lead-out flow path 16 leads to the inner space of the closed culture container 10A (container portion 100). The fluid outlet 161 faces a direction substantially perpendicular to the vertical direction (first direction). The inner diameter dimensions of the fluid inlet 151 and the fluid outlet 161 formed on the base member 12 are larger than the inner diameter dimensions of the fluid inlet 151 and the fluid outlet 161 formed on the seal member 13 in the above embodiment. The base member 12 and the seal member 13 are integrally molded by, for example, insert molding.

The attachment 11L has a bottom wall 121 (protruding portion). As a result, the storage space of the culture solution in the culture container 10A (container portion 100) is reduced in the state where the attachment 11L is attached to the culture container 10A. The ratio of the volume of the storage space of the container 100 when the attachment 11L is attached to the volume of the inner space of the container 100 is, for example, 5 to 50%, preferably 5 to 25%, and more preferably 5. ~ 15%. The dimension L1 (see FIG. 33) between the upper surface of the bottom plate 101 of the container portion 100 and the lower surface of the bottom wall 121 is, for example, about 1 to 9 mm, preferably 1 to 3 mm. The dimension L1 is, for example, 5 to 50%, preferably 5 to 25%, of the height dimension L2 of the container portion 100 in the vertical direction.

In the attachment 11L, the lower surface of the bottom wall 121 of the base member 12 (the surface facing the other side in the first direction) is surface-modified by, for example, plasma treatment or corona discharge treatment. This surface modification is performed to prevent air bubbles from adhering to the lower surface of the bottom wall 121. As the surface modification treatment, a hydrophilic treatment or a hydrophobic treatment is appropriately selected according to the gap (dimension L1) between the bottom plate 101 of the container portion 100 and the bottom wall 121 of the base member 12. For example, when the dimension L1 is relatively small, the hydrophilization treatment is considered to be suitable, and when the dimension L1 is relatively large, the hydrophobic treatment is considered to be suitable. Further, plasma treatment is preferable from the viewpoint of ensuring the transparency of the bottom wall 121. Preferably, the lower surface of the bottom wall 121 is hydrophilized or hydrophobized by plasma treatment.

The attachment 11L of the present embodiment accommodates cultured cells and a culture solution in a closed culture container unit 1L configured by mounting on an open culture container 10A, and the culture solution is maintained in a cell culture state. Used to make a replacement.

In the culture vessel unit 1L, cells can be cultured in a closed state by attaching the attachment 11L to the existing open culture vessel 10A. The attachment 11L (base member 12 and seal member 13) closes the opening 103 of the culture container 10A (container portion 100), and the flexible seal member 13 plays a role of sealing the inner space of the container portion 100. .. Further, the base member 12 is formed with a fluid introduction flow path 15 and a fluid lead-out flow path 16 (flow path in the seal member), and the culture container is formed by using the fluid introduction flow path 15 and the fluid lead-out flow path 16. By connecting the culture fluid container 2 and the vacuum vessel unit 3 to the unit 1L, perfusion culture in a closed state is possible.

The attachment 11L has a bottom wall 121 (protruding portion), and the bottom wall 121 projects downward (the other side in the first direction) inside the culture container 10A (container portion 100). According to such a configuration, the storage space of the culture solution in the culture container 10A (container portion 100) is reduced, and the amount of the culture solution used during cell culture can be reduced.

In the present embodiment, the bottom plate 101 of the culture vessel 10A and the bottom wall 121 of the base member 12 are transparent. Thereby, it is possible to observe the situation in the culture vessel unit 1L from either the upper part or the lower portion of the culture vessel unit 1L through the transparent bottom wall 121 or the bottom plate 101. With such a configuration, irradiation light from upper and lower light sources, lasers, echoes, etc. can be transmitted, and cells can be transmitted using an optical microscope, a phase contrast microscope, a laser microscope, an ultrasonic microscope, and various other observation devices. Monitoring and shooting are possible.

In the present embodiment, the lower surface of the bottom wall 121 of the base member 12 is subjected to a hydrophilic treatment or a hydrophobic treatment. This makes it possible to prevent air bubbles from adhering to the lower surface of the bottom wall 121. According to such a configuration, it is possible to avoid the risk that air bubbles stay near the culture surface in the culture container unit 1L and hinder the culture or hinder monitoring and photographing.

In the present embodiment, the base member 12 and the seal member 13 are integrally molded by, for example, insert molding. Further, the fluid introduction flow path 15 and the fluid lead-out flow path 16 are formed in a base member 12 made of a hard plastic material. As shown in FIG. 1 and the like, during perfusion culture, the ends of the tubes (partial flow path 411 and partial flow path 421) having flexibility in the fluid introduction flow path 15 and the fluid out-flow flow path 16 are Be connected. According to the configuration in which the fluid introduction flow path 15 and the fluid lead-out flow path 16 are formed on the hard base member 12, the base member 12 and the tubes can be accurately joined by adhesion or the like. Therefore, leakage of the contents (culture solution, etc.) of the culture container 10A (container portion 100) between the base member 12 and the seal member 13 or from the connection portion of the tubes is more reliably prevented.

Further, according to the configuration in which the base member 12 and the seal member 13 are integrally molded as described above, when handling the attachment 11L, the peripheral edge portion of the hard base member 12 can be grasped. Therefore, the attachment 11L is also excellent in handleability by a robot.

In addition, the attachment 11L and the culture vessel unit 1L of the present modification have the same effects as those of the attachment 11A and the culture vessel unit 1A described above.

Although the specific embodiment of the present invention has been described above, the present invention is not limited to this, and various modifications can be made without departing from the idea of the invention. The specific configuration of each part of the perfusion culture attachment according to the present invention can be freely redesigned.

In the attachment 11J of the fourth embodiment described with reference to FIGS. 26 to 30, the seal member 13 is configured to have a plurality of communication flow paths 17, but the present invention is not limited thereto. For example, in the seal member 13 of the attachment 11J, a plurality of pairs of fluid introduction flow paths 15 and fluid lead-out flow paths 16 corresponding to the plurality of container portions 100 of the culture container 10B may be provided without providing the plurality of communication flow paths 17. Good.

Further, in the attachment 11J, there is a gap between the cylindrical portion 131 of the seal member 13 and the side plate 102 in the container portion 100, and the cylindrical portion 131 is not press-fitted, but the cylindrical portion 131 is press-fitted into the container portion 100. It may be configured to do so. When the cylindrical portion 131 is press-fitted into the container portion 100, the outer diameter dimension of each of the plurality of cylindrical portions 131 in the natural state may be slightly larger than the inner diameter dimension of the side plate 102 (container portion 100). Even when each cylindrical portion 131 is press-fitted into the container portion 100, a closed system leak occurs due to variations in the dimensions and press-fitting depth of the cylindrical portion 131 and the container portion 100, which are valuable cells. There is a considerable risk of losing. Therefore, if pressure is applied to the attachment and the culture vessel in the vertical direction using a pressing holder such as a holder or a clip, the pressing portion 120A of the base member 12 presses the entire plate portion 130A of the sealing member 13. As a result, the opening peripheral edges of the culture container 10B (plurality of container portions 100) are sealed in a well-balanced manner, and the sealing between the sealing member 13 and each of the plurality of container portions 100 can be made more reliable.

In the above embodiment, the case where the culture container unit 1A provided with the attachment 11A is connected to the vacuum container unit 3 and used has been described, but the present invention is not limited thereto. The attachment for perfusion culture according to the present invention can be used in various culture devices. As a means for supplying, collecting, or perfusing the culture solution to the culture container unit in which the perfusion culture attachment according to the present invention is attached to the culture container, a liquid feeding unit using various pumps such as a tube pump and a syringe pump is used. It is preferably available.

A10 Perfusion culture system M1 Culture solution 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1J, 1L Culture container unit 10A, 10B Culture container 100 Container part 101 Bottom plate 102 Side plate 103 Opening 11A, 11B, 11C, 11D , 11E, 11F, 11G, 11H, 11J, 11L attachment (attachment for perfusion culture)
12 Base member 120A Holding part 120B First bottomed cylinder part 121 Bottom wall (protruding part)
122 Side wall 123 Flange 124 Holding cylinder part 125 Extension piece 126 Through hole 127 Extension part 128 Extension piece 129 Projection 13 Sealing member 130A Plate part 130B Second bottomed cylinder part 131 Cylindrical part (cylindrical part)
132 Plate part 133 Circular protrusion 134 Bottom wall part (protruding part)
135 Thin-walled part 15 Fluid introduction flow path (flow path in attachment)
15A Seal member inner flow path 15B Base member inner flow path 151 Fluid inflow port 16 Fluid lead-out flow path (attachment inner flow path)
16A Seal member inner flow path 16B Base member inner flow path 161 Fluid outlet 17 Communication flow path 171 Hole 172 Groove 2 Culture solution container 21 Outlet port 3 Vacuum vessel unit 31 Tube body 311 Open end 32 Plug 41 First flow path 411 Partial flow path 412 Partial flow path 42 Second flow path 421 Partial flow path 422 Partial flow path 43 Puncture needle 44 Holder 51 First connection means 511 Female lure connector 512 Male lure connector 52 Second connection means 521 Female lure connector 522 Male lure Connector 6 constriction

Claims (21)

It is attached to a culture vessel having an opening at one end in the first direction and having at least one bottomed tubular container portion extending in the first direction, and the opening is closed to close the inner space of the container portion. It is an attachment for closed and perfused culture.
A seal member that is flexible and comes into contact with the container portion, and a base member that is made of a hard material separate from the seal member and that comes into contact with the seal member.
A protruding portion that fits inside the container portion and protrudes toward the other side in the first direction.
Perfusion, which is formed on one or both of the seal member and the base member, and has a plurality of in-attachment channels, one end of which leads to the outside and the other end of which leads to the inner space of the container portion. Attachment for culture. The base member has a bottom wall forming the protrusion, a tubular side wall that stands up from the bottom wall, and an annular flange that extends radially outward from the end of the side wall.
The perfusion culture according to claim 1, wherein the sealing member has a plate portion that is interposed between the flange and the peripheral edge of the opening in the first direction and can be in contact with all the peripheral edges of the opening. attachment. The sealing member has a tubular portion extending from the plate portion to the other side in the first direction.
The attachment for perfusion culture according to claim 2, wherein the tubular portion is press-fitted between the container portion and the side wall. The base member has a bottom wall forming the protrusion and a tubular side wall that rises from the bottom wall.
The attachment for perfusion culture according to claim 1, wherein the seal member is press-fitted between the container portion and the side wall. The seal member has a seal member bottom wall portion that constitutes the protrusion, and has a seal member bottom wall portion.
The bottom wall portion of the sealing member has a transparent thin-walled portion having a thickness relatively smaller than that of other portions.
According to any one of claims 2 to 4, a through hole is formed in the bottom wall of the base member so as to overlap the thin-walled portion in the first direction and penetrates in the first direction. The attachment for perfusion culture described. The seal member has an annular shape that extends outward in the radial direction from the bottom wall portion of the seal member constituting the protruding portion, the tubular portion that stands up from the bottom wall portion of the seal member, and the end portion of the tubular portion. With a plate part,
The bottom wall portion of the sealing member has a transparent thin-walled portion having a thickness relatively smaller than that of other portions.
The attachment for perfusion culture according to claim 1, wherein the base member has an annular flange that can come into contact with the entire upper surface of the plate portion. The attachment for perfusion culture according to any one of claims 2 to 4, wherein the surface of the bottom wall facing the other side in the first direction is subjected to a hydrophilic treatment or a hydrophobic treatment. The attachment for perfusion culture according to any one of claims 1 to 7, wherein the base member is transparent. The attachment for perfusion culture according to any one of claims 1 to 8, wherein one end of the flow path in each of the attachments faces a direction substantially perpendicular to the first direction. The plurality of attachment inner flow paths are a fluid introduction flow path for introducing a fluid from the outside into the inner space of the container portion and a fluid derivation flow for guiding the fluid from the inner space of the container portion to the outside. The attachment for perfusion culture according to any one of claims 1 to 9, which comprises a tract. The culture vessel includes a plurality of the vessel portions arranged apart from each other in the in-plane direction perpendicular to the first direction.
The base member includes a plurality of first bottomed tubular portions each having a bottom wall that fits inside the container portion and constitutes the protruding portion, and a tubular side wall that rises from the bottom wall.
It has a plate-shaped holding portion that extends outward from the end of each of the side walls and is integrated.
The first aspect of the present invention, wherein the sealing member has a plate portion that is interposed between the pressing portion and the peripheral edge portion of each of the openings in the first direction and is capable of contacting all the peripheral edges of the respective openings. Attachment for perfusion culture. Each of the sealing members has a plurality of tubular portions extending from the plate portion to the other side in the first direction and into which the first bottomed tubular portion can be inserted.
The attachment for perfusion culture according to claim 11, wherein the tubular portion is press-fitted between the container portion and the side wall. The attachment for perfusion culture according to claim 11 or 12, wherein the base member is transparent. The culture vessel includes a plurality of the vessel portions arranged apart from each other in the in-plane direction perpendicular to the first direction.
The base member has a plate-shaped pressing portion and has a plate-shaped pressing portion.
The seal member is interposed between the holding portion and the peripheral edge portion of each opening in the first direction, and is capable of contacting all the peripheral edges of the respective openings, and the first plate portion from the plate portion. The attachment for perfusion culture according to claim 1, further comprising a tubular portion extending to the other side in the direction and a plurality of second bottomed tubular portions each having a bottom wall portion of a seal member constituting the protruding portion. .. Each of the seal member bottom wall portions has a transparent thin-walled portion whose thickness is relatively smaller than that of other portions.
The 14th aspect of the present invention, wherein each of the holding portions is positioned so as to overlap with any of the thin-walled portions in the first direction view, and a plurality of through holes penetrating in the first direction are formed. Attachment for perfusion culture. The attachment for perfusion culture according to any one of claims 11 to 15, wherein one end of the flow path in each of the attachments faces a direction substantially perpendicular to the first direction. Each of the attachment inner flow paths is formed in the seal member, and one end thereof is formed in the seal member inner flow path facing one side in the first direction and the holding portion. The attachment for perfusion culture according to any one of claims 11 to 15, further comprising a flow path in the base member that communicates with one end and penetrates in the first direction. The plurality of attachment inner flow paths are the plurality of fluid introduction flow paths for introducing a fluid into the inner space of each of the plurality of container portions from the outside, and the outside from the inner space of each of the plurality of container portions. The attachment for perfusion culture according to any one of claims 11 to 17, further comprising a plurality of fluid derivation channels for deriving the fluid. The attachment for perfusion culture according to any one of claims 11 to 17, wherein the seal member has at least one communication flow path for communicating the inner spaces of the container portion with each other. The plurality of attachment inner flow paths include a fluid introduction flow path for introducing a fluid from the outside into the inner space of the first container portion among the plurality of container portions, and the plurality of container portions. Includes a fluid lead-out flow path for leading the fluid out of the inner space of the second container portion.
The claim is provided so that the connecting flow path is sequentially provided from the inner space of the first container portion to the inner space of the adjacent container portion and sequentially to the inner space of the second container portion. 19. The attachment for perfusion culture according to 19. The connecting flow path is configured to include a groove formed on a surface of the plate portion facing the one side in the first direction.
The perfusion culture according to claim 19 or 20, wherein the groove forms a part of the connecting flow path by overlapping the holding portion on the plate portion and blocking the one side end in the first direction. Attachment for.

Images