Cell culture apparatus

Abstract

To provide a cell culture tool that can be easily attached / detached in order to perform cell culture on both faces of a cell culture membrane.SOLUTION: A cell culture tool has: cylindrical members each having a body cylindrical part and a culture membrane arranged for an opening end of the body cylindrical part; and a cylindrical covering member installed to the body cylindrical part having (i) a first cylindrical part which covers an outer peripheral surface of the body cylindrical part in the case of being installed to the cylindrical member, and (ii) a second cylindrical part which is adjacent to the first cylindrical part in an axial direction of the covering member, and forms a container having the culture membrane as a bottom face in the case of being installed to the cylindrical member, where the first cylindrical part has a first area in which a length in the axial direction is a first length, and a second area in which a length is a second length shorter than the first length.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a cell culture apparatus. [Background technology]

[0002] In recent years, cell culture has been performed on both sides of a cell culture membrane. Therefore, a cell culture device for performing double-sided culture has been proposed (for example, Patent Document 1 and Non-Patent Document 1). In the cell culture device of Non-Patent Document 1, a cylindrical optional ring is attached to the outside of the insert in order to perform cell culture on the surface of the cell culture membrane that faces the outside of the insert. The optional ring is attached and detached using tweezers. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. WO2012 / 045368 [Non-patent literature]

[0004] [Non-Patent Document 1] "Kanto Chemical Co., Ltd. | Product Information | ad-MED Vidrigel (registered trademark) 2", [online], [searched on November 9, 2021], Internet<URL:https: / / products.kanto.co.jp / web / index.cgi?c=t_product_table&pk=55> Summary of the Invention [Problem to be solved by the invention]

[0005] However, with the option ring described in Non-Patent Document 1, it may be difficult to attach and detach the option ring to and from the insert. [Means for solving the problem]

[0006] The present disclosure can be realized in the following forms. According to one embodiment of the present disclosure, there is provided a cell culture instrument, comprising: a cylindrical member having a main cylindrical portion and a culture membrane disposed at an open end of the main cylindrical portion; and a cylindrical covering member attached to the main cylindrical portion, the covering member having (i) a first cylindrical portion that covers at least the entire outer periphery of the open end when attached to the cylindrical member, and (ii) a second cylindrical portion that is adjacent to the first cylindrical portion in the axial direction of the covering member and forms a container having the culture membrane as a bottom surface when attached to the cylindrical member, the first cylindrical portion having a first portion having a first length in the axial direction and a second portion having a second length shorter than the first length, the covering member being separate from the cylindrical member, and an end surface of the first cylindrical portion having an elliptical shape and inclined with respect to a central axis of the first cylindrical portion.

[0007] (1) According to one embodiment of the present disclosure, there is provided a cell culture instrument. The cell culture instrument includes a cylindrical member having a cylindrical main body portion and a culture membrane disposed at an open end of the cylindrical main body portion, and a cylindrical covering member attached to the cylindrical main body portion, the covering member having (i) a first cylindrical portion that covers an outer circumferential surface of the cylindrical main body portion when attached to the cylindrical member, and (ii) a second cylindrical portion that is adjacent to the first cylindrical portion in the axial direction of the covering member and forms a container with the culture membrane as a bottom surface when attached to the cylindrical member, the first cylindrical portion having a first portion having a first length in the axial direction and a second portion having a second length shorter than the first length. According to this embodiment, the first cylindrical portion has a second portion and a first portion that are different in length from each other. Therefore, the central axis of the covering member is tilted with respect to the central axis of the main cylindrical portion, and the second portion is brought into contact with the cylindrical portion before the first portion. From this position, the covering member can be attached to the cylindrical portion by moving the covering member around the vicinity of the second portion as a fulcrum so that the central axis of the covering member is parallel to the central axis of the cylindrical portion. According to this attachment method, the range of the inner peripheral surface of the covering member that rubs against the outer peripheral surface of the cylindrical portion during the process of moving the covering member is limited to a part of the inner peripheral surface around the first portion. Therefore, compared to the case where a covering member having the same axial length is attached over the circumferential direction, the range where friction occurs between the inner peripheral surface of the covering member and the outer peripheral surface of the cylindrical portion during attachment can be reduced. Therefore, the force required for attaching and detaching the covering member can be reduced, making it easier to attach and detach, and improving workability. (2) In the cell culture instrument of the above embodiment, the inner diameter of the first cylindrical portion may be larger than the inner diameter of the second cylindrical portion, and the covering member may further have a step portion at a boundary between an inner circumferential surface of the first cylindrical portion and an inner circumferential surface of the second cylindrical portion, the step portion being formed by the difference between the inner diameter of the first cylindrical portion and the inner diameter of the second cylindrical portion. According to this embodiment, when the covering member is attached to the cylindrical member, the open end of the cylindrical member can be abutted against the step portion, thereby facilitating positioning. (3) In the cell culture instrument of the above embodiment, the covering member may be flexible, and the radial thickness of the first cylindrical portion may be thinner than the radial thickness of the second cylindrical portion. According to this embodiment, the covering member can be easily attached to and detached from the cylindrical member by the first cylindrical portion having good flexibility, while maintaining stability when used as a container with a culture membrane as a bottom surface. (4) In the cell culture instrument of the above embodiment, the inner diameter of the main cylindrical portion and the inner diameter of the second cylindrical portion may be the same. According to this embodiment, when cells are cultured on both sides of the culture membrane, the areas on which the cells are cultured can be made uniform on both sides. (5) In the cell culture instrument of the above embodiment, the cell culture instrument may have a groove extending in the circumferential direction on an outer circumferential surface of the second cylindrical portion. According to this embodiment, when the cylindrical member is attached or detached using the instrument, the tip of the instrument can be hooked into the groove, so that the cylindrical member can be stably held. (6) In the cell culture instrument of the above embodiment, the cell culture instrument may include a plurality of the covering members and a holder for holding the plurality of covering members. According to this embodiment, the plurality of covering members can be attached to the plurality of cylindrical members at the same time. Furthermore, since the covering members can be moved by holding the holder by hand without touching the covering members, contamination in the cell culture process can be suppressed. (7) The cell culture instrument of the above embodiment may further include a gripping instrument for gripping the second cylindrical portion, the gripping instrument having a pair of gripping parts that face each other in the radial direction sandwiching the second cylindrical portion and have gripping inner surfaces along the outer circumferential surface of the second cylindrical portion when gripping the second cylindrical portion. According to this embodiment, the gripping inner surfaces of the pair of gripping parts can be aligned along the outer circumferential surface of the second cylindrical portion, so that the cylindrical member can be stably gripped using the gripping instrument. The present disclosure can be realized in various forms, and in addition to the forms described above, can be realized in the form of a cell culture method, etc. [Brief description of the drawings]

[0008] [Figure 1] FIG. [Diagram 2] FIG. [Diagram 3] FIG. 3 is a cross-sectional view of the cell culture instrument in an attached state. [Figure 4] FIG. [Diagram 5] 5A to 5C are diagrams illustrating a method of attaching the covering member to the cylindrical member. [Figure 6] 1 is a flow chart showing a cell culture process. [Figure 7] FIG. 1 is a schematic diagram illustrating a cell culture process. [Figure 8] FIG. 11 is a perspective view of a cell culture instrument according to a second embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] A. First embodiment: A1. Cell culture equipment consists of: FIG. 1 is a perspective view of a cell culture instrument 100. FIG. 2 is a cross-sectional view of the cell culture instrument 100. FIG. 3 is a cross-sectional view of the cell culture instrument 100 in a state in which the covering member 50 is attached to the cylindrical member 10. FIG. 4 is a perspective view of a gripping instrument 80. FIG. 5 is a diagram for explaining a method for attaching the covering member 50 to the cylindrical member 10. In FIG. 1 to FIG. 3 and FIG. 5, an X-axis, a Y-axis, and a Z-axis are shown, which are orthogonal to each other. The cell culture instrument 100 is typically used by being disposed such that the Z-direction is aligned with the vertical direction. The directions in which the arrows of the X-axis, Y-axis, and Z-axis point indicate the positive directions along the X-axis, Y-axis, and Z-axis, respectively. The positive directions along the X-axis, Y-axis, and Z-axis are respectively referred to as the +X direction, +Y direction, and +Z direction. The directions opposite to the directions in which the arrows of the X-axis, Y-axis, and Z-axis point are the negative directions along the X-axis, Y-axis, and Z-axis, respectively. The negative directions along the X-axis, Y-axis, and Z-axis are called the -X direction, -Y direction, and -Z direction, respectively. Directions along the X-axis, Y-axis, and Z-axis, regardless of whether they are positive or negative, are called the X direction, Y direction, and Z direction, respectively. This also applies to the following figures and explanations. The +Z direction may be called the up direction, and the -Z direction may be called the down direction.

[0010] The cell culture instrument 100 includes a cylindrical member 10, a covering member 50, and a gripping instrument 80 (FIG. 4). As shown in FIG. 1, the cylindrical member 10 includes a main cylindrical portion 11, a culture membrane 12, and a flange 13. The main cylindrical portion 11 and the flange 13 are integrally molded using, for example, a resin material. Examples of the resin material that can be used include high-density polyethylene, polypropylene, ABS resin, acrylic resin, polycarbonate, polystyrene, polyester, and the like. The flange 13 has an annular shape and extends radially from one end in the axial direction, which is a direction along the central axis CX1 of the cylindrical member 10. The culture membrane 12 is disposed at an opening end 11a of the main cylindrical portion 11. Specifically, the culture membrane 12 is disposed at the opening end 11a opposite to the end where the flange 13 is formed, out of the two axial ends of the cylindrical member 10. The circular culture membrane 12 is joined to the open end 11a of the cylindrical main body portion 11 so as to cover the open end 11a of the cylindrical main body portion 11. The culture membrane 12 has a first surface 12a facing the inside of the cylindrical main body portion 11 and a second surface 12b which is the surface opposite to the first surface 12a. In the cell culture process, cells are cultured on both the first surface 12a and the second surface 12b.

[0011] The culture membrane 12 is a membrane that serves as a scaffold for cell culture when cell culture is performed. As the culture membrane 12, a porous membrane made of synthetic polymer or a porous membrane made of natural polymer, which has a large number of pores penetrating in the thickness direction of the membrane, can be used. Specifically, the porous membrane can be a track-etched membrane, a nanofiber membrane, a collagen vitrigel membrane, a honeycomb membrane, a porous polyurethane membrane, etc. As the porous polyurethane membrane, a porous polyurethane membrane manufactured by a manufacturing method including a first step and a second step described below can be used. The first step is a step of forming a layer of uncured polyurethane raw material on a substrate. The second step is a step of supplying water vapor to an exposed surface of the polyurethane raw material layer formed on the substrate, which is spaced from the substrate, to advance the curing of the polyurethane raw material in the polyurethane raw material layer and form a porous shape having a plurality of irregularities on the exposed surface.

[0012] The covering member 50 has a cylindrical shape. The covering member 50 is attached to the cylindrical member 10 and used in the cell culture process. The covering member 50 is made of a material that is flexible and has a small dynamic friction coefficient with respect to the cylindrical member 10. Specifically, low-density polyethylene (LDPE), polypropylene, tetrafluoroethylene-perfluoroether copolymer (PFA), etc. can be used as the material with a small dynamic friction coefficient. The covering member 50 may be formed by subjecting the surface of a flexible material to a surface treatment for reducing the dynamic friction coefficient. Specifically, the covering member 50 may be formed by coating the surface of a flexible material with diamond-like carbon (DLC) or 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer. This can improve the workability in the process of attaching and detaching the covering member 50 to the cylindrical member 10, as described in detail later.

[0013] The covering member 50 has a first cylindrical portion 51 and a second cylindrical portion 52. As shown in FIG. 3, the first cylindrical portion 51 is a portion of the main body of the covering member 50 that covers the outer circumferential surface of the main body cylindrical portion 11 when the covering member 50 is attached to the cylindrical member 10. The second cylindrical portion 52 is a portion of the main body of the covering member 50 that does not cover the outer circumferential surface of the main body cylindrical portion 11 when the covering member 50 is attached to the cylindrical member 10. The second cylindrical portion 52 is adjacent to the first cylindrical portion 51 in the axial direction along the central axis CX2 of the covering member 50. When the second cylindrical portion 52 is attached to the cylindrical member 10, the second cylindrical portion 52 forms a container with the second cylindrical portion 52 as a side surface and the culture membrane 12 as a bottom surface. As shown in FIG. 2, the first cylindrical portion 51 has a first portion 53 having a length in the axial direction of a first length L1 and a second portion 54 having a length in the axial direction of a second length L2. The second length L2 is shorter than the first length L1. This improves workability in the process of attaching and detaching the covering member 50 to and from the cylindrical member 10, as described in detail later. The first portion 53 and the second portion 54 face each other in a radial direction perpendicular to the axial direction. The first cylindrical portion 51 has a shape in which the length in the axial direction gradually shortens from the first portion 53 toward the second portion 54 in a circumferential direction centered on the axial direction. That is, in this embodiment, the end face of the first cylindrical portion 51 of the covering member 50 Is, inThe shape is inclined with respect to the axis CX2. End The shape of the surface is elliptical.

[0014] The covering member 50 further has a step portion 55 on the inner circumferential surface. The radial thickness of the first cylindrical portion 51 is thinner than the radial thickness of the second cylindrical portion 52. The outer diameter of the first cylindrical portion 51 is the same as the outer diameter of the second cylindrical portion 52. The inner diameter D3 of the first cylindrical portion 51 is larger than the inner diameter D1 of the second cylindrical portion 52. A step portion 55 is formed at the boundary between the inner circumferential surface of the first cylindrical portion 51 and the inner circumferential surface of the second cylindrical portion 52 due to the difference in the inner diameter of the first cylindrical portion and the inner diameter of the second cylindrical portion 52. The step portion 55 is a surface formed in the circumferential direction at the boundary between the first cylindrical portion 51 and the second cylindrical portion 52. This step portion 55 protrudes radially inward from the inner circumferential surface of the first cylindrical portion 51 at the boundary. This makes it easier to position the covering member 50 relative to the cylindrical member 10 in the process of attaching the covering member 50 to the cylindrical member 10, as described in detail later. The inner diameter D1 of the second cylindrical portion 52 is the same as the inner diameter D1 of the main body cylindrical portion 11. This makes it possible to align the areas in which cells are cultured on the first surface 12a and the second surface 12b in the cell culture process. Note that "the inner diameter D1 of the second cylindrical portion 52 is the same as the inner diameter D1 of the main body cylindrical portion 11" does not necessarily mean that the inner diameter of the second cylindrical portion 52 and the inner diameter of the main body cylindrical portion 11 are the same, and may mean, for example, that the inner diameter of the second cylindrical portion 52 is within a range of ±10% based on the inner diameter of the main body cylindrical portion 11.

[0015] In this embodiment, the inner diameter D1 of the main body cylindrical portion 11 is about 6 mm, and the outer diameter D2 is about 9 mm. The inner diameter D3 of the first cylindrical portion 51 is smaller than the outer diameter D2 of the main body cylindrical portion 11 by about 0.1 mm or more and 0.3 mm or less. Therefore, the covering member 50 is stretched so that the diameter becomes larger and is attached to the cylindrical member 10. This allows the covering member 50 to be attached closely to the cylindrical member 10. In addition, the thickness of the first cylindrical portion 51 is about 0.3 mm or more and 2 mm or less. This allows the flexibility of the first cylindrical portion 51 to be well maintained, and therefore the workability can be improved in the process of attaching and detaching the covering member 50 to the cylindrical member 10. In the case of a flexible material, the thicker the material, the more difficult it tends to deform. Therefore, by setting the thickness of the first cylindrical portion 51 within the above range, the flexibility can be well maintained.

[0016] The covering member 50 further has a plurality of grooves 56 extending in the circumferential direction on the outer circumferential surface. The plurality of grooves 56 are formed recessed from the outer circumferential surface of the second cylindrical portion 52 toward the central axis CX2. As a result, as will be described in detail later, the protrusions 83 of the grasping tool 80 can be fitted into the grooves 56, so that the cylindrical member 10 can be stably grasped using the grasping tool 80. Even if the grasping tool 80 is not used, the covering member 50 can be easily grasped using a tool such as tweezers by hooking the tip of the tool into the grooves 56.

[0017] FIG. 4 is a perspective view of the gripping tool 80. The gripping tool 80 is a tool for gripping the second cylindrical portion 52 of the covering member 50. The gripping tool 80 has a pair of gripping parts 81, 81 and a pair of plate parts 82, 82. The pair of plate parts 82, 82 have a plate shape, and the end opposite to the end where the pair of gripping parts 81, 81 are arranged in the long side direction is joined to each other. In the pair of plate parts 82, 82, the pair of gripping parts 81, 81 are separated from each other as shown in FIG. 4 when no external force is applied. Then, when an external force is applied, the pair of plate parts 82, 82 are reversibly deformed so as to approach each other. As a result, the worker can grip the pair of plate parts 82, 82 with the second cylindrical portion 52 sandwiched between the pair of gripping parts 81, 81, and deform the gripping tool 80 to grip the covering member 50 using the gripping tool 80.

[0018] When the pair of gripping parts 81, 81 grip the second cylindrical part 52, they face each other in the radial direction of the second cylindrical part 52 with the second cylindrical part 52 therebetween. Each of the pair of gripping parts 81, 81 has a gripping inner surface 81a that is a curved surface along the outer circumferential surface of the second cylindrical part 52 (FIG. 1). This allows the cylindrical member 10 to be stably gripped. The gripping inner surface 81a has a plurality of protrusions 83 extending in the circumferential direction at positions corresponding to the grooves 56 of the second cylindrical part 52. Specifically, the plurality of protrusions 83 protrude outward from the gripping inner surface 81a in the radial direction. The intervals between the plurality of protrusions 83 are the same as the intervals between the plurality of grooves 56. In this embodiment, the gripping inner surface 81a is covered with a sheet-like member, and the sheet-like member and the protrusions 83 are integrally formed. The sheet-like member and the protrusions 83 are formed of a material having a high static friction coefficient against the covering member 50. The static friction coefficient of the material forming the sheet-like member and the protrusions 83 is preferably greater than 1. Examples of materials with a high static friction coefficient include isobutylene-isoprene rubber (IIR), natural rubber, ethylene propylene rubber (EPDM), and vinylidene fluoride rubber (FKM). This allows the covering member 50 to be stably grasped using the grasping tool 80.

[0019] 5, when the covering member 50 is attached to the cylindrical member 10, the covering member 50 is first placed in a position in which the central axis CX2 of the covering member 50 is inclined with respect to the central axis CX1 of the cylindrical member 10. The covering member 50 is grasped by a grasping tool 80. Next, the inner peripheral surface of the second portion 54 is brought into close contact with the outer peripheral surface of the cylindrical member 10 before the first portion 53. Here, since the covering member 50 has a step portion 55, the opening end 11a of the main cylindrical portion 11 is brought into contact with the step portion 55, so that the covering member 50 can be easily positioned with respect to the cylindrical member 10.

[0020] Next, the covering member 50 is moved in the direction of the arrow, with the vicinity of the second portion 54 as a fulcrum, so that the central axis CX2 of the covering member 50 is parallel to the central axis CX1 of the cylindrical member 10. As shown by the two-dot chain line, the covering member 50 is moved until the opening end 11a of the cylindrical main body portion 11 is in a position where it abuts against the step portion 55. As in the above, since the covering member 50 has the step portion 55, the opening end 11a of the cylindrical main body portion 11 can be abutted against the step portion 55, and therefore the covering member 50 can be easily positioned relative to the cylindrical member 10.

[0021] Since the first cylindrical portion 51 has the second portion 54 and the first portion 53, which are different in length, the covering member 50 can be attached to the cylindrical member 10 by the above procedure. According to this attachment method, the range of the inner circumferential surface of the first cylindrical portion 51 that rubs against the outer circumferential surface of the cylindrical member 10 in the process of moving the covering member 50 is limited to a very small part of the inner circumferential surface around the first portion 53. Therefore, the range where friction occurs between the inner circumferential surface of the covering member 50 and the outer circumferential surface of the cylindrical member 10 during attachment can be reduced. Therefore, the force required to attach the covering member 50 can be reduced, making it easier to attach and improving workability.

[0022] 3, the first cylindrical portion 51 of the covering member 50 is attached to the cylindrical member 10 in a deformed state so that the outer diameter and the inner diameter are increased. Here, the covering member 50 has flexibility, and since the thickness of the first cylindrical portion 51 is thinner than the thickness of the second cylindrical portion 52, the flexibility of the second cylindrical portion 52 is good. Therefore, the covering member 50 is easy to attach, and in the attached state, the covering member 50 can be closely attached to the first cylindrical portion 51.

[0023] When the covering member 50 is removed from the cylindrical member 10, the above procedure is carried out in reverse. That is, first, the covering member 50 is moved around the vicinity of the second portion 54 as a fulcrum so that the central axis CX2 of the covering member 50 is inclined with respect to the central axis CX1 of the cylindrical member 10. After the first portion 53 is separated from the cylindrical main body portion 11, the covering member 50 is moved so that the second portion 54 is separated from the cylindrical main body portion 11. When the covering member 50 is removed from the cylindrical member 10, the force required to attach the covering member 50 can be reduced, as in the case of attachment, so that the covering member 50 can be easily removed, and workability can be improved.

[0024] A2. Cell culture method using cell culture equipment: FIG. 6 is a flow chart showing a cell culture process for culturing cells on both sides of a culture membrane 12 using a cell culture instrument 100. FIG. 7 is a schematic diagram for explaining the cell culture process, showing the steps and the states of the cell culture instrument 100 in correspondence with each other. In step P10 shown in FIG. 6, the cell culture instrument 100 is prepared. In step P20, the cylindrical member 10 is inserted into a well 502 (FIG. 7) of a well plate 500 (FIG. 7). Specifically, as shown in step P30 in FIG. 7, the cylindrical member 10 is in an orientation that is upside down from the orientation shown in FIG. 1, and the flange 13 is engaged with an upper surface 501 that connects the wells 502 of the well plate 500. As a result, the cylindrical member 10 is suspended in the well 502.

[0025] In step P30 shown in FIG. 6, a first cell suspension B1 containing first cells C1 and a culture solution for the first cells is injected into the cylindrical member 10. By injecting the first cell suspension B1, the first cells C1 are seeded on the first surface 12a of the culture membrane 12. In this embodiment, the culture solution is also injected into the wells 502. After this operation is completed, the well plate 500 is covered. In step P40, the culture solution is appropriately replaced as necessary, and the first cells C1 are cultured until they are fixed on the first surface 12a of the culture membrane 12. The culture is performed under the conditions of temperature, humidity, and CO 2 This is done in an incubator, which has a constant concentration and a stable environment, allowing cells to be cultured under conditions closer to physiological conditions.

[0026] In step P50, a storage jig 800 (FIG. 7) for storing the culture solution inside the main body cylindrical portion 11 is attached to the cylindrical member 10. As shown in step P50 in FIG. 7, the storage jig 800 is flexible and formed in a cylindrical shape. The outer diameter of the storage jig 800 is large enough to be in close contact with the inner circumferential surface of the main body cylindrical portion 11. The storage jig 800 is inserted into the main body cylindrical portion 11 with the central axis of the storage jig 800 aligned with the central axis CX1 of the cylindrical member 10. The storage jig 800 is inserted until the lower end, which is the insertion direction of the storage jig 800, reaches a position where it does not touch the culture membrane 12. As a result, even if the cylindrical member 10 is turned upside down in the next step P60, the culture solution can be stored inside the main body cylindrical portion 11. Therefore, the culture of the first cell C1 can be continued.

[0027] 6, the cylindrical member 10 is turned upside down, and the covering member 50 is attached to the outer circumferential surface of the cylindrical member 10 using a gripping tool 80. By attaching the covering member 50, a container for storing culture fluid is formed, with the culture membrane 12 as the bottom surface and the inner circumferential surface of the covering member 50 as the side surface. Here, the first cylindrical portion 51 is in close contact with the cylindrical member 10, and therefore leakage of the second cell suspension B2 to be injected into the second cylindrical portion 52 in the next step P70 can be suppressed.

[0028] In step P70, a second cell suspension B2 containing second cells C2 and a culture medium for the second cells is injected into the covering member 50. As shown in step P70 of FIG. 7, the second cells C2 are seeded on the second surface 12b of the culture membrane 12 by injecting the second cell suspension B2.

[0029] In step P80 shown in FIG. 6, the culture medium is appropriately replaced as necessary until the second cells C2 contained in the second cell suspension B2 are fixed to the second surface 12b of the culture membrane 12, and the second cells C2 are cultured. The culture is performed in an incubator. It is preferable to perform the culture in a wide-mouthed container with a lid, for example. In this way, evaporation of the culture medium seeded inside the covering member 50 during culture can be suppressed, and contamination during culture can be suppressed. Here, as described above, the inner diameter D1 of the second cylindrical portion 52 and the inner diameter D1 of the main body cylindrical portion 11 are the same, so that the culture area of ​​the second cells C2 can be made equal to the culture area of ​​the first cells C1.

[0030] In step P90, first, the culture medium is removed from the inside of the covering member 50, and the covering member 50 is removed from the cylindrical member 10 using the grasping tool 80. Thereafter, the cylindrical member 10 is turned upside down and inserted again into the wells 502 of the well plate 500. After all the cylindrical members 10 have been inserted, the well plate 500 is covered.

[0031] In step P100, the culture of the second cells C2 continues. The culture is entirely carried out in an incubator. Specifically, as shown in step P100 of FIG. 7, a culture medium for the second cells C2 that have been fixed to the second surface 12b is poured into the well 502, and a culture medium for the first cells C1 that have been fixed to the first surface 12a is poured into the cylindrical member 10. The culture medium is replaced as necessary, and the cells are cultured until the culture operation is completed. When the culture of the second cells C2 is completed, this cell culture process is completed. By the cell culture process, cells can be cultured on both the first surface 12a and the second surface 12b of the culture membrane 12.

[0032] According to the first embodiment described above, a cell culture instrument 100 is provided that has a cylindrical member 10 and a covering member 50. When the covering member 50 is attached to the cylindrical main body portion 11, the covering member 50 has a first cylindrical portion 51 that covers the outer peripheral surface of the cylindrical main body portion 11, and a second cylindrical portion 52 that forms a container with the culture membrane 12 as the bottom surface. The first cylindrical portion 51 has a first portion 53 that has a first length L1 in the axial direction, and a second portion 54 that has a second length L2 in the axial direction that is shorter than the first length L1. Since the first cylindrical portion 51 has the second portion 54 and the first portion 53, which are different in length, the central axis CX2 of the covering member 50 is tilted with respect to the central axis CX1 of the cylindrical member 10, and the second portion 54 is brought into contact with the cylindrical member 10 before the first portion 53. Then, the covering member 50 is moved around the vicinity of the second portion 54 as a fulcrum so that the central axis CX2 of the covering member 50 is parallel to the central axis CX1 of the cylindrical member 10, whereby the covering member 50 can be attached to the cylindrical member 10. According to this attachment method, the range of the inner circumferential surface of the covering member 50 that rubs against the outer circumferential surface of the cylindrical member 10 in the process of moving the covering member 50 is limited to a part of the inner circumferential surface around the first portion 53. Therefore, the range where friction occurs between the inner circumferential surface of the covering member 50 and the outer circumferential surface of the cylindrical member 10 during attachment can be reduced, so that the force required to attach the covering member 50 can be reduced. Therefore, the covering member 50 can be easily attached and detached, and the workability can be improved.

[0033] Additionally, the covering member 50 has a step portion 55 on its inner circumferential surface formed by the difference between the inner diameter D3 of the first cylindrical portion 51 and the inner diameter D1 of the second cylindrical portion 52. This allows the opening end 11a to abut against the step portion 55 when attaching the covering member 50 to the cylindrical member 10, facilitating positioning.

[0034] Moreover, the covering member 50 has flexibility, and the radial thickness of the first cylindrical portion 51 is thinner than the radial thickness of the second cylindrical portion 52. This allows the covering member 50 to be easily attached to and detached from the cylindrical member 10 by the first cylindrical portion 51 having good flexibility, while maintaining stability when the covering member 50 is attached to the cylindrical member 10 and used as a container with the culture membrane 12 as the bottom surface.

[0035] Furthermore, the inner diameter D1 of the main body cylindrical portion 11 is the same as the inner diameter D1 of the second cylindrical portion 52. Therefore, when cells are cultured on both sides of the culture membrane 12, the areas on which the cells are cultured can be made uniform between the first surface 12a and the second surface 12b.

[0036] In addition, the second cylindrical portion 52 has a groove portion 56 extending in the circumferential direction on the outer circumferential surface. Therefore, when the cylindrical member 10 is attached using the gripping tool 80, the cylindrical member 10 can be stably gripped by fitting the convex portion 83 of the gripping tool 80 into the groove portion 56. Also, when tweezers are used instead of the gripping tool 80, the tip of the tweezers can be hooked into the groove portion 56, so that the cylindrical member 10 can be stably gripped.

[0037] Furthermore, when the gripping instrument 80 grips the second cylindrical portion 52, it has a pair of gripping parts 81, 81 that face each other in the radial direction across the second cylindrical portion 52 and have gripping inner surfaces 81a that are aligned with the outer circumferential surface of the second cylindrical portion 52. This allows the gripping inner surfaces 81a of the pair of gripping parts 81, 81 to be aligned with the outer circumferential surface of the second cylindrical portion 52, so that the cylindrical member 10 can be stably gripped using the gripping instrument 80.

[0038] B. Second embodiment: 8 is a perspective view of a cell culture instrument 1100 according to the second embodiment. The cell culture instrument 1100 according to this embodiment differs from the cell culture instrument 100 according to the first embodiment in that it has a holding unit 90 that holds a plurality of covering members 50, a connecting unit 95 that connects a plurality of cylindrical members 10, and a knob 91. The same components as those in the first embodiment are denoted by the same reference numerals, and detailed descriptions thereof will be omitted as appropriate.

[0039] The holding portion 90 is a plate-like member extending in the direction in which the multiple covering members 50 are arranged. The holding portion 90 has multiple holes formed therein that penetrate in the thickness direction. Each of the multiple covering members 50 is inserted into each of the multiple holes formed in the holding portion 90, and is fixed to the holding portion 90 with the end of the second cylindrical portion 52 exposed from the holding portion 90. The multiple cylindrical members 10 arranged on the same line are connected to each other by the connecting portion 95. The arrangement interval of the cylindrical members 10 is the same as the arrangement interval of the covering members 50. Since the multiple covering members 50 are held by the holding portion 90, the multiple covering members 50 can be attached to the multiple cylindrical members 10 arranged on the same line at the same time. In addition, knobs 91 are attached to both ends of the holding portion 90 in the long side direction. As a result, when an operator attaches or detaches the covering member 50 to or from the cylindrical member 10, the operator can hold the knob 91 with a gloved hand and attach or detach the covering member 50 without directly touching it, thereby preventing contamination during the cell culture process.

[0040] According to the second embodiment described above, the cell culture instrument 1100 includes a holder 90 that holds a plurality of covering members 50. This allows a plurality of covering members 50 to be attached to a plurality of cylindrical members 10 at the same time. Furthermore, since the covering members 50 can be moved by holding the holder 90 without touching the covering members 50 with the hands, contamination in the cell culture process can be suppressed.

[0041] C. Other embodiments: (C1) In the first embodiment, the end face of the first cylindrical portion 51 of the covering member 50 has a shape that draws an oblique line with respect to the central axis CX2 when viewed from the radial direction, and the shape of the end face is an ellipse. The shape of the end face of the first cylindrical portion 51 is not limited to an ellipse. For example, the end face of the first cylindrical portion 51 of the covering member 50 may have a curve having multiple inflection points midway when viewed from the radial direction. Regardless of the shape of the end face, since the first cylindrical portion 51 has the first portion 53 and the second portion 54, the covering member 50 can be easily attached to and detached from the cylindrical member 10.

[0042] (C2) In the above first embodiment, a case has been described in which the covering member 50 is attached to the cylindrical member 10 by an operator, but the covering member 50 may be attached to the cylindrical member 10 by a machine. Even when the covering member 50 is attached by a machine, the force required to attach the covering member 50 can be reduced, making it easier to attach and remove, and improving workability.

[0043] The present disclosure is not limited to the above-mentioned embodiment, and can be realized in various configurations without departing from the spirit of the present disclosure. For example, the technical features of the embodiments corresponding to the technical features in each aspect described in the Summary of the Invention can be appropriately replaced or combined to solve some or all of the above-mentioned problems or to achieve some or all of the above-mentioned effects. Furthermore, if a technical feature is not described as essential in this specification, it can be appropriately deleted. [Explanation of symbols]

[0044] 10...Cylindrical member, 11...Main body cylindrical portion, 11a...Open end, 12...Culture membrane, 12a...First surface, 12b...Second surface, 13...Flange, 50...Covering member, 51...First cylindrical portion, 52...Second cylindrical portion, 53...First portion, 54...Second portion, 55...Step portion, 56...Groove portion, 80...Gripping device, 81...Gripping portion, 81a...Gripping inner surface, 82...Plate portion, 83...Convex portion, 90...Holding portion, 91 ...knob, 95...connecting part, 100, 1100...cell culture device, 500...well plate, 501...upper surface, 502...well, 800...storage jig, B1...first cell suspension, B2...second cell suspension, C1...first cell, C2...second cell, CX1, CX2...center axis, D1, D3...inner diameter, D2...outer diameter, L1...first length, L2...second length, P10 to P100...process

Claims

1. A cell culture device comprising: A cylindrical member having a main body cylindrical portion and a culture membrane disposed at an open end of the main body cylindrical portion; a cylindrical covering member to be attached to the main cylindrical portion, the covering member having: (i) a first cylindrical portion that covers at least the entire outer periphery of the open end when attached to the cylindrical portion; and (ii) a second cylindrical portion that is adjacent to the first cylindrical portion in the axial direction of the covering member and that forms a container having the culture membrane as a bottom surface when attached to the cylindrical portion; the first cylindrical portion has a first portion having a first length in the axial direction and a second portion having a second length that is shorter than the first length, the covering member is separate from the cylindrical member, The cell culture instrument, wherein an end surface of the first cylindrical portion has an elliptical shape and is inclined with respect to a central axis of the first cylindrical portion.

2. The cell culture instrument according to claim 1, The inner diameter of the first cylindrical portion is larger than the inner diameter of the second cylindrical portion, and the covering member further comprises: A cell culture instrument having a step portion at the boundary between an inner circumferential surface of the first cylindrical portion and an inner circumferential surface of the second cylindrical portion, the step portion being formed by the difference in inner diameter between the first cylindrical portion and the second cylindrical portion.

3. The cell culture instrument according to claim 1 or 2, The covering member has flexibility, A cell culture instrument, wherein a radial thickness of the first cylindrical portion is smaller than a radial thickness of the second cylindrical portion.

4. The cell culture instrument according to claim 1 , The cell culture instrument, wherein an inner diameter of the main cylindrical portion and an inner diameter of the second cylindrical portion are the same.

5. The cell culture instrument according to claim 1 , further comprising: The cell culture instrument has a groove extending in a circumferential direction on an outer circumferential surface of the second cylindrical portion.

6. The cell culture instrument according to claim 1 , A plurality of the covering members are provided, A cell culture instrument comprising a holder for holding the plurality of covering members.

7. The cell culture instrument according to claim 1 , further comprising: A cell culture device comprising: a gripping device for gripping the second cylindrical portion, the gripping device having a pair of gripping portions that, when gripping the second cylindrical portion, are radially opposed across the second cylindrical portion and have gripping inner surfaces that follow the outer peripheral surface of the second cylindrical portion.

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