Cell culture container

Abstract

Provided is a cell culture vessel suitable for improving problems occurring in perfusion culture in a closed system.　The cell culture vessel is equipped with: a bottomed cylindrical vessel body 1 that has an opening 110 at one end x1 in a first direction and a vessel bottom 14 closing the other end x2 in the first direction; and a closing body 2 that seals the inner space of the vessel body 1 by closing the opening 110. The closing body 2 has a filter press 22 and a membrane filter 23 that is positioned on the other side x2 in the first direction relative to the filter press 22. A first closed area S1 sandwiched between the membrane filter 23 and the vessel bottom 14, and a second closed area S2 sandwiched between the filter press 22 and the membrane filter 23 are formed. The closing body 2 has a first flow channel 211 and a second flow channel 223. The first flow channel 211 communicates with the outside at one end and with the first closed area S1 at the other end. The second flow channel 223 communicates with the outside at one end and with the second closed area S2 at the other end.

Description

Technical Field

[0001] The present disclosure relates to a cell culture container for culturing cells, and particularly to a cell culture container used for perfusion culture that automatically supplies a culture solution at a constant low speed at all times and simultaneously discharges the same amount of the culture solution.

Background Art

[0002] When culturing cells, culture containers such as dishes, well plates, and flasks are widely used. Dishes and well plates have gaps even when a lid is placed, whether there is a lid or not, and cannot be sealed. In the case of a flask, it can be sealed, but in all of these conventional culture containers, opening and closing the lid is required for operations such as replacing the culture solution. Therefore, although these culture containers are highly versatile for cell culture applications for research purposes, for cell culture applications for medical purposes, there are problems such as the risk of contamination, problems of personnel with specialized skills and costs, and difficulty in uniformizing the quality of cultured cells.

[0003] Recently, various automatic culture devices have been developed to solve these problems (see, for example, Patent Document 1). In a culture container used in an automatic culture device, for example, a pair of ports are provided. These ports are provided, for example, on the lid of the culture container, and each extends downward from the lid and is disposed inside the culture container. When culturing cells, a new culture solution is supplied to the culture container through one port, and the culture solution inside the culture container is discharged to the outside through the other port. In order to supply the culture solution from one port and discharge it from the other port, a pump suitable for each port may be connected. In that case, even if there is a layer of culture solution and gas in the container, the liquid will flow. However, if the amount of the culture solution supplied from one port and the amount of the culture solution discharged from the other port do not exactly match, the liquid in the container will run out or overflow when the culture is carried out over a long period. In order to avoid this, when using a water level sensor or the like, the device becomes large-scale.

[0004] To avoid these inconveniences, a simple method is effective: using one pump per container, supplying the culture medium under positive pressure from one port and discharging it through the other port via overflow, or discharging the culture medium under negative pressure from one port and drawing it in from the other. This is perfusion culture, where the culture area inside the container is filled with culture medium, and the culture medium is continuously supplied at a constant low rate while the same amount of culture medium is simultaneously discharged. In this perfusion culture, it is important that the system is closed and that no gas layer or bubbles are created in the culture area of ​​the culture vessel in order to achieve a smooth liquid flow. However, even when trying to fill the inside of a closed culture vessel with culture medium, a small amount of air (bubbles) may remain inside the container. For example, if the port extends downward from the lid and is located inside the container, it was not possible to discharge the air remaining inside the container to the outside. Furthermore, a challenge of perfusion culture is that while there is no problem if the target cells are adherent cells that proliferate using the bottom surface of the container as a scaffold, if they are suspension cells, there is a risk that they will be washed away along with the discharged liquid. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2017-79633 [Overview of the project] [Problems that the invention aims to solve]

[0006] This disclosure was conceived under these circumstances, and its primary objective is to provide a cell culture vessel suitable for improving the disadvantages of performing perfusion culture in a closed system. [Means for solving the problem]

[0007] To address the above challenges, this disclosure employs the following technical measures.

[0008] The cell culture vessel provided by this disclosure comprises a bottomed cylindrical container body having an opening at one end in a first direction and a container bottom that closes the other end in the first direction, and a closure body that closes the opening and seals the inner space of the container body, wherein the closure body has a first ceiling portion that separates the outside from the inner space of the container body, and a membrane filter disposed on the other side in the first direction relative to the first ceiling portion and through which liquid can pass, forming a first closed area sandwiched between the membrane filter and the container bottom portion, and a second closed area sandwiched between the first ceiling portion and the membrane filter, wherein the closure body has at least one first channel and one second channel, the first channel having one end open to the outside and the other end open to the first closed area, and the second channel having one end open to the outside and the other end open to the second closed area.

[0009] In a preferred embodiment, the first ceiling portion has a ceiling recess formed on one side in the first direction, and the ceiling recess is connected to the second flow path.

[0010] In a preferred embodiment, the occluding body has a second ceiling portion that separates the outside from the inner space of the container body, and a gas permeable membrane that is spaced apart from the second ceiling portion on the other side in the first direction, and a third closed area is formed between the second ceiling portion and the gas permeable membrane, and the gas permeable membrane partitions the first closed area and the third closed area, and the occluding body has a plurality of third channels, each of which has one end open to the outside and the other end open to the third closed area.

[0011] In a preferred embodiment, the container body further comprises an annular sealing member interposed between the container body and the closing body, the first cylindrical portion extending in the first direction and having the opening, and an outer lid having an annular top plate portion and a second cylindrical portion extending from the outer peripheral edge of the top plate portion to the other side in the first direction and fitted onto the first cylindrical portion, wherein at least one of the container body and the outer lid is provided with locking means for preventing relative movement between the first cylindrical portion and the second cylindrical portion while the top plate portion is in contact with the closing body.

[0012] In a preferred embodiment, the bottom of the container has a first bottom surface facing one side in the first direction, and the first bottom surface is provided with a plurality of recesses, which are densely arranged along the in-plane direction of the first bottom surface.

[0013] Other features and advantages of this disclosure will become more apparent from the detailed description below, with reference to the accompanying drawings. [Brief explanation of the drawing]

[0014] [Figure 1] This is a perspective view showing a cell culture vessel according to the first embodiment of this disclosure. [Figure 2] Figure 1 is a plan view of the cell culture vessel shown. [Figure 3] This is a cross-sectional view along line III-III in Figure 2. [Figure 4] This is a cross-sectional view along the line IV-IV in Figure 2. [Figure 5] This is a cross-sectional view along the VV line in Figure 2. [Figure 6] This is a cross-sectional view similar to Figure 3, showing the state of use of a cell culture vessel according to the first embodiment of this disclosure. [Figure 7] This is a cross-sectional view similar to Figure 3, showing the state of use of a cell culture vessel according to the first embodiment of this disclosure. [Figure 8] This is a cross-sectional view similar to Figure 3, showing a cell culture vessel according to a modified example of the first embodiment. [Figure 9]It is a view showing the arrangement state of a plurality of recesses formed on the first bottom surface of the container bottom, and is a view seen in the first direction.

Mode for Carrying Out the Invention

[0015] Hereinafter, preferred embodiments of the present disclosure will be specifically described with reference to the drawings.

[0016] In the present disclosure, terms such as "first", "second", "third", etc. are merely used as labels and do not necessarily intend to assign an order to those objects.

[0017] <First Embodiment> Figs. 1 to 5 show a cell culture container according to the first embodiment of the present disclosure. The cell culture container A1 of the present embodiment includes a container body 1, a closing body 2, a sealing member 3, and an outer lid 4. Fig. 1 is a perspective view of the cell culture container A1. Fig. 2 is a plan view of the cell culture container A1. Fig. 3 is a cross-sectional view taken along line III-III of Fig. 2. Fig. 4 is a cross-sectional view taken along line IV-IV of Fig. 2. Fig. 5 is a cross-sectional view taken along line V-V of Fig. 2.

[0018] As shown in Figs. 3 to 5, in the present embodiment, the container body 1 includes a first cylindrical portion 11, a connecting portion 12, a cylindrical portion 13, and a container bottom portion 14. The first cylindrical portion 11 extends in the vertical direction (first direction x). The container body 1 has an opening 110 at the upper end 112 which is one end of the first cylindrical portion 11 in the first direction x (hereinafter, appropriately referred to as "one end x1 in the first direction"). The first cylindrical portion 11 is generally cylindrical, and a male screw 111 is formed on the outer peripheral surface of the first cylindrical portion 11. The connecting portion 12 is connected to the other end of the first cylindrical portion 11 in the first direction x (hereinafter, appropriately referred to as "the other end x2 in the first direction"), and is an annular portion protruding radially inward. The cylindrical portion 13 is connected to the radially inner side of the connecting portion 12 and extends in the first direction x.

[0019] One end of the cylindrical portion 13 on one side x1 in the first direction is located on the other side x2 in the first direction with respect to one end of the first cylindrical portion 11 on one side x1 in the first direction, and the other end of the cylindrical portion 13 on the other side x2 in the first direction is located on the other side x2 in the first direction with respect to the other end of the first cylindrical portion 11 on the other side x2 in the first direction. The surface of the connecting portion 12 facing one side x1 in the first direction constitutes a part of the concave groove 121. The concave groove 121 is an annular groove formed by the surface of the connecting portion 12 facing one side x1 in the first direction, the inner peripheral surface of the first cylindrical portion 11 connected thereto, and the outer peripheral surface of the cylindrical portion 13, and is a part for arranging the seal member 3.

[0020] The container bottom 14 is connected to the lower end (the other end x2 in the first direction) of the cylindrical portion 13 and closes the inside of the cylindrical portion 13. In the present embodiment, an extension piece 131 is provided at an appropriate position at the lower end of the cylindrical portion 13. The extension piece 131 is a portion that is connected to the cylindrical portion 13 and extends radially outward.

[0021] The container body 1 is formed of, for example, a translucent or transparent plastic material. As the plastic material, for example, in addition to polystyrene, materials having preferably transparency such as polycarbonate, cycloolefin polymer, cycloolefin copolymer, etc. are used, but are not limited thereto.

[0022] The container bottom 14 is flat and has a first bottom surface 14a. The first bottom surface 14a faces one side x1 in the first direction and is a flat cell culture surface for culturing cells. A surface treatment for improving cell adhesion is appropriately applied to this cell culture surface (the first bottom surface 14a of the container bottom 14) as needed. Examples of the surface treatment include hydrophilic treatments such as corona discharge treatment and plasma treatment.

[0023] The closing body 2 is for closing the opening 110 of the container body 1 and sealing the inner space of the container body 1. As shown in FIGS. 3 to 5, the closing body 2 includes a first annular plate portion 21, a filter presser 22, a membrane filter 23, a bulging portion 24, and a gas permeable membrane 25.

[0024] The first annular plate portion 21 is annular when viewed in the first direction x, and in this embodiment, it is composed of a substantially annular plate-like portion. The first annular plate portion 21 has a first flow path 211, a discharge cylindrical portion 212, and an inner locking portion 213. The discharge cylindrical portion 212 is cylindrical and extends in one direction x1, and is the portion for positioning the filter retainer 22. The inner locking portion 213 is an annular projection that protrudes radially inward from the lower end (the other end x2 in the first direction) of the discharge cylindrical portion 212. The first flow path 211 will be described later.

[0025] The filter retainer 22 is a component that holds the membrane filter 23 and is fitted into the discharge cylindrical portion 212. The filter retainer 22 has a cylindrical portion 221, a ceiling recess 222, and a second flow path 223. The cylindrical portion 221 is fitted inside the discharge cylindrical portion 212. The ceiling recess 222 is connected to the cylindrical portion 221 and closes the inside of the cylindrical portion 221. The ceiling recess 222 is recessed on one side x1 in the first direction. The first annular plate portion 21 and the filter retainer 22 (cylindrical portion 221) hold the membrane filter 23 from both sides in the first direction x. Detailed illustrations are omitted, but an O-ring is interposed between the cylindrical portion 221 and the discharge cylindrical portion 212. As a result, the fitting portion between the first annular plate portion 21 and the filter retainer 22 is sealed, and the space x2 on the other side in the first direction from the filter retainer 22 (ceiling recess 222) is separated from the outside. The second flow path 223 will be described later.

[0026] The membrane filter 23 is a liquid-permeable membrane through which liquids can pass. For example, liquids such as culture medium can pass through the membrane filter 23. Gases such as air can also pass through the membrane filter 23. On the other hand, non-liquid contents such as cultured cells are trapped and cannot pass through the membrane filter 23. The membrane filter 23 is positioned on the other side x2 of the first direction relative to the filter retainer 22. The provision of this membrane filter 23 creates a first closed area S1 and a second closed area S2 within the cell culture vessel A1, which are partitioned from each other. The first closed area S1 is sandwiched between the membrane filter 23 and the bottom of the container 14. The second closed area S2 is sandwiched between the filter retainer 22 and the membrane filter 23. The constituent material of the membrane filter 23 is not particularly limited; for example, a hydrophilic filter made of PET (polyester) or PC (polycarbonate) can be used. The filter retainer 22 described above corresponds to an example of the "first ceiling portion".

[0027] The bulging portion 24 is connected to the inner periphery of the first annular plate portion 21. The bulging portion 24 closes the inside of the first annular plate portion 21 when viewed in the first direction x. The bulging portion 24 also protrudes from the first annular plate portion 21 to one side x1 in the first direction. The bulging portion 24 has a plurality of third channels 241. The third channels 241 will be described later.

[0028] The gas permeable membrane 25 is a thin membrane that is permeable to gas. On the other hand, the gas permeable membrane 25 is impermeable to liquids, as liquids cannot pass through it. The gas permeable membrane 25 is attached to the first annular plate portion 21. Specifically, the gas permeable membrane 25 is fixed in close contact with the lower surface of the first annular plate portion 21 (the surface facing the other side x2 in the first direction) by appropriate means such as adhesive. With the provision of this gas permeable membrane 25, a third closed area S3 is formed inside the cell culture vessel A1, separated from the first closed area S1. The third closed area S3 is sandwiched between the bulging portion 24 and the gas permeable membrane 25. The first closed area S1 is sandwiched between the gas permeable membrane 25 and the bottom portion 14 of the container. The constituent material of the gas permeable membrane 25 is not particularly limited, and examples include hydrophobic filters made of PET (polyester) or PC (polycarbonate). The bulging portion 24 mentioned above corresponds to an example of the "second ceiling portion".

[0029] The sealing member 3 is interposed between the container body 1 and the closure body 2, and separates the inner space of the container body 1 from the outside. The sealing member 3 is annular in shape and is sandwiched between the cylindrical portion 13 of the container body 1 and the first annular plate portion 21 of the closure body 2 in the first direction x. In addition, a part of the sealing member 3 is positioned in the groove 121.

[0030] The sealing member 3 is made of a flexible and elastic soft material. Examples of materials that make up the sealing member 3 include silicone rubber and elastomer resin. As will be described in detail later, considering the contact between the sealing member 3 and the contents (e.g., culture medium), medical-grade silicone rubber, which is non-cytotoxic and biocompatible, is more preferable as the material for the sealing member 3. Furthermore, the hardness of the sealing member 3 is preferably such that the rubber hardness is around 20 to 40 degrees.

[0031] The outer cover 4 has a top plate portion 41 and a second cylindrical portion 42. The top plate portion 41 is roughly annular in shape and is the portion that sandwiches the sealing member 3 between the first annular plate portion 21 (closing body 2) and the cylindrical portion 13 (container body 1). A through hole 411 is formed on the radially inner side of the top plate portion 41. The through hole 411 is surrounded by the top plate portion 41 when viewed in the thickness direction (first direction x) of the top plate portion 41.

[0032] The second cylindrical portion 42 extends from the outer peripheral edge of the top plate portion 41 in the thickness direction of the top plate portion 41 (the first direction x, which is the vertical direction in the figure), and is generally cylindrical in shape. A female thread 421 is formed on the inner circumferential surface of the second cylindrical portion 42, and this female thread 421 can be screwed onto the male thread 111 of the container body 1 (first cylindrical portion 11).

[0033] The outer lid 4 is formed from, for example, an opaque, translucent, or transparent plastic material. Examples of such plastic materials include, but are not limited to, polyethylene, polypropylene, polystyrene, and polycarbonate.

[0034] The first channel 211, the second channel 223, and the multiple third channels 241 are cylindrical passages that connect to the outside and to the internal space of the cell culture vessel A1. As shown in Figure 3, one end of the first channel 211 (end x1 in the first direction) leads to the outside, and the other end (end x2 in the first direction) leads to the first closed area S1. One end of the second channel 223 (end x1 in the first direction) leads to the outside, and the other end (end x2 in the first direction) leads to the second closed area S2. The ceiling recess 222 of the filter retainer 22 that defines the second closed area S2 is connected to the second channel 223. As shown in Figure 5, each of the multiple third channels 241 has one end (end x1 in the first direction) leading to the outside, and the other end (end x2 in the first direction) leads to the third closed area S3.

[0035] Next, the method of use and function of cell culture vessel A1 will be explained with reference to Figures 6 and 7.

[0036] Cell culture vessel A1 is used for perfusion culture, in which culture medium is supplied into the vessel and the same amount of culture medium is simultaneously discharged from the vessel. The cultured cells and culture medium contained in cell culture vessel A1 are not particularly limited.

[0037] In cell culture using cell culture vessel A1, the vessel body 1, occluding body 2, sealing member 3, and outer lid 4 are assembled in advance. Next, culture medium is supplied to cell culture vessel A1 and air is discharged via the first channel 211 and the second channel 223, filling the inner space of the vessel body 1 (first closed area S1) with culture medium. Here, as shown in Figure 6, tubes 8 are connected to the first channel 211 and the second channel 223, respectively. A perfusion pump (not shown) is connected to one end of the first channel 211 (one end in the first direction x1) via tube 8, and a drainage container (not shown) is connected to one end of the second channel 223 (one end in the first direction x1) via tube 8. Before filling the first closed area S1 with culture medium, cultured cells are supplied to the first closed area S1 along with the culture medium.

[0038] The other end of the first channel 211 (the other end x2 in the first direction) leads to the first closed area S1. As a result, the first closed area S1 is filled with culture medium supplied through the first channel 211. Cultured cells supplied through the first channel 211 also flow into the first channel 211. Air in the first closed area S1 passes through a membrane filter 23 that allows gas to pass through, and is discharged to the outside through the second channel 223 via the second closed area S2. When performing perfusion culture, new culture medium C is supplied to the first closed area S1 through the first channel 211. The culture medium C in the first closed area S1 passes through the membrane filter 23 and flows into the second closed area S2. The culture medium C in the second closed area S2 is then discharged to the outside through the second channel 223. In the perfusion culture process, the culture medium C in the cell culture vessel A1 (first closed area S1 and second closed area S2) is continuously replaced little by little.

[0039] In the cell culture vessel A1, the occluding body 2 has a membrane filter 23. This membrane filter 23 divides the inside of the cell culture vessel A1 into a first closed area S1 and a second closed area S2. As described above, the culture medium C supplied from the first channel 211 into the cell culture vessel A1 (first closed area S1) passes through the membrane filter 23 and is discharged to the outside via the second closed area S2 and the second channel 223. On the other hand, since the perfusion speed is extremely slow, cultured cells are unlikely to float against their own weight and be drawn to the membrane filter 23 located on the ceiling recess 222 side. Even if cultured cells in the culture medium C reach the membrane filter 23, they cannot pass through the membrane filter 23. As a result, even if cultured cells float in the culture medium C in the first closed area S1, it is prevented that cultured cells in the culture medium C are improperly discharged from the second channel 223.

[0040] A small amount of air may remain in the first closed area S1 of the cell culture vessel A1. The filter retainer 22, which forms the second closed area S2 located above the membrane filter 23 (one side x1 in the first direction), has a ceiling recess 222 that is recessed on one side x1 in the first direction, and this ceiling recess 222 is connected to the second channel 223. With this configuration, when the cell culture vessel A1 is tilted so that the second channel 223 is positioned above, the air bubbles in the first closed area S1 move through the membrane filter 23 to the second closed area S2. By operating the perfusion pump in this state, the air bubbles remaining in the second closed area S2 move into the second channel 223 through the ceiling recess 222 and can be discharged to the outside of the cell culture vessel A1.

[0041] The occluding body 2 includes a bulge 24 and a gas permeable membrane 25. In the cell culture vessel A1, a third closed area S3 is formed between the bulge 24 and the gas permeable membrane 25, and the gas permeable membrane 25 divides the first closed area S1 and the third closed area S3. The bulge 24 (occluding body 2) has a plurality of third channels 241. Each third channel 241 has one end (first direction one side x1 end) that is open to the outside and the other end (first direction other side x2 end) that is open to the third closed area S3. With this configuration, the third closed area S3 can be maintained in a predetermined gas atmosphere by supplying and discharging a predetermined gas to the bulge 24 through the plurality of third channels 2411 as needed. The contents of the first closed area S1 (culture medium C and cultured cells) are able to maintain a permeable state with the third closed area S3 via the gas permeable membrane 25, and can take in gas from the third closed area S3. Furthermore, by changing the type of gas supplied to the third channel 241, the gas atmosphere in the third closed area S3 can be easily and quickly changed. Compared to creating a predetermined gas atmosphere using, for example, a CO2 incubator, it is possible to aerate the desired gas atmosphere with a simpler configuration.

[0042] Cell culture vessel A1 comprises a container body 1 and a closure body 2, as well as a sealing member 3 and an outer lid 4. The sealing member 3 is interposed between the container body 1 and the closure body 2. The second cylindrical portion 42 of the outer lid 4, which is fitted onto the first cylindrical portion 11 of the container body 1, has a female thread 421. By tightening the female thread 421 onto the male thread 111 of the container body 1 (first cylindrical portion 11), the sealing member 3 is sandwiched between the upper end of the cylindrical portion 13 of the container body 1 and the first annular plate portion 21 of the closure body 2, maintaining the assembled state. As a result, the contents (cultured cells and culture medium C) contained in the container body 1 are sealed. Therefore, during the perfusion culture process, the culture medium perfusion operation can be performed while maintaining the closed state of the cell culture vessel A1, thus avoiding the risk of contamination. Consequently, with cell culture vessel A1, the state of the culture medium C can be kept in good condition, ensuring the quality of the cells. The male thread 111 of the container body 1 (first cylindrical part 11) and the female thread 421 of the outer lid 4 (second cylindrical part 42) are examples of "locking means".

[0043] <Modified form of the first embodiment> Figures 8 and 9 show a cell culture vessel according to a modified example of the first embodiment. Figure 8 is a cross-sectional view similar to that shown in Figure 3 of the above embodiment. In the drawings from Figure 8 onward, elements that are the same as or similar to the cell culture vessel A1 of the above embodiment are denoted by the same reference numerals as in the above embodiment, and their descriptions are omitted as appropriate.

[0044] In the cell culture vessel A11 of this example of modification, a plurality of recesses 141 are formed on the first bottom surface 14a of the vessel body 1 (bottom surface 14). As shown in Figure 9, the plurality of recesses 141 are densely arranged along the in-plane direction of the first bottom surface 14a. The plurality of recesses 141 are depressions that facilitate the three-dimensional aggregation of cultured cells and the formation of spheroids.

[0045] In the modified cell culture vessel A11, the occlusion body 2 has a membrane filter 23. This membrane filter 23 divides the inside of the cell culture vessel A11 into a first closed area S1 and a second closed area S2. The culture medium C supplied from the first channel 211 into the cell culture vessel A11 (first closed area S1) passes through the membrane filter 23 and is discharged to the outside via the second closed area S2 and the first channel 211. On the other hand, since the perfusion speed is extremely slow, cultured cells are hardly attracted to the membrane filter 23 located on the ceiling recess 222 side by floating against their own weight. Even if cultured cells in the culture medium C reach the membrane filter 23, they cannot pass through the membrane filter 23. As a result, even if cultured cells float in the culture medium C in the first closed area S1, it is prevented that cultured cells in the culture medium C are improperly discharged from the second channel 223.

[0046] In this modified example, the first bottom surface 14a of the container body 1 (container bottom 14) is provided with a plurality of recesses 141. The container bottom 14 (first bottom surface 14a) including the plurality of recesses 141 may be coated with a cell-non-adhesion coating that is advantageous for spheroid formation. With this configuration, cells aggregate three-dimensionally, and spheroids are easily formed. According to this disclosure, air bubbles are less likely to remain in the perfusion liquid, and the perfusion speed is extremely slow, so spheroids are less likely to float up unjustly from the recesses 141, and spheroids are not discharged by the membrane filter 23 provided on the ceiling recess 222 side. When the formed spheroids are to be collected in a closed manner, they can be collected by suction from the first channel 211 side. In addition, the cell culture container A11 of this modified example exhibits the same effects as the cell culture container A1 of the above embodiment.

[0047] Although specific embodiments of the present disclosure have been described above, the cell culture vessel relating to the present disclosure is not limited to the above embodiments. The specific configuration of each part of the cell culture vessel relating to the present disclosure can be modified in various ways. [Explanation of symbols]

[0048] A1, A11, A2: Cell culture vessel, 1: Vessel body, 11: First cylindrical part, 110: Opening, 111: Male screw (locking means), 112: Upper end (one side end of the first cylindrical part in the first direction), 12: Connecting part, 121: Groove, 13: Cylindrical part, 131: Extension piece, 14: Bottom of the vessel, 14a: First bottom surface, 141: Recess, 2: Closure body, 21: First annular plate part, 211: First flow path, 212: Discharge cylindrical part, 213: Inner locking part, 22: Filter retainer (first ceiling part), 221: Cylindrical section, 222: Ceiling recess, 223: Second channel, 23: Membrane filter, 24: Bulging section (second ceiling section), 241: Third channel, 25: Gas permeable membrane, 3: Sealing member, 4: Outer cover, 41: Top plate section, 411: Through hole, 42: Second cylindrical section, 421: Female screw (locking means), 8: Tube, C: Culture medium, S1: First closed area, S2: Second closed area, S3: Third closed area, x: First direction, x1: One side of the first direction, x2: Other side of the first direction

Claims

1. A bottomed cylindrical container body having an opening at one end in the first direction and a container bottom that closes the other end in the first direction, It comprises a closing body that closes the opening and seals the inner space of the container body, The sealing body comprises a first ceiling portion that separates the outside from the inner space of the container body, and a membrane filter positioned on the other side in the first direction relative to the first ceiling portion, through which liquid can pass. The inner space of the container body has a first closed area sandwiched between the membrane filter and the bottom of the container, and a second closed area sandwiched between the first ceiling and the membrane filter. The occluding body has at least one first channel and one second channel, The first channel has one end that is open to the outside and the other end that is open to the first closed area. A cell culture vessel in which the second channel has one end that is open to the outside and the other end that is open to the second closed area.

2. The first ceiling portion has a ceiling recess formed on one side in the first direction, The cell culture vessel according to claim 1, wherein the ceiling recess is connected to the second channel.

3. The sealing body comprises a second ceiling portion that separates the outside from the inner space of the container body, and a gas permeable membrane that is spaced apart from the second ceiling portion on the other side in the first direction and is gas permeable. A third closed area is formed in the inner space of the container body, sandwiched between the second ceiling portion and the gas permeable membrane, and the gas permeable membrane separates the first closed area and the third closed area. The aforementioned occluding body has a plurality of third channels, The cell culture vessel according to claim 1 or 2, wherein one end of the third channel is open to the outside and the other end is open to the third closed area.

4. The container body includes a first cylindrical portion that extends in the first direction and has the opening, An annular sealing member interposed between the container body and the closure body, The outer cover further comprises an annular top plate portion and a second cylindrical portion extending from the outer peripheral edge of the top plate portion to the other side in the first direction and fitted onto the first cylindrical portion, The cell culture container according to any one of claims 1 to 3, wherein at least one of the container body and the outer lid is provided with a locking means for preventing relative movement between the first cylindrical portion and the second cylindrical portion while the top plate portion is in contact with the closing body.

5. The bottom of the container has a first bottom surface facing one side in the first direction, The first bottom surface is provided with a plurality of recesses, The cell culture vessel according to any one of claims 1 to 4, wherein the plurality of recesses are densely arranged along the in-plane direction of the first bottom surface.

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