Cell culture device and cell culture method

Abstract

To provide a cell culture tool having good workability which can perform cell culture onto both faces of a culture membrane.SOLUTION: A cell culture tool comprises: cylindrical members each having a cylindrical part, a culture membrane arranged for one end of the cylindrical part in an axial direction, and a first engaging part formed at the other end in the axial direction; and holding members each having a bottomed cylindrical part capable of housing the cylindrical part and a second engaging part capable of being engaged with the first engaging part, where the second engaging part is engaged with the first engaging part in either a first state where the cylindrical part is housed in the bottomed cylindrical part or a second state where a posture of the cylindrical part is in the opposite direction in the axial direction from the first state.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a cell culture instrument and a cell culture method. [Background technology]

[0002] Generally, in cell culture, a well plate with multiple wells is used to simultaneously culture small amounts of multiple samples. An insert with a cell culture membrane attached is inserted into the well, and cells are cultured on one side of the membrane (for example, Patent Document 1). The insert in Patent Document 1 is suspended by a flange extending radially of the insert abutting against the upper surface of the well plate (culture vessel), and the insert is held in a state where the membrane is separated from the bottom surface of the well.

[0003] In recent years, cell culture has been performed on both sides of a membrane. Therefore, a cell culture device for performing double-sided culture has been proposed (for example, Patent Document 2). The cell culture device of Patent Document 2 includes a hanging component and a complementary tubular body in addition to an outer tubular body to which a membrane is attached. In a first step of performing cell culture on one side of the membrane, the complementary tubular body is fitted into the outer tubular body to form a space for storing a culture medium outside the outer tubular body. In a second step of performing cell culture on the other side of the membrane after the first step, the hanging component is fitted into the outer tubular body to suspend the outer tubular body from a well. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Publication No. H7-75553 [Patent Document 2] International Publication No. WO2012 / 045368 Summary of the Invention [Problem to be solved by the invention]

[0005] However, in the cell culture device of Patent Document 2, in the first step of culturing cells on one side of the membrane, the device is configured to be self-supporting, which may result in poor operability when culturing multiple samples. [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 cylindrical portion, a culture membrane disposed at one axial end of the cylindrical portion, and a first engagement portion formed at the other axial end, and a holding member having a bottomed cylindrical portion capable of accommodating the cylindrical portion, and a second engagement portion engageable with the first engagement portion, the second engagement portion engaging with the first engagement portion both in a first state in which the cylindrical portion is accommodated in the bottomed cylindrical portion and in a second state in which the orientation of the cylindrical portion is opposite to that in the first state in the axial direction, and the second engagement portion has an axial regulation portion that regulates both the movement of the cylindrical member from the one end to the other end in the axial direction and the movement of the cylindrical member from the other end to the one end in the axial direction by engaging with the first engagement portion.

[0007] (1) According to one embodiment of the present disclosure, a cell culture instrument is provided. The cell culture instrument includes a cylindrical member having a cylindrical portion, a culture membrane disposed at one axial end of the cylindrical portion, and a first engagement portion formed at the other axial end, and a holding member having a bottomed cylindrical portion capable of accommodating the cylindrical portion, and a second engagement portion capable of engaging with the first engagement portion. The second engagement portion engages with the first engagement portion in either a first state in which the cylindrical portion is accommodated in the bottomed cylindrical portion, or a second state in which the attitude of the cylindrical portion is opposite to that in the first state in the axial direction. According to this embodiment, the second engagement portion can engage with the first engagement portion in either the first state or the second state. By bringing the cylindrical member into a state in which it is engaged with the holding member, the cylindrical member and the holding member can be handled as a unit, thereby improving the operability of the cell culture instrument. Furthermore, even when the holding member is tilted, it is possible to suppress the cylindrical member from shifting relative to the holding member, and stable cell culture can be performed. (2) The cell culture instrument of the above embodiment may include a plurality of the holding members, and may further include a connecting portion that connects two adjacent holding members of the plurality of holding members. According to this embodiment, the plurality of holding members can be handled as a unit, thereby further improving workability. (3) In the cell culture instrument of the above aspect, the second engagement portion may have a horizontal restriction portion that restricts movement of the cylindrical member in a horizontal direction perpendicular to the axial direction by engaging with the first engagement portion. According to this aspect, it is possible to restrict movement of the cylindrical member in a horizontal direction perpendicular to the axial direction relative to the holding member. (4) In the cell culture instrument of the above embodiment, the first engagement portion may have a protrusion protruding outward from an outer circumferential surface of the cylindrical portion, and the horizontal regulating portion may have a recess into which the protrusion is inserted in the axial direction, and the protrusion abuts against the recess in the first state and the second state to regulate the movement in the horizontal direction. According to this embodiment, the protrusion is inserted into the recess to regulate the movement of the cylindrical member in the horizontal direction relative to the holding member. (5) In the cell culture instrument of the above aspect, the second engagement portion may have an axial restriction portion that restricts the movement of the cylindrical member in the axial direction by engaging with the first engagement portion. According to this aspect, the axial movement of the cylindrical member relative to the holding member can be restricted. (6) In the cell culture instrument of the above embodiment, the first engagement portion has a protrusion protruding outward from an outer circumferential surface of the cylindrical portion, and the axial restriction portion has (i) a first insertion portion into which the protrusion is inserted in the axial direction, and (ii) a second insertion portion extending from the first insertion portion in a circumferential direction of the holding member and into which the protrusion is inserted in the circumferential direction, the second insertion portion being partitioned by a pair of peripheral screens opposed to each other with a gap in the axial direction, and in the first state and the second state, the pair of peripheral screens may restrict the movement in the axial direction by abutting against the protrusion inserted therebetween. According to this embodiment, the axial movement of the cylindrical member relative to the holding member can be restricted by inserting the protrusion into the second insertion portion. (7) In the cell culture instrument of the above embodiment, the first and second insertion parts may be formed to radially penetrate a side wall of the bottomed cylindrical part, and the protrusion may protrude from an outer circumferential surface of the side wall in the first and second states. According to this embodiment, when the first and second engagement parts are engaged with each other, the part of the protrusion protruding from the outer circumferential surface of the side wall can be gripped, thereby improving workability when engaging the first and second engagement parts. (8) In the cell culture instrument of the above embodiment, a plurality of the cylindrical members may be provided, and two adjacent cylindrical members of the plurality of cylindrical members may be connected by the protrusion. According to this embodiment, the plurality of cylindrical members can be handled as a unit, thereby further improving workability. (9) In the cell culture instrument of the above embodiment, the first engagement portion may have an extension portion extending outward from an outer circumferential surface of the cylindrical portion, the axial regulation portion may have an outer circumferential protrusion portion protruding outward from an outer circumferential surface of the bottomed cylindrical portion, the extension portion having a claw portion covering the outer circumferential protrusion portion and abutting against the outer circumferential protrusion portion in the axial direction, and the claw portion may abut against the outer circumferential protrusion portion to regulate the movement in the axial direction in the first state and the second state. According to this embodiment, the abutment of the claw portion against the outer circumferential protrusion portion can regulate the movement in the axial direction of the cylindrical member relative to the holding member. (10) According to another aspect of the present disclosure, there is provided a cell culture method, comprising: a first step of preparing a cell culture instrument comprising: a cylindrical member having a cylindrical portion, a culture membrane disposed at one axial end of the cylindrical portion, and a first engagement portion formed at the other axial end; a holding member having a bottomed cylindrical portion capable of accommodating the cylindrical portion, and a second engagement portion engageable with the first engagement portion, the second engagement portion engaging with the first engagement portion in either a first state in which the cylindrical portion is accommodated in the bottomed cylindrical portion, or a second state in which the attitude of the cylindrical portion is opposite to that in the first state in the axial direction; a second step of culturing cells on a first surface of the culture membrane facing the inside of the cylindrical portion in the first state; and a third step of culturing cells on a second surface of the culture membrane opposing the first surface in the second state. According to this embodiment, in the cell culture step, by engaging the cylindrical member with the holding member, the cylindrical member and the holding member can be handled as one unit, improving the operability of the cell culture instrument. Also, even if the holding member is tilted in the second and third steps, the cylindrical member can be prevented from shifting relative to the holding member, allowing stable cell culture. The present disclosure can be realized in various forms, and in addition to the forms described above, the present disclosure can be realized in the form of a method for manufacturing a cell culture instrument, etc. [Brief description of the drawings]

[0008] [Figure 1] FIG. [Diagram 2] FIG. [Diagram 3] FIG. 2 is a perspective view of the cell culture instrument in a first state. [Figure 4] FIG. 4 is a perspective view of the cell culture instrument in a second state. [Diagram 5] 1 is a flow chart showing a cell culture process. [Figure 6] FIG. 11 is a perspective view of a cell culture instrument according to a second embodiment. [Figure 7] FIG. 13 is a diagram showing the cell culture instruments according to the third to sixth embodiments. 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. In FIG. 1, a part of a cylindrical member 10 is shown in cross section. FIG. 2 is a plan view of the cell culture instrument 100. FIG. 3 is a perspective view of the cell culture instrument 100 in a first state. FIG. 4 is a perspective view of the cell culture instrument 100 in a second state. FIGS. 1 to 4 show an X-axis, a Y-axis, and a Z-axis that 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 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 defined 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 respectively negative directions along the X-axis, Y-axis, and Z-axis. The negative directions along the X-axis, Y-axis, and Z-axis are respectively defined as the -X direction, -Y direction, and -Z direction. The 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 also be called the upward direction, and the -Z direction may also be called the downward direction.

[0010] The cell culture instrument 100 includes a cylindrical member 10 and a holding member 50. As shown in FIG. 2, in this embodiment, the cell culture instrument 100 has six holding members 50 arranged on the same line. Two adjacent holding members 50 are connected by a connecting portion 90. This allows the multiple holding members 50 to be handled as a single unit. Similarly, in this embodiment, the cell culture instrument 100 has six cylindrical members 10 arranged on the same line. Two adjacent cylindrical members 10 are connected by a first engaging portion 13. This allows the multiple cylindrical members 10 to be handled as a single unit. The direction in which the multiple cylindrical members 10 are arranged and the direction in which the multiple holding members 50 are arranged are parallel to the X direction.

[0011] As shown in FIG. 1, the cylindrical member 10 has a cylindrical portion 11, a culture membrane 12, and two first engagement portions 13. Except for the cylindrical member 10 arranged at the end, each of the two first engagement portions 13 is shared with the adjacent cylindrical portion 11. The culture membrane 12 is arranged at one end in the axial direction, which is a direction along the central axis CX1 of the cylindrical member 10. Specifically, the circular culture membrane 12 is joined to the end of the cylindrical portion 11 so as to cover the opening of the cylindrical portion 11. The culture membrane 12 has a first surface 12a facing the inside of the cylindrical portion 11 and a second surface 12b facing the first surface 12a. In the cell culture process, cells are cultured on both the first surface 12a and the second surface 12b. The two first engagement portions 13 are arranged at the other end in the axial direction of the cylindrical member 10.

[0012] 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 manufacturing step and a second manufacturing step, which will be described below, can be used. The first manufacturing step is a step of forming a layer of uncured polyurethane raw material on a substrate. The second manufacturing 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.

[0013] The cylindrical portion 11 is made of a resin material. Examples of the resin material that can be used include high-density polyethylene, polypropylene, ABS resin, acrylic resin, polycarbonate, polystyrene, and polyester. The two first engagement portions 13 are disposed facing each other in the radial direction of the cylindrical portion 11 across the central axis CX1. The first engagement portions 13 have a protrusion 14. The protrusion 14 extends in the radial direction from the outer circumferential surface of the cylindrical portion 11. The protrusion 14 has a flat plate shape with the thickness direction being the axial direction and the length direction being the X direction.

[0014] The holding member 50 has a bottomed cylindrical portion 51 and two second engagement portions 52. The holding member 50 is made of a resin material. Examples of the resin material that can be used include high-density polyethylene, polypropylene, ABS resin, acrylic resin, polycarbonate, polystyrene, and polyester. The bottomed cylindrical portion 51 has a cylindrical side wall 51a and a bottom surface 51b that closes the opening of the side wall 51a. In Figs. 1, 3, and 4, the central axis CX1 of the cylindrical member 10 and the central axis CX2 of the bottomed cylindrical portion 51 are drawn coaxially. The inner diameter of the side wall 51a is larger than the outer diameter of the cylindrical portion 11. Therefore, the bottomed cylindrical portion 51 can accommodate the cylindrical portion 11. The above-mentioned holding member 50 is a member called a well, a well plate, a microplate, or the like.

[0015] The two second engagement parts 52 are arranged facing each other in the X direction across the central axis CX2 of the bottomed cylindrical part 51. The two second engagement parts 52 have a plane-symmetric shape with a plane that passes through the central axis CX2 and is parallel to the YZ plane as a symmetry plane. The second engagement part 52 can be engaged with the first engagement part 13. The second engagement part 52 has an axis regulating part 53. The axis regulating part 53 has a first insertion part 54 and a second insertion part 55. The first insertion part 54 and the second insertion part 55 have a shape in which a part of the side wall 51a is cut out. Specifically, the first insertion part 54 is formed by penetrating the side wall 51a in the radial direction. The first insertion part 54 extends from the open end of the side wall 51a toward the bottom surface 51b in the axial direction that is the direction along the central axis CX2. The first insertion part 54 is partitioned by a pair of first insertion part partition surfaces 54a, 54b. The pair of first insertion section partition faces 54a, 54b face each other with a gap therebetween in the Y direction. The gap between the pair of first insertion section partition faces 54a, 54b is greater than the width of the protrusion 14. Thus, the first insertion section 54 can receive the protrusion 14 along the axial direction.

[0016] The second insertion portion 55 extends in the circumferential direction of the holding member 50 from the axial end of the first insertion portion 54. The second insertion portion 55 is formed by penetrating the side wall 51a in the radial direction, similar to the first insertion portion 54. The second insertion portion 55 is defined by a pair of peripheral screens 55a, 55b. The pair of peripheral screens 55a, 55b extend in the circumferential direction. The pair of peripheral screens 55a, 55b face each other with a gap in the axial direction. The gap between the pair of peripheral screens 55a, 55b is slightly larger than the thickness of the protrusion 14. Therefore, the second insertion portion 55 can receive the protrusion 14 inserted into the first insertion portion 54 along the Y direction.

[0017] The connecting portion 90 connects the side walls 51a of the adjacent bottomed cylindrical portions 51. In this embodiment, the connecting portion 90 is formed integrally with the holding member 50.

[0018] In the cell culture process, the cell culture instrument 100 is used in a first state in which the cylindrical portion 11 is housed in the bottomed cylindrical portion 51 as shown in Fig. 3, and in a second state in which the culture membrane 12 is exposed to the outside of the bottomed cylindrical portion 51 as shown in Fig. 4. In the second state, the orientation of the cylindrical portion 11 is axially opposite to that in the first state.

[0019] As shown in FIG. 3, in the first state, a part of the protrusion 14 of the cylindrical member 10 is inserted into the second insertion portion 55 of the holding member 50. The transition from the non-engagement state of the cell culture instrument 100 in which the first engagement portion 13 of the cylindrical member 10 and the second engagement portion 52 of the holding member 50 are not engaged with each other as shown in FIG. 1 to the first state is performed in two stages. That is, the transition is performed in a first stage in which the protrusion 14 is inserted into the first insertion portion 54 along the axial direction up to the end of the first insertion portion 54, and a second stage in which the protrusion 14 is inserted into the second insertion portion 55 along the Y direction. In FIG. 2, the cylindrical member 10 in the first stage is drawn by a two-dot chain line, and the cylindrical member 10 in the first state is drawn by a solid line. In the first stage, the central axis CX1 of the cylindrical member 10 is shifted in the −Y direction with respect to the central axis CX2 of the holding member 50. Then, in the second stage, the cylindrical member 10 is moved in the +Y direction, so that the central axis CX1 of the cylindrical member 10 and the central axis CX2 of the bottomed cylindrical portion 51 are substantially aligned with each other. As shown in Fig. 3, the protruding portion 14 is inserted into the second insertion portion 55, so that the protruding portion 14 fits into the second insertion portion 55, and the first engagement portion 13 and the second engagement portion 52 are engaged with each other. In the state where the first engagement portion 13 and the second engagement portion 52 are engaged with each other, the protruding portion 14 comes into contact with the peripheral surfaces 55a and 55b, so that the axial movement of the cylindrical member 10 relative to the holding member 50 is restricted.

[0020] When the cell culture instrument 100 transitions from the first state to the disengaged state, that is, when the engagement between the first engagement portion 13 and the second engagement portion 52 is released, the above procedure is carried out in reverse, that is, in a first stage in which the protrusion 14 is pulled out from the second insertion portion 55 along the -Y direction, and in a second stage in which the protrusion 14 is pulled out from the first insertion portion 54 upward along the axial direction.

[0021] 2, in the first state and the second state, the protruding portion 14 protrudes from the outer peripheral surface of the side wall 51a. Therefore, when the first engaging portion 13 and the second engaging portion 52 are engaged with each other, the portion of the protruding portion 14 protruding from the outer peripheral surface of the side wall 51a can be gripped. This improves the workability when engaging the first engaging portion 13 and the second engaging portion 52.

[0022] As shown in FIG. 4, in the second state, the protrusion 14 of the cylindrical member 10 is inserted into the second insertion portion 55 of the holding member 50, as in the first state. The posture of the cylindrical member 10 is different between the first state and the second state. In the first state, the cylindrical member 10 is in a posture in which the culture membrane is downward with respect to the first engagement portion 13. In contrast, in the second state, the cylindrical member 10 is in a posture in which the culture membrane is upward with respect to the first engagement portion 13. The first state is a posture in which the culture membrane 12 is disposed between the first engagement portion 13 and the bottom surface 51b of the holding member 50 in the axial direction. The second state is a posture in which the bottom surface 51b of the holding member 50 and the cylindrical portion 11 face each other with the first engagement portion 13 interposed therebetween. The transition of the cell culture instrument 100 from the non-engaged state to the second state is performed in the same manner as the transition to the first state. That is, the transition is performed in a first stage in which the protrusion 14 is inserted into the first insertion portion 54 along the axial direction up to the end of the first insertion portion 54, and a second stage in which the protrusion 14 is inserted into the second insertion portion 55 along the Y direction. The transition from the first state to the disengaged state is also performed in the same manner as the transition from the first state to the disengaged state when the first engagement portion 13 and the second engagement portion 52 are released from engagement with each other.

[0023] A2. Cell culture method using cell culture equipment: FIG. 5 is a flow chart showing cell culture steps for achieving a cell culture method using the cell culture instrument 100. In a first step P10, the cell culture instrument 100 is prepared. In a step P20, the cylindrical portion 11 is housed in the holding member 50, and the first engagement portion 13 and the second engagement portion 52 are engaged. This brings the cell culture instrument 100 into the first state shown in FIG. 3. In a step P30, a first cell suspension containing first cells and a culture medium for the first cells is injected into the cylindrical member 10. By injecting the first cell suspension, the first cells are seeded on the first surface 12a of the culture membrane 12. In a step P30, the culture medium may or may not be injected into the holding member 50.

[0024] In the second step P40, the culture medium is appropriately replaced, and the first cells are cultured until they are fixed on the first surface 12a of the culture membrane 12. Specifically, the culture medium is extracted from the cylindrical member 10 and injected into the cylindrical member 10 using a pipette. When the culture medium is injected into the holding member 50, the culture medium is also extracted from and injected into the holding member 50. When the culture medium is extracted and injected, the operator may tilt the cell culture instrument 100 so that the central axes CX1 and CX2 are inclined relative to the vertical direction in order to move the culture medium to the vicinity of the boundary between the cylindrical portion 11 and the culture membrane 12. Here, in this embodiment, even when the cell culture instrument 100 is tilted, the first engagement portion 13 and the second engagement portion 52 are engaged with each other, so that the positional deviation of the cylindrical member 10 with respect to the holding member 50 is suppressed. Therefore, the workability of the culture medium replacement can be improved, and stable cell culture can be performed. Furthermore, since the multiple holding members 50 are connected by the connecting portions 90, the operator can tilt the multiple holding members 50 simultaneously. The operator can then hold the multiple holding members 50 in a tilted state and extract the culture medium from the multiple cylindrical members 10 in succession. This can improve the workability of replacing the culture medium.

[0025] In step P50, the first jig 800 shown in FIG. 3 is attached to the cylindrical member 10 to store the culture solution inside the cylindrical portion 11. The first jig 800 is flexible and formed in a cylindrical shape. The outer diameter of the first jig 800 is large enough to be in close contact with the inner circumferential surface of the cylindrical portion 11. The first jig 800 is inserted into the cylindrical portion 11 with the central axis of the first jig 800 aligned with the central axis CX1 of the cylindrical member 10. The first jig 800 is inserted until the lower end, which is the insertion direction of the first jig 800, is separated from the culture membrane 12. This allows the culture solution to be stored inside the cylindrical portion 11 even when the cylindrical member 10 is turned upside down in the next step P60. Therefore, the culture of the first cells can be continued. It is preferable that the culture solution is stored in a state in which the inside of the cylindrical portion 11 closed by the first jig 800 is liquid-tight.

[0026] In step P60, after the first engagement portion 13 and the second engagement portion 52 are disengaged, the cylindrical member 10 is turned upside down and the first engagement portion 13 and the second engagement portion 52 are engaged with each other. This brings the cell culture instrument 100 into the second state shown in FIG.

[0027] In step P70, the second jig 900 shown in Fig. 4 is attached to the outer circumferential surface of the cylindrical member 10 so as to cover the periphery of the culture membrane 12. The second jig 900 is a flexible cylindrical member. By attaching the second jig 900, a container for storing the culture solution is formed, with the culture membrane 12 as the bottom surface and the inner circumferential surface of the second jig 900 as the side surface.

[0028] In step P80, a second cell suspension containing second cells and a culture medium for the second cells is injected into the second jig 900. By injecting the second cell suspension, the second cells are seeded on the second surface 12b of the culture membrane 12.

[0029] In the third step P90, the culture medium is appropriately exchanged and the second cells are cultured until the second cells contained in the second cell suspension are fixed on the second surface 12b of the culture membrane 12. The culture in the third step P90 is performed in an incubator in an environment with constant and stable temperature, humidity, and CO2 concentration, that is, in order to culture the cells in a state closer to physiological conditions. When exchanging the culture medium in the third step P90, the cell culture instrument 100 may be tilted by an operator, as in the second step P40. Even when the cell culture instrument 100 is tilted, the first engagement portion 13 and the second engagement portion 52 are engaged with each other, as in the second step P40, so that the positional deviation of the cylindrical member 10 with respect to the holding member 50 is suppressed. This can improve the workability. In addition, the first engagement portion 13 and the second engagement portion 52 are engaged with each other, so that the cell culture instrument 100 can be prevented from shaking during transportation.

[0030] In step P100, first, the culture medium is drained from the inside of the second jig 900, and the second jig 900 is removed from the cylindrical member 10. At this time, the culture medium may be drained one row at a time by tilting the cell culture instrument 100. Then, after the engagement between the first engagement portion 13 and the second engagement portion 52 is released, the cylindrical member 10 is turned upside down, and the first engagement portion 13 and the second engagement portion 52 are engaged. As a result, the cell culture instrument 100 is again in the first state shown in FIG. 3.

[0031] In step P110, the culture of the second cells is continued. The culture is performed in an incubator in the same manner as in the culture in the third step P90 described above. Specifically, a culture medium for the second cells fixed on the second surface 12b is poured into the bottomed cylindrical portion 51, and a culture medium for the first cells fixed on the first surface 12a is poured into the cylindrical portion 11. As in the second step P40 and the third step P90, the culture medium is appropriately replaced, and the second cells are cultured until they become confluent. In step P110, as in the second step P40, even when the cell culture instrument 100 is tilted, the first engagement portion 13 and the second engagement portion 52 are engaged with each other, so that the positional deviation of the cylindrical member 10 with respect to the holding member 50 is suppressed. This can improve the workability. When the culture of the second cells is completed, this cell culture step is completed. By the cell culture step, cells can be cultured on each of the first surface 12a and the second surface 12b of the culture membrane 12.

[0032] In the cell culture process described above, the cylindrical member 10 and the holding member 50 can be handled as one unit by engaging the first engaging portion 13 with the second engaging portion 52. Generally, in cell culture, a small amount of a plurality of samples are simultaneously cultured. In order to identify the plurality of samples, an identification number is assigned to each sample. Here, in this embodiment, since the cylindrical member 10 and the holding member 50 can be handled as one unit, each sample can be identified by assigning an identification number to either the cylindrical member 10 or the holding member 50. In addition, since the cylindrical member 10 can be engaged with the holding member 50 in any of the second step P40, the third step P90, and the step P110, it is possible to prevent the cylindrical member 10 that is associated with the holding member 50 in the second step P40 from being attached to an incompatible holding member 50 in the subsequent third step P90 or step P110. Therefore, the workability in the cell culture process can be improved.

[0033] According to the first embodiment described above, the cylindrical member 10 has the cylindrical portion 11, the culture membrane 12, and the first engagement portion 13. The holding member 50 has the bottomed cylindrical portion 51 and the second engagement portion 52 that can be engaged with the first engagement portion 13. The second engagement portion 52 engages with the first engagement portion 13 in a first state in which the cylindrical portion 11 is accommodated in the bottomed cylindrical portion 51 and in a second state in which the attitude of the cylindrical portion 11 is axially opposite to that in the first state. In both the first state and the second state, the second engagement portion 52 can be engaged with the first engagement portion 13, so that the cylindrical member 10 and the holding member 50 can be handled as a single unit. This improves the operability of the cell culture instrument 100 in the cell culture process. Furthermore, by engaging the first engagement portion 13 with the second engagement portion 52, even when the holding member 50 is tilted, shifting of the cylindrical member 10 relative to the holding member 50 can be suppressed, enabling stable cell culture to be performed.

[0034] Furthermore, the cell culture instrument 100 includes a plurality of holding members 50, and the plurality of holding members 50 are connected by connecting portions 90. Therefore, the plurality of holding members 50 can be handled as a unit, which can further improve the workability in the cell culture process.

[0035] In addition, the second engagement portion 52 has an axial restriction portion 53 that restricts the axial movement of the cylindrical member 10 by engaging with the first engagement portion 13. This makes it possible to restrict the axial movement of the cylindrical member 10 relative to the holding member 50.

[0036] Moreover, the first engagement portion 13 has a protrusion 14. The axial restriction portion 53 has a first insertion portion 54 and a second insertion portion 55. In the first state and the second state, a pair of peripheral partition surfaces 55a, 55b that define the second insertion portion 55 abut against the protrusion 14 inserted therebetween, thereby restricting axial movement. Thus, by inserting the protrusion 14 into the second insertion portion 55, the axial movement of the cylindrical member 10 relative to the holding member 50 can be restricted.

[0037] Moreover, the first insertion portion 54 and the second insertion portion 55 are formed to radially penetrate the side wall 51a of the bottomed cylindrical portion 51. In the first state and the second state, the protrusion 14 protrudes from the outer circumferential surface of the side wall 51a. This allows the portion of the protrusion 14 protruding from the outer circumferential surface of the side wall 51a to be gripped when engaging the first engagement portion 13 and the second engagement portion 52, thereby improving the workability when engaging them.

[0038] Furthermore, the multiple cylindrical members 10 are connected by protruding portions 14. This allows the multiple cylindrical members 10 to be handled as a unit, improving workability.

[0039] The cell culture process includes a first step P10 of preparing the cell culture instrument 100, a second step P40 of culturing a first cell on the first surface 12a of the culture membrane 12 in the first state, and a third step P90 of culturing a second cell on the second surface 12b of the culture membrane 12 in the second state. In the cell culture process, the cylindrical member 10 is engaged with the holding member 50, so that the cylindrical member 10 and the holding member 50 can be handled as a unit, thereby improving the workability of the cell culture instrument 100. Even when the holding member 50 is tilted in the second step P40 and the third step P90, the displacement of the cylindrical member 10 with respect to the holding member 50 can be suppressed, and stable cell culture can be performed.

[0040] B. Second embodiment: 6 is a perspective view of a cell culture instrument 2100 according to the second embodiment. The cell culture instrument 2100 differs from the cell culture instrument 100 according to the first embodiment in the shapes of first engagement portion 13 and second engagement portion 52. The same components as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof will be omitted as appropriate.

[0041] The cell culture instrument 2100 includes a cylindrical member 210 and a holding member 250. In this embodiment, unlike the first embodiment, the holding member 250 has a structure in which the multiple holding members 250 are not connected, but they may be connected as in the first embodiment. The same applies to the third to sixth embodiments described below. The cylindrical member 210 has a cylindrical portion 11, a culture membrane 12, and four first engagement portions 213.

[0042] The four first engagement portions 213 are arranged at equal angular intervals in the circumferential direction at one open end of the cylindrical portion 11. The first engagement portions 213 have a protrusion 214. The protrusion 214 protrudes outward from the outer circumferential surface of the cylindrical member 10. The protrusion 214 is rod-shaped with a rectangular cross section and extends in the radial direction of the cylindrical portion 11.

[0043] The holding member 250 has a bottomed cylindrical portion 51 and four second engagement portions 252. The four second engagement portions 252 are arranged at equal angular intervals in the circumferential direction at one open end of the side wall 51a. The radial thickness of the side wall 51a is not uniform, and the thickness of the side wall 51a at the open end is thicker than the thickness of the lower side wall 51a connected to the bottom surface 51b. Specifically, the inner diameter of the side wall 51a is uniform, and the outer diameter of the side wall 51a at the open end is larger than the outer diameter of the bottom surface 51b. The second engagement portions 252 are arranged on the thick side wall 51a.

[0044] The second engagement portion 252 has a horizontal regulation portion 253 that regulates the horizontal movement of the cylindrical member 210 by engaging with the first engagement portion 213. The horizontal regulation portion 253 has a recess 254 that extends in the axial direction along the central axis CX2 of the bottomed cylindrical portion 51. The recess 254 into which the protrusion 214 is axially inserted has a shape in which a part of the inner circumferential surface of the side wall 51a is cut out. Specifically, the radial length of the recess 254 is shorter than the thickness of the side wall 51a. In addition, the axial length of the recess 254 is longer than the axial thickness of the protrusion 214 of the cylindrical member 210. This makes it possible to suppress the positional deviation of the cylindrical member 210 with respect to the holding member 250 even when the cell culture instrument 100 is tilted in a state in which the first engagement portion 213 and the second engagement portion 252 are engaged with each other.

[0045] In both the first state and the second state, the protrusion 214 fits into the recess 254, so that the first engagement portion 213 and the second engagement portion 252 are engaged with each other. In the state in which the first engagement portion 213 and the second engagement portion 252 are engaged with each other, the protrusion 214 abuts against the recess 254, so that the movement of the cylindrical member 10 in the horizontal direction perpendicular to the axial direction relative to the holding member 50 is restricted. This allows the cylindrical member 210 and the holding member 250 to be handled as a single unit, as in the first embodiment. This improves the workability of the cell culture instrument 2100 in the cell culture step. In addition, by engaging the first engagement portion 213 with the second engagement portion 252, even when the holding member 250 is tilted, it is possible to suppress the displacement of the cylindrical member 210 relative to the holding member 250, so that stable cell culture can be performed.

[0046] According to the second embodiment described above, the second engagement portion 252 has the horizontal restriction portion 253 that restricts the horizontal movement of the cylindrical member 210 by engaging with the first engagement portion 213. This makes it possible to restrict the horizontal movement of the cylindrical member 210 relative to the holding member 250.

[0047] Moreover, the first engagement portion 213 has a protruding portion 214. The horizontal restriction portion 253 has a recessed portion 254 into which the protruding portion 214 is axially inserted. In the first state and the second state, the protruding portion 214 abuts against the recessed portion 254, thereby restricting horizontal movement. As a result, by inserting the protruding portion 214 into the recessed portion 254, it is possible to restrict the horizontal movement of the cylindrical member 210 relative to the holding member 250.

[0048] C. Third embodiment: 7 is a diagram showing cell culture instruments according to the third to sixth embodiments. The same components as those in the first or second embodiment are given the same reference numerals, and detailed description will be omitted as appropriate. A cylindrical member 310 according to the third embodiment differs from the cylindrical member 10 according to the first embodiment in the shape of a first engagement portion 313. A holding member 350 according to the third embodiment differs from the holding member 50 according to the first embodiment in the shape of a second engagement portion 352.

[0049] The cylindrical member 310 has a cylindrical portion 11, a culture membrane 12, and four first engagement portions 313. The four first engagement portions 313 are arranged at equal angular intervals in the circumferential direction at one open end of the cylindrical portion 11. The first engagement portions 313 have a protruding portion 314. The protruding portion 314 protrudes outward from the outer circumferential surface of the cylindrical member 10. The protruding portion 314 is rod-shaped with a rectangular cross section and extends in the radial direction of the cylindrical portion 11.

[0050] The holding member 350 has a bottomed cylindrical portion 51 and four second engagement portions 352. The four second engagement portions 352 are arranged at equal angular intervals in the circumferential direction at one end where the side wall 51a opens. The four second engagement portions 352 are arranged at equal angular intervals in the circumferential direction at one end where the bottomed cylindrical portion 51 opens. The second engagement portions 352 have an axis regulating portion 353. The axis regulating portion 353 has a first insertion portion 354 and a second insertion portion 355. The second insertion portion 355 according to this embodiment has a different direction of extension from the first insertion portion 354 than the second insertion portion 55 according to the first embodiment. Specifically, the second insertion portion 55 according to the first embodiment extends in the Y direction from the first insertion portion 54, whereas the second insertion portion 355 according to this embodiment extends counterclockwise from the first insertion portion 354 around the central axis CX2 when viewed from above.

[0051] As in the first embodiment, the first insertion portion 354 is partitioned by a pair of first insertion portion partition surfaces. Also, the second insertion portion 355 is partitioned by a pair of peripheral partition surfaces. In this embodiment, the procedure of engagement between the first engagement portion 313 and the second engagement portion 352 is different from that of the first embodiment. Specifically, the first stage is the same as that of the first embodiment, but in the second stage, the cylindrical member 310 is rotated counterclockwise around the central axis CX1 relative to the holding member 350. As a result, the first engagement portion 313 is inserted into the second insertion portion 355. As in the first embodiment, the protrusion 314 abuts against the pair of peripheral partition surfaces of the second insertion portion 355, thereby restricting the axial movement of the cylindrical member 310 relative to the holding member 350.

[0052] D. Fourth embodiment: The cylindrical member 410 according to the fourth embodiment is different from the cylindrical member 10 according to the first embodiment in the shape of the first engagement portion 413. The holding member 450 according to the fourth embodiment is different from the holding member 50 according to the first embodiment in the shape of the second engagement portion 452. The cylindrical member 410 has a cylindrical portion 11, a culture membrane 12, and four first engagement portions 413. The four first engagement portions 413 are arranged at equal angular intervals in the circumferential direction at one end where the cylindrical portion 11 opens. The first engagement portion 413 has an extension portion 414. The extension portion 414 protrudes outward from the outer circumferential surface of the cylindrical member 10. The extension portion 414 has a base portion 415 and two claw portions 416. The base portion 415 is rod-shaped with a rectangular cross section and extends radially from the outer circumferential surface of the cylindrical portion 11. Two claw portions 416 are formed at the tip of the base portion 415. The two claws 416 have a shape that is symmetrical with respect to a plane that passes through the center position of the base 415 in the axial direction and is parallel to the culture membrane 12.

[0053] The holding member 450 has a bottomed cylindrical portion 51 and a second engagement portion 452. The second engagement portion 452 has an axis regulating portion 453. The axis regulating portion 453 has an outer peripheral protrusion 454 that protrudes outward from the bottomed cylindrical portion 51. The outer peripheral protrusion 454 has a circular ring shape that is coaxial with the bottomed cylindrical portion 51, and is formed around the entire circumference of the bottomed cylindrical portion 51. The outer peripheral protrusion 454 has a surface that is parallel to the central axis CX2 and faces outward from a cut surface cut along a plane along the radial direction, and is a curved surface that is convex outward in the radial direction.

[0054] Of the two claws 416 of the extension 414, one claw 416 closer to the culture membrane 12 draws an arc and bends downward toward the tip. The other claw 416 draws an arc and bends upward toward the tip. The arc drawn by the claw 416 is an arc along the outer circumferential surface of the outer circumferential protrusion 454. When the first engagement portion 413 and the second engagement portion 452 are engaged with each other, the cylindrical member 410 is brought closer to the holding member 450 in the axial direction with the central axis CX1 aligned with the central axis CX2 of the holding member 450. The tip of the claw 416 rides over the outer circumferential protrusion 454 and fits in a state of covering the outer circumferential protrusion 454. When the first engagement portion 413 and the second engagement portion 452 are engaged with each other, the claw 416 covers the outer circumferential protrusion 454 and abuts against the outer circumferential protrusion 454 in the axial direction. In the first state, of the two claw portions 416, one claw portion 416 closer to the culture membrane 12 engages with the outer circumferential protrusion 454. In contrast, in the second state, of the two claw portions 416, the other claw portion 416 engages with the outer circumferential protrusion 454. In this embodiment, the claw portion 416 abuts against the outer circumferential protrusion 454 in the axial direction, thereby restricting the axial movement of the cylindrical member 410 relative to the holding member 450.

[0055] According to the fourth embodiment described above, the first engagement portion 413 has the extension portion 414. The shaft regulating portion 453 has the outer peripheral protrusion 454. The extension portion 414 has the claw portion 416 that covers the outer peripheral protrusion 454 and abuts against the outer peripheral protrusion 454 in the axial direction. In the first state and the second state, the claw portion 416 abuts against the outer peripheral protrusion 454 to regulate the axial movement of the cylindrical member 410. By engaging the claw portion 416 with the outer peripheral protrusion 454, the axial movement of the cylindrical member 410 relative to the holding member 450 can be regulated.

[0056] E. Fifth embodiment: The cylindrical member 510 according to the fifth embodiment is different from the cylindrical member 210 according to the second embodiment in the shape of the first engagement portion 513. The holding member 550 according to the fifth embodiment is different from the holding member 250 according to the second embodiment in the shape of the second engagement portion 552. The four first engagement portions 513 are arranged at equal angular intervals in the circumferential direction at one end where the cylindrical portion 11 opens. The first engagement portion 513 has a protruding portion 514. The protruding portion 514 has a base portion 515 and two protruding portions 516. The protruding portion 514 protrudes outward from the outer circumferential surface of the cylindrical portion 11. The base portion 515 has a flat plate shape and extends from the outer circumferential surface of the cylindrical portion 11 in the radial direction of the cylindrical portion 11. The two protruding portions 516 extend in the axial direction from the surface facing upward and the surface facing downward of the base portion 515, respectively. The protruding portion 516 is rod-shaped with a circular cross section.

[0057] The holding member 550 has a bottomed cylindrical portion 51 and four second engagement portions 552. The four second engagement portions 552 are arranged at equal angular intervals in the circumferential direction at one end where the side wall 51a opens. The second engagement portion 552 has a horizontal regulation portion 553. The horizontal regulation portion 553 has a recess 554 that extends from the end where the side wall 51a opens toward the bottom surface 51b in the axial direction. The cross-sectional shape of the recess 554, cut along a plane parallel to the bottom surface 51b, is circular. The diameter of the recess 554 is larger than the outer diameter of the protrusion 516.

[0058] In both the first state and the second state, the protrusion 516 is inserted into the recess 554, and the protrusion 516 fits into the recess 554, thereby engaging the first engagement portion 513 with the second engagement portion 552. In the first state, of the two protrusions 516, one protrusion 516 closer to the culture membrane 12 fits into the recess 554. In contrast, in the second state, of the two protrusions 516, the other protrusion 516 fits into the recess 554. In a state in which the first engagement portion 513 and the second engagement portion 552 are engaged, the protrusion 514 abuts against the recess 554, thereby restricting horizontal movement of the cylindrical member 510 relative to the holding member 550.

[0059] F. Sixth embodiment: The cylindrical member 610 according to the sixth embodiment is different from the cylindrical member 210 according to the second embodiment in the shape of the first engagement portion 213. The holding member 650 according to the sixth embodiment is different from the holding member 250 according to the second embodiment in the shape of the second engagement portion 652. The cylindrical member 610 has a cylindrical portion 11, a culture membrane 12, and four first engagement portions 613. The first engagement portions 613 have protrusions 614. The shape of the first engagement portions 613 is similar to the shape of the first engagement portions 313 according to the third embodiment, and therefore description thereof will be omitted.

[0060] The holding member 650 according to the sixth embodiment is different from the holding member 250 according to the second embodiment in the shape of the second engagement portion 652. The holding member 650 has a bottomed cylindrical portion 51 and four second engagement portions 652. The four second engagement portions 652 are arranged at equal angular intervals in the circumferential direction at one end where the side wall 51a opens. The second engagement portion 652 has a horizontal regulation portion 653. The horizontal regulation portion 653 has a recess 654 extending in the axial direction from the end where the side wall 51a opens. The recess 654 has a shape in which a part of the side wall 51a is cut out. Specifically, the recess 654 is formed penetrating the side wall 51a in the radial direction. The recess 654 extends in the axial direction, which is the direction along the central axis CX2, from the end where the side wall 51a opens toward the bottom surface 51b. The axial length of the recess 654 is longer than the axial thickness of the protrusion 614. In this embodiment, the axial length of the recess 654 is about three times the axial thickness of the protrusion 614. This makes it difficult for the engagement between the protrusion 614 and the recess 654 to be released in both the first state and the second state.

[0061] In both the first state and the second state, the protruding portion 614 is inserted into the recessed portion 654, and the protruding portion 614 fits into the recessed portion 654, thereby engaging the first engaging portion 613 with the second engaging portion 652. In the state in which the first engaging portion 613 and the second engaging portion 652 are engaged, the protruding portion 614 abuts against the recessed portion 654, thereby restricting horizontal movement of the cylindrical member 610 relative to the holding member 650.

[0062] G. Other embodiments: (G1) In the first embodiment, the cell culture instrument 100 includes a plurality of holding members 50, which are arranged in a straight line. In addition to this form, the plurality of holding members 50 may be arranged in a matrix. The matrix arrangement may be appropriately selected from those having an appropriate number of holding members in the vertical and horizontal directions, such as 2×3, 4×6, or 8×12. By arranging in a matrix, the cell culture instrument 100 can be more stably placed on the workbench. Note that the plurality of holding members 50 arranged in a matrix may be connected by a plurality of connecting parts such as the connecting parts 90, or may be arranged inside a box-shaped planar plate with an open top.

[0063] (G2) In the above fifth embodiment, the cylindrical member 510 is not connected to other cylindrical members 510. Alternatively, a configuration in which a plurality of cylindrical members 510 are connected via the first engagement portion 513 may be adopted. This allows the plurality of cylindrical members 510 to be handled as a single unit, thereby improving workability. The same applies to the sixth embodiment.

[0064] (G3) The cell culture instrument 100 of the above embodiment may include a lid that fastens to the holding member 50 to close the cell culture instrument 100 in the first state and the second state. The lid is used to maintain the culture environment in the above steps. The lid may be composed of two types of lid: a lid for the first state and a lid for the second state. Alternatively, in order to have only one type of lid, the side wall 51a of the holding member 50 may be extended so that the cylindrical member 10 is not exposed from the holding member 50 even in the second state.

[0065] 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]

[0066] 10,210,310,410,510,610...Cylindrical member, 11...Cylindrical portion, 12...Culture membrane, 12a...First surface, 12b...Second surface, 13,213,313,413,513,613...First engagement portion, 14,214,314,514,614...Protruding portion, 50,250,350,450,550,650...Holding member, 51...Bottomed cylindrical portion, 51a...Side wall, 51b...Bottom surface, 52,252,352,452,552,652...Second engagement portion, 53,353,453...Axis regulating portion, 54,354...First insertion portion, 54a,54b... First insertion section screen, 55,355...second insertion section, 55a,55b...peripheral section screen, 90...connecting section, 100,2100...cell culture device, 253,553,653...horizontal regulation section, 254,554,654...recess, 414...extension section, 415,515...base section, 416...claw section, 454...peripheral protrusion section, 516...projection section, 800...first jig, 900...second jig, CX1,CX2...center axis, P10...first step, P40...second step, P90...third step, P20,P30,P50,P60,P70,P80,P100,P110...steps

Claims

1. A cell culture device comprising: A cylindrical member having a cylindrical portion, a culture membrane disposed at one end of the cylindrical portion in an axial direction, and a first engagement portion formed at the other end of the cylindrical portion in the axial direction; a holding member having a bottomed cylindrical portion capable of accommodating the cylindrical portion and a second engaging portion capable of engaging with the first engaging portion, the second engagement portion engages with the first engagement portion both in a first state in which the cylindrical portion is housed in the bottomed cylindrical portion and in a second state in which the orientation of the cylindrical portion is opposite to that in the first state in the axial direction, The second engagement portion has an axial regulating portion that engages with the first engagement portion to regulate both movement of the cylindrical member in the axial direction from the one end to the other end and movement of the cylindrical member in the axial direction from the other end to the one end.

2. The cell culture instrument according to claim 1, The holding member and the cylindrical member are each provided in a plurality of pieces, and a connecting portion is provided to connect two adjacent holding members of the plurality of holding members, The first engagement portion has a protrusion that protrudes outward from an outer circumferential surface of the cylindrical portion, The axial regulating portion has: (i) a first insertion portion into which the protruding portion is inserted in the axial direction; and (ii) a second insertion portion extending from the first insertion portion in a circumferential direction of the holding member and into which the protruding portion is inserted in the circumferential direction, the second insertion portion being partitioned by a pair of circumferential partition surfaces opposed to each other with a gap in the axial direction, The first insertion portion and the second insertion portion are formed by radially penetrating a side wall of the bottomed cylindrical portion, In the first state and the second state, the protrusion protrudes from an outer circumferential surface of the side wall, A cell culture instrument, wherein two adjacent cylindrical members of the plurality of cylindrical members are connected to each other by the protrusion.

3. The cell culture instrument according to claim 1, The first engagement portion has an extension portion extending outward from an outer circumferential surface of the cylindrical portion, the shaft restricting portion has an outer peripheral protruding portion protruding outward from an outer peripheral surface of the bottomed cylindrical portion, the extension portion covers the outer circumferential protrusion and has a claw portion that abuts against the outer circumferential protrusion in the axial direction, In the first state and the second state, the claw portion restricts movement in the axial direction by abutting against the outer peripheral protrusion.

4. 1. A cell culture method comprising: a first step of preparing a cell culture instrument comprising: a cylindrical member having a cylindrical portion, a culture membrane disposed at one axial end of the cylindrical portion, and a first engagement portion formed at the other axial end; and a holding member having a bottomed cylindrical portion capable of accommodating the cylindrical portion, and a second engagement portion engageable with the first engagement portion, wherein the second engagement portion engages with the first engagement portion both in a first state in which the cylindrical portion is accommodated in the bottomed cylindrical portion and in a second state in which the orientation of the cylindrical portion is opposite to that in the first state in the axial direction, and the second engagement portion engages with the first engagement portion to thereby regulate both movement of the cylindrical member from one end to the other end in the axial direction and movement of the cylindrical member from the other end to the one end in the axial direction; A second step of culturing cells on a first surface of the culture membrane facing the inside of the cylindrical portion in the first state; A cell culture method comprising: a third step of culturing cells on a second surface of the culture membrane opposite the first surface in the second state.

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