JP7505431B2 - Cell culture vessel and cell culture method - Google Patents

Description

This disclosure relates to a cell culture vessel and a cell culture method.

Conventionally, there is a cell culture instrument for culturing cells on both sides of a culture membrane (for example, Patent Document 1). The cell culture instrument of Patent Document 1 includes a first inner cylinder, an outer cylinder, and a cell culture membrane arranged to cover the end of the first inner cylinder. When cells are cultured, the cell culture instrument is placed in a well, and the well is filled with culture medium, so that the cell culture membrane is immersed in the culture medium. After cells are cultured on one side of the cell culture membrane, the cell culture instrument is removed from the well, the cell culture instrument is inverted, and the cell culture instrument is placed in the well, and cells are cultured on the other side of the cell culture membrane.

JP 2015-208283 A

However, in the cell culture instrument described in Patent Document 1, the culturing process requires the step of placing the cell culture instrument in the well as described above, and further requires the step of removing the cell culture instrument from the well in order to invert the cell culture instrument, which can make the process complicated.

The present disclosure can be realized in the following forms.
According to a first aspect of the present disclosure, there is provided a cell culture vessel, comprising: an inner cylinder having an inner cylinder one end, an inner cylinder other end facing the inner cylinder one end, a cylindrical inner cylinder side wall connecting the inner cylinder one end and the inner cylinder other end, and an inner cylinder recess formed as a notch at the inner cylinder one end, a liquid stop pin detachably attached to the inner cylinder recess and closing the inner cylinder recess, a culture membrane part disposed at the inner cylinder one end and closing an inner cylinder opening formed at the inner cylinder one end, and an outer cylinder detachably attached to the inner cylinder from the inner cylinder one end side, the outer cylinder having the outer cylinder one end, the outer cylinder other end facing the outer cylinder one end, and a cylindrical outer cylinder side wall connecting the outer cylinder one end and the outer cylinder other end, and the culture membrane part closes the outer cylinder opening at the outer cylinder other end when the outer cylinder is attached to the inner cylinder.
According to a second aspect of the present disclosure, a cell culture method using a plurality of cell culture vessels is provided, each of the plurality of cell culture vessels includes an inner cylinder having one end of an inner cylinder, an other end of the inner cylinder facing the one end of the inner cylinder, a cylindrical inner cylinder side wall connecting the one end of the inner cylinder and the other end of the inner cylinder, and an inner cylinder recess formed as a notch in the one end of the inner cylinder, a culture membrane part disposed in the one end of the inner cylinder and closing an inner cylinder opening formed in the one end of the inner cylinder, and an outer cylinder detachably attached to the inner cylinder from the one end side of the inner cylinder, the one end of the outer cylinder, the other end of the outer cylinder facing the one end of the outer cylinder, and a wall of the outer cylinder between the one end of the outer cylinder and the other end of the outer cylinder. and an outer cylinder having a cylindrical outer cylinder side wall connecting one end of the outer cylinder to the other end of the inner cylinder, the culture membrane part closes an outer cylinder opening at the other end of the outer cylinder when the outer cylinder is attached to the inner cylinder, and the culture membrane part is joined to each of the multiple inner cylinders. The cell culture method includes a fixing jig capable of simultaneously fixing the multiple inner cylinders of the multiple cell culture vessels, an inversion jig that holds the multiple inner cylinders and can simultaneously invert the multiple inner cylinders, and a reversing jig that is detachably attached to the multiple inner cylinder recesses of the multiple cell culture vessels and can invert the multiple inner cylinders. and a liquid stopping jig for closing an inner tube recess, the method comprising the steps of: preparing the inner tubes to which the liquid stopping jig is attached; injecting a first cell suspension containing a first cell into the inner tubes fixed on the fixing jig so that one end of the inner tube is below the other end of the inner tube, and culturing the first cell on the culture membrane portion; and sucking out the first cell suspension from the inner tubes and simultaneously inverting the inner tubes using the inversion jig so that one end of the inner tube is above the other end of the inner tube. the step of attaching the outer tube to each of the plurality of inner tubes and removing the liquid stopping device from the plurality of inner tubes; the step of arranging the plurality of inner tubes in a well plate in which a plurality of wells containing a first culture medium for the first cells are arranged, such that each of the plurality of inner tubes with the outer tube attached thereto enters each of the plurality of wells; and the step of placing a second cell suspension containing second cells different from the first cells into each of the plurality of outer tubes and culturing the second cells on the culture membrane portion.

(1) According to one embodiment of the present disclosure, there is provided a cell culture vessel, comprising: an inner cylinder having an inner cylinder one end, an inner cylinder other end opposite the inner cylinder one end, a cylindrical inner cylinder side wall connecting the inner cylinder one end and the inner cylinder other end, and an inner cylinder recess formed as a notch in the inner cylinder other end, a liquid stop pin detachably attached to the inner cylinder recess and closing the inner cylinder recess, a culture membrane part disposed at the inner cylinder one end and closing an inner cylinder opening formed at the inner cylinder one end, and an outer cylinder detachably attached to the inner cylinder from the inner cylinder one end side, the outer cylinder having the outer cylinder one end, the outer cylinder other end opposite the outer cylinder one end, and a cylindrical outer cylinder side wall connecting the outer cylinder one end and the outer cylinder other end, and the culture membrane part closes the outer cylinder opening at the outer cylinder other end when the outer cylinder is attached to the inner cylinder. According to this embodiment, in the first culture step of culturing the first cells, the inner cylinder can be used as a container for storing the first cell liquid medium by posing the culture membrane part as the bottom surface of the inner cylinder. This eliminates the need to insert the cell culture vessel into the well or remove the cell culture vessel from the well, improving workability. In addition, in the second culture step of culturing the second cells, the outer cylinder can be used as a container for storing the second cell liquid medium by posing the culture membrane part as the bottom surface of the outer cylinder. Thus, co-culture can be performed using the cell culture vessel. In addition, in the second culture step, residual air in the first cell liquid medium can be discharged from the inner cylinder recess.
(2) In the cell culture vessel of the above embodiment, the outer cylinder may further have an outer cylinder recess formed as a notch in a part of the outer cylinder side wall at a position overlapping with the inner cylinder recess in the radial direction of the inner cylinder when in the attached state. According to this embodiment, when the inner cylinder is immersed in liquid in the second culture step, air remaining in the liquid can be discharged through the inner cylinder recess where the liquid stop pin is not attached and the outer cylinder recess. Therefore, culture can be performed well.
(3) In the cell culture vessel of the above embodiment, the outer cylinder may further have a convex portion that protrudes from the inner peripheral surface of the side wall of the outer cylinder and that faces and abuts against the culture membrane portion in a direction along the central axis of the inner cylinder when the outer cylinder is in the attached state. According to this embodiment, it is possible to easily fix the outer cylinder to the inner cylinder in which the culture membrane portion is disposed.
(4) In the cell culture vessel of the above embodiment, the convex portion may have a smooth surface that contacts the culture membrane portion. According to this embodiment, leakage of the liquid placed in the outer cylinder through a gap between the convex portion and the culture membrane portion in the second culture step can be suppressed.
(5) In the cell culture vessel of the above embodiment, the culture membrane part may further include a ring-shaped support frame arranged to cover an end face of one end of the inner tube on the side wall of the inner tube, and a cell culture membrane joined to the support frame. According to this embodiment, the support frame is arranged on the end face, so that the support frame can bear external forces, thereby reducing stress on the inner tube recess and reducing stress on the cell culture membrane. Therefore, it is possible to suppress breakage or damage to the cell culture membrane 12 and deterioration of quality due to damage to the first cells.
(6) According to one embodiment of the present disclosure, there is provided a cell culture method using a plurality of cell culture vessels, each of the plurality of cell culture vessels including an inner tube having an inner tube one end, an inner tube other end facing the inner tube one end, a cylindrical inner tube side wall connecting the inner tube one end and the inner tube other end, and an inner tube recess formed as a notch in the inner tube other end, a culture membrane part disposed in the inner tube one end and closing an inner tube opening formed in the inner tube one end, and an outer tube detachably attached to the inner tube from the inner tube one end side, the outer tube having the outer tube one end, the outer tube other end facing the outer tube one end, and a cylindrical outer tube side wall connecting the outer tube one end and the outer tube other end, and the culture membrane part closes the outer tube opening of the outer tube other end when the outer tube is attached to the inner tube. The cell culture method includes a fixing jig capable of simultaneously fixing the inner cylinders of the cell culture vessels, an inversion jig capable of gripping the inner cylinders and simultaneously inverting the inner cylinders, and a liquid stopper jig that is detachably attached to the inner cylinder recesses of the cell culture vessels and blocks the inner cylinder recesses. According to this embodiment, since the work is performed using the fixing jig, the inversion jig, and the liquid stopper jig that can work on the multiple cell culture vessels simultaneously, the cell culture can be performed more efficiently than by working on each cell culture vessel individually.

The present disclosure can also be realized in various forms other than cell culture vessels and cell culture methods. For example, it can be realized in the form of a cell culture method using a cell culture vessel, a fixing jig, an inversion jig, a liquid stopping jig, etc.

FIG. 2 is an exploded perspective view of the cell culture vessel. FIG. 2 is a perspective view of a cell culture vessel. FIG. 3 is a cross-sectional view taken along line III-III of FIG. 1 is a flow chart showing steps of a cell culture method. FIG. 2 is a schematic diagram of the inner cylinder in the first culture step. FIG. 2 is a schematic diagram of a cell culture vessel in a second culture step. FIG. FIG. FIG. This is a cross-sectional view taken along the line XX in FIG. 9 .

A. First embodiment:
A1. Configuration of cell culture vessel:
FIG. 1 is an exploded perspective view of the cell culture vessel 100. FIG. 2 is a perspective view of the cell culture vessel 100. FIG. 3 is a cross-sectional view taken along line III-III of FIG. 2. FIGS. 1 to 3 show an X-axis, a Y-axis, and a Z-axis, which are perpendicular to each other. The cell culture vessel 100 is typically used by being disposed such that the Z-direction is aligned with the vertical direction. In the following description, the +Z direction may be referred to as the upward direction, and the -Z direction may be referred to as the downward direction. The cell culture vessel 100 includes a culture membrane part 10, an inner cylinder 20, an outer cylinder 40, and a liquid stop pin 60. The cell culture vessel 100 is a vessel used for co-culture in which cells are cultured on both sides of the culture membrane part 10.

As shown in FIG. 1, the culture membrane part 10 has a disk shape having a central axis CX. The culture membrane part 10 has a support frame 11 and a cell culture membrane 12. The culture membrane part 10 is joined to an inner tube 20. The support frame 11 is an annular frame. A circular cell culture membrane 12 is joined to cover the end face of the support frame 11. The support frame 11 is formed of a resin material. Examples of the resin material include high-density polyethylene, polypropylene, ABS (Acrylonitrile Butadiene Styrene), acrylic, polyester, polymethylpentene, and the like. The cell culture membrane 12 is a membrane that serves as a scaffold for cell culture when performing cell culture. As the cell 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, examples of the porous membrane include a track-etched membrane, a nanofiber membrane, a collagen vitrigel membrane, and a porous polyurethane membrane.

The inner tube 20 is cylindrical. Specifically, the inner tube 20 has a cylindrical shape with a central axis CX. The outer diameter of the inner tube 20 is approximately the same as the outer diameter of the culture membrane section 10. The inner tube 20 is formed of a resin material. Examples of the resin material include polyethylene, polypropylene, polystyrene, acrylic resin, etc.

The inner tube 20 has an inner tube one end 21, an inner tube other end 22, an inner tube side wall 23, and an inner tube recess 24. The inner tube one end 21 is one end of the inner tube 20 in the direction of the central axis CX. The inner tube other end 22 faces the inner tube one end 21 in the direction of the central axis CX. The inner tube side wall 23 connects the inner tube one end 21 and the inner tube other end 22. The inner tube recess 24 is formed as a notch in the inner tube one end 21 along the central axis CX direction from the end face of the inner tube one end 21. In the first culture step P20 described later in which cells are cultured using the inner tube 20, a liquid stop pin 60 is attached to the inner tube recess 24. The inner tube recess 24 is formed by two first recess surfaces 24a along the central axis CX direction and a second recess surface 24b along the circumferential direction of the inner tube side wall 23. That is, the inner tube recess 24 is recessed from the end face 21a, and includes a second recess surface 24b that is the bottom of the recess, and two first recess surfaces 24a that rise from the second recess surface 24b toward the end face 21a. The inner tube recess 24 has a tapered shape in which the opening area increases from the radially inward to the radially outward direction of the inner tube 20. In this embodiment, as shown in FIG. 3, the second recess surface 24b has a tapered shape in the radial direction of the inner tube 20 by inclining away from the inner tube one end 21 from the inner peripheral surface to the outer peripheral surface of the inner tube 20. This makes it easier to attach the liquid stop pin 60.

2, the culture membrane part 10 is placed on one end 21 of the inner tube. The culture membrane part 10 covers the inner tube opening 25 formed on one end 21 of the inner tube. Specifically, as shown in FIG. 3, the culture membrane part 10 is placed so as to cover the end face 21a of the inner tube one end 21 and contact the end face 21a with the support frame 11. The end face 21a of the inner tube one end 21 and the culture membrane part 10 are then joined. By placing the support frame 11 on the end face 21a, the inner tube 20 on which the culture membrane part 10 is placed can receive external forces through the support frame 11. This reduces the stress on the inner tube recess 24 and reduces the stress on the cell culture membrane 12. This can suppress damage or scratches to the cell culture membrane 12 and deterioration of quality due to damage to the first cells.

As shown in FIG. 1, the liquid stop pin 60 is detachably attached to the inner tube recess 24 along the radial direction and closes the inner tube recess 24. As a result, in the first culture process P20, the inner tube 20 can be used as a container with the inner tube 20 as the side surface and the culture membrane part 10 as the bottom surface. The liquid stop pin 60 is formed of an elastic material. Examples of elastic materials include silicone rubber, fluororubber, urethane rubber, styrene-based elastomer, olefin-based elastomer, urethane-based elastomer, ester-based elastomer, and amide-based elastomer. The liquid stop pin 60 has a pin body 61 and a pin gripping part 62. The size of the pin body 61 is larger than the size of the inner tube recess 24. The pin body 61 is reduced by elastic deformation and fitted into the inner tube recess 24 to be attached. As a result, when the inner tube 20 is used as a container, liquid leakage from the inner tube recess 24 is suppressed.

3, when the liquid stop pin 60 is attached to the inner tube 20, the pin gripping portion 62 is disposed outside the inner tube side wall 23. As shown in FIG. 1, when viewed from the radial direction of the inner tube 20, the outer shape of the pin gripping portion 62 is larger than the outer shape of the pin body 61. Specifically, when viewed from the radial direction of the inner tube 20, the widthwise sides of the pin gripping portion 62 (the faces on the +Y side and the -Y side) and the heightwise lower face of the pin gripping portion 62 (the faces on the -Z side) are located outwardly of the widthwise sides of the pin body 61 (the faces on the +Y side and the -Y side) and the heightwise lower face of the pin body 61 (the faces on the -Z side). This allows an instrument such as tweezers to be inserted between the outer circumferential surface of the inner tube side wall 23 and the pin gripping portion 62, making it easier to remove the liquid stop pin 60. When viewed from the radial direction of the inner tube 20, the upper surface (+Z surface) of the pin gripping portion 62 in the height direction is flush with the upper surface (+Z surface) of the pin body 61 in the height direction. This makes it possible to suppress interference between the liquid stop pin 60 and the outer tube 40 when the outer tube 40 is attached to the inner tube 20 to which the liquid stop pin 60 is attached. Furthermore, as shown in FIG. 1 and FIG. 3, a pin recess 62a recessed in the circumferential direction is formed on the end surface of the pin gripping portion 62 along the central axis CX of the inner tube 20. When removing the liquid stop pin 60, the tip of a tool such as tweezers can be placed in the pin recess 62a to make it easier to grip the liquid stop pin 60 with the tool. This makes it easier to remove the liquid stop pin 60 from the inner tube 20.

As shown in FIG. 1, the outer tube 40 is cylindrical. Specifically, the outer tube 40 has a cylindrical shape with a central axis CX. The outer tube 40 is formed of the same resin material as the inner tube 20. The inner diameter of the outer tube 40 is larger than the outer diameter of the inner tube 20. The outer tube 40 has an outer tube one end 41, an outer tube other end 42, an outer tube side wall 43, an outer tube recess 44, and a protrusion 45. The outer tube one end 41 is one end of the outer tube 40 in the central axis CX direction. The outer tube other end 42 faces the outer tube one end 41 in the central axis CX direction. The outer tube side wall 43 connects the outer tube one end 41 and the outer tube other end 42. The outer tube recess 44 is formed as a notch in the outer tube other end 42 from the end face of the outer tube other end 42 along the central axis CX direction. In step P40 of the culture process described below, the outer cylinder 40 is attached to the inner cylinder 20 in which the culture membrane section 10 is placed, from the inner cylinder one end 21 side so that the outer cylinder other end 42 covers the inner cylinder one end 21.

2, the outer tube recess 44 has a shape that overlaps with the inner tube recess 24 when viewed from the radial direction. The convex portion 45 is formed to protrude radially from the inner peripheral surface 43a of the outer tube side wall 43. When the outer tube 40 is attached to the inner tube 20 in which the culture membrane portion 10 is arranged, the convex portion 45 abuts against the culture membrane portion 10 arranged in the inner tube 20 in the central axis CX direction. The shape of the convex portion 45 when viewed from the central axis CX direction is annular. The convex portion 45 is formed at a position away from the end face of the outer tube other end 42 in the central axis CX direction. In the central axis CX direction, the outer tube recess 44 is formed between the convex portion 45 and the end face of the outer tube other end 42. In the second culture step P60 described later, when the outer tube 40 is attached to the inner tube 20 in which the culture membrane portion 10 is arranged, the culture membrane portion 10 blocks the outer tube opening 46 of the outer tube other end 42. The outer cylinder 40 functions as a container with the outer cylinder side wall 43 of the outer cylinder 40 as a side surface and the culture membrane part 10 as a bottom surface. As shown in FIG. 3, the convex part 45 has a smooth surface 45a that abuts the inner cylinder 20. As a result, when the outer cylinder 40 is used as a container in the second culture step P60, as described in detail later, leakage of the liquid contained in the outer cylinder 40 can be suppressed. The inner diameter of the other end part 42 of the outer cylinder is formed so as to increase from the boundary with the smooth surface 45a toward the side where the inner cylinder 20 is located (-Z direction side). As a result, a gap is formed between the outer peripheral surfaces of the culture membrane part 10 and the inner cylinder 20 and the inner peripheral surface 43a of the other end part 42 of the outer cylinder, making it easier to attach the outer cylinder 40 to the inner cylinder 20 in which the culture membrane part 10 is arranged.

A2. Cell culture method using cell culture vessel:
A cell culture method using the cell culture vessel 100 will be described. FIG. 4 is a flow chart showing steps of the cell culture method using the cell culture vessel 100. FIG. 5 is a schematic diagram of the inner cylinder 20 in a first culture step P20. FIG. 6 is a schematic diagram of the cell culture vessel 100 in a second culture step P60. FIG. 5 and FIG. 6 show the X-axis, Y-axis, and Z-axis in a usage mode of the cell culture vessel 100. The Z direction indicates the vertical direction, and the XY plane indicates the horizontal plane. The +Z direction is also called the up direction, and the -Z direction is also called the down direction.

First, in step P10 of FIG. 4, the inner tube 20 to which the culture membrane part 10 is joined and the liquid stop pin 60 is attached is prepared. In the following description, unless otherwise specified, the inner tube 20 refers to the inner tube 20 to which the culture membrane part 10 is joined. In the first culture step P20 of FIG. 4, as shown in FIG. 5, the first cell suspension 401 is injected into the inner tube 20 placed on the plate 500 so that the inner tube one end 21 is below the inner tube other end 22, and the first cells 402 are cultured. The first cell suspension 401 contains the first cells 402 and the first culture medium 403, which is a liquid for the first cells 402. By injecting the first cell suspension 401, the first cells 402 are seeded on the cell culture membrane 12. The surface of the plate 500 facing the inner tube 20 is smooth. This makes it difficult for gaps to form between the culture membrane part 10 and the plate 500, and can suppress leakage of the first cell suspension 401 from the inner tube 20. Furthermore, the plate 500 is formed of a material with low cell adhesion. This can suppress the first cells 402 from settling on the surface of the cell culture membrane 12 facing the plate 500, even if the first cell suspension 401 leaks from the inner tube 20.

4, the first cell suspension 401 is aspirated from the inner tube 20 using, for example, a pipette, and the inner tube 20 is inverted so that one end 21 of the inner tube is above the other end 22 of the inner tube. In step P40, the outer tube 40 is attached to the inner tube 20, and the liquid stop pin 60 is removed from the inner tube 20. Specifically, the outer tube 40 is fitted into the inner tube 20 from the inner tube one end 21 side of the inner tube 20 to which the liquid stop pin 60 is attached, until the convex portion 45 of the outer tube 40 abuts the culture membrane portion 10. Furthermore, the outer tube 40 is fitted into the inner tube 20 so that the position of the outer tube recess 44 overlaps the position of the liquid stop pin 60 when viewed from the direction of the central axis CX. At this time, the pin gripping portion 62 of the liquid stop pin 60 is located outside the inner tube side wall 23, so the outer tube 40 is attached to the inner tube 20 so that the outer tube recess 44 fits into the protruding pin gripping portion 62. This allows the outer tube 40 to be attached to the inner tube 20 so that the inner tube recess 24 and the outer tube recess 44 overlap when viewed from the radial direction of the inner tube 20. By removing the liquid stop pin 60 from the inner tube 20, the inside and outside of the inner tube 20 are connected via the inner tube recess 24.

In step P50, the inner cylinder 20 with the outer cylinder 40 attached is placed in the well 501 containing the first culture medium 403 for the first cells 402. As a result, the surface of the cell culture membrane 12 on the inner cylinder 20 side on which the first cells 402 are fixed is immersed in the first culture medium 403. In the second culture step P60, as shown in FIG. 6, the second cell suspension 404 is placed in the outer cylinder 40 and the second cells 405 are cultured. Specifically, the second cells 405 are seeded by placing the second cell suspension 404, and then the second cells 405 are cultured on the cell culture membrane 12 over time. The second cell suspension 404 contains the second cells 405 and a second culture medium, which is a liquid for the second cells 405. The second cell suspension 404 is injected, and the second cells 405 are seeded on the cell culture membrane 12. Since the inside and outside of the inner tube 20 are in communication with each other through the inner tube recess 24 and the outer tube recess 44, the residual air in the first culture medium 403 can be discharged to the outside of the inner tube 20 through the inner tube recess 24 and the outer tube recess 44. This allows the first cells 402 to be cultured well. Since the convex portion 45 has a smooth surface 45a, the formation of a gap between the convex portion 45 and the culture membrane portion 10 is reduced, and leakage of the second cell suspension 404 placed in the outer tube 40 can be suppressed. Thus, the second cells 405 can be prevented from being mixed into the first culture medium 403. During the culture of the second cells 405, the second culture medium may be replaced as appropriate. When the second culture medium is replaced, the second cell suspension 404 is sucked out of the outer tube 40 using an instrument such as a pipette. At this time, by positioning the tip of the instrument on the convex portion 45, damage to the cell culture membrane 12 caused by the tip of the instrument hitting the cell culture membrane 12 can be suppressed. When the culture of the second cells 405 is completed, this process ends.

According to the first embodiment described above, the cell culture vessel 100 includes the inner tube 20, the liquid stop pin 60 that closes the inner tube recess 24, the culture membrane part 10 arranged at the inner tube one end 21 of the inner tube side wall 23, and the outer tube 40 that is detachably attached to the inner tube 20 from the inner tube one end 21 side. Therefore, in the first culture step P20, the inner tube 20 can be used as a container for storing the first culture medium 403 for the first cell, so there is no need to put the cell culture vessel 100 into the well or remove the cell culture vessel 100 from the well, and workability can be improved. In addition, in the second culture step P60, the outer tube 40 can be used as a container for storing the second culture medium for the second cell. Therefore, co-culture can be performed using the cell culture vessel 100. In addition, since the inner tube 20 includes the inner tube recess 24, the residual air in the first culture medium 403 can be discharged from the inner tube recess 24 in the second culture step P60.

The outer cylinder 40 has an outer cylinder recess 44 at a position overlapping with the inner cylinder recess 24 in the radial direction of the inner cylinder 20. This makes it easier to discharge the air remaining in the first culture medium 403 when the inner cylinder 20 is immersed in the first culture medium 403 in the second culture step P60. This allows culture to be performed well. The convex portion 45 has a smooth surface 45a that abuts the culture membrane portion 10. This makes it possible to suppress leakage of the second cell suspension 404 placed in the outer cylinder 40 from the gap between the convex portion 45 and the culture membrane portion 10 in the second culture step P60. The culture membrane portion 10 also has a circular support frame 11 that is arranged to cover the end surface 21a of the inner cylinder one end portion 21, and a cell culture membrane 12. This allows the support frame 11 to bear external forces, thereby reducing the stress on the inner cylinder recess 24 and reducing the stress on the cell culture membrane 12. This makes it possible to prevent damage or scratches to the cell culture membrane 12 and deterioration of the quality of the first cells due to damage.

B. Second embodiment:
B1. Jig configuration:
In the second embodiment, cell culture is performed using a plurality of cell culture vessels 100. FIG. 7 is a perspective view of a liquid stopper jig 510 and a fixing jig 520 shown together with the inner cylinder 20. FIG. 8 is a perspective view of an inversion jig 530 shown together with the inner cylinder 20. FIG. 9 is a plan view of the inversion jig 530 shown together with the inner cylinder 20. FIG. 10 is a cross-sectional view taken along the X-X line in FIG. 9. In the cell culture vessel 100 according to the second embodiment, the liquid stopper jig 510 is used instead of the liquid stopper pin 60 according to the first embodiment. In FIGS. 7 to 10, an X-axis, a Y-axis, and a Z-axis are shown, which are orthogonal to each other. The X-direction and the Y-direction are the arrangement directions of the inner cylinder 20 in the fixing jig 520. The Z-direction is the direction of the central axis CX of the inner cylinder 20. The cell culture vessel 100 is typically used in such a manner that the Z-direction is aligned with the vertical direction. In the following description, the +Z direction may be the upward direction, and the -Z direction may be the downward direction. Since the configurations of the culture membrane part 10, the inner cylinder 20, and the outer cylinder 40 are the same as those in the first embodiment, the same reference numerals are used and detailed description is omitted.

In this embodiment, a case will be described in which a total of 24 cell culture vessels 100 are arranged in a matrix of 4 rows and 6 columns as shown in FIG. 7 for cell culture. Note that the number of cell culture vessels 100 in which cells are cultured simultaneously is not limited to 24.

The fixing jig 520 is a jig for fixing the multiple inner cylinders 20 placed thereon and culturing the first cells 402. The fixing jig 520 can simultaneously fix multiple inner cylinders 20 of multiple cell culture vessels 100. The fixing jig 520 is formed of a resin material or a metal material. Examples of the resin material include polypropylene, polystyrene, acrylic resin, etc. Examples of the metal material include stainless steel and titanium. Furthermore, the fixing jig 520 is preferably a metal used in medical equipment. Examples of the metal used in medical equipment include titanium, Ti-6Al-4V alloy, stainless steel, Co-Cr alloy, etc. The fixing jig 520 has a bottom surface portion 521 and 24 fixing protrusions 522. In this embodiment, the bottom surface portion 521 has a box shape having a rectangular plate and four side surfaces erected upward from the four sides of the rectangular plate. As described later, when culturing the first cell 402, a lid (not shown) is placed on the fixing jig 520 to suppress contamination during the culture. The plate of the bottom surface portion 521 has a smooth surface and low cell adhesion, similar to the plate 500 according to the first embodiment. Methods for reducing cell adhesion include, for example, forming a plate using a material with low cell adhesion, such as polystyrene, and coating the surface of the resin forming the plate with a material with low cell adhesion, such as polyethylene glycol or agarose gel. The fixing convex portion 522 has a C-shaped shape that is missing a part of the circumferential direction of the ring. The fixing convex portions 522 are arranged at equal intervals along the Y direction. Similarly, the fixing convex portions 522 are arranged at equal intervals along the X direction. The inner diameter of the fixing convex portion 522 is larger than the outer diameter of the inner cylinder 20. In addition, the opening portion of the fixing convex portion 522 is larger than the pin body 511 of the liquid stopping jig 510, which will be described later. This allows the pin body 511 to be placed in the opening of the fixing convex portion 522, and multiple inner tubes 20 with the liquid stopping jig 510 attached to them to be placed on the fixing jig 520. Alternatively, the liquid stopping jig 510 can be attached to the inner tube 20 that has been placed with the inner tube recess 24 placed in the opening of the fixing convex portion 522.

The liquid stopper 510 is detachably attached to the inner tube recesses 24 of the cell culture vessels 100, and blocks the inner tube recesses 24. The liquid stopper 510 has six pin bodies 511 and a connecting plate 512. The pin body 511 has a shape similar to that of the pin body 61 according to the first embodiment. That is, the size of the pin body 511 is larger than the size of the inner tube recess 24, and the pin body 511 is elastically deformed and reduced in size to be fitted into the inner tube recess 24 and attached. The six pin bodies 511 are provided corresponding to each of the inner tube recesses 24 of the six inner tubes 20 to be attached. Specifically, the intervals between the six pin bodies 511 are the same as the intervals between the fixing protrusions 522. The connecting plate 512 has a flat shape that is long in the Y direction, which is the arrangement direction of the inner tubes 20, and connects the six pin bodies 511 on the surface on the +X direction side facing the inner tube 20.

The inversion jig 530 shown in FIG. 8 is formed of an elastic resin material or metal material. Examples of the resin material include polyetherketone, polypropylene, polystyrene, acrylic resin, etc. Examples of the metal material include stainless steel and titanium. The metal material is preferably a metal used in medical equipment. Examples of the metal used in medical equipment include titanium, Ti-6Al-4V alloy, stainless steel, Co-Cr alloy, etc. The inversion jig 530 is a jig that can hold multiple inner tubes 20 of multiple cell culture vessels 100 and invert multiple inner tubes 20 simultaneously. The inversion jig 530 has a first flat plate 531, a second flat plate 532, multiple first semicircular portions 533, multiple second semicircular portions 534, a first bottom portion 535, a second bottom portion 536, and a locking portion 537. The first flat plate 531 is a flat plate that is long in the Y direction, which is the arrangement direction of the inner tubes 20. The first flat plate 531 is disposed so that the width direction is along the central axis CX direction of the inner tube 20 and the thickness direction is along the radial direction of the inner tube 20. A first flat plate recess 531a is formed at the end of the first flat plate 531 in the +Y direction. The first flat plate recess 531a is formed on the end face of the first flat plate 531 on the +Z direction side, recessed along the Z direction. The second flat plate 532 is a flat plate that is elongated in the Y direction similar to the first flat plate 531. The second flat plate 532 is disposed so that the width direction is along the central axis CX direction of the inner tube 20 and the thickness direction is along the radial direction of the inner tube 20. The second flat plate 532 faces the first flat plate 531 in the X direction, which is the radial direction of the inner tube 20, with the first semicircular portion 533 and the second semicircular portion 534 sandwiched therebetween. As shown in FIG. 9, the first semicircular portion 533 and the second semicircular portion 534 have a semicircular ring shape formed by cutting a cylindrical shape surrounding the outer periphery of the inner cylinder 20 along the diameter direction of the inner cylinder 20. The ends of the first semicircular portions 533 on the -X direction side that contact the first flat plate 531 are joined to the first flat plate 531. The ends of the second semicircular portions 534 on the +X direction side that contact the second flat plate 532 are joined to the second flat plate 532.

The first bottom surface portion 535 and the second bottom surface portion 536 have a flat plate shape and connect the first flat plate 531 and the second flat plate 532. The first bottom surface portion 535 is disposed at the -Y direction end of the first flat plate 531 and the second flat plate 532. The second bottom surface portion 536 is disposed at the +Y direction end of the first flat plate 531 and the second flat plate 532. The first flat plate 531 is fixed to the first bottom surface portion 535 and the second bottom surface portion 536. In contrast, the second flat plate 532 is attached so as to be movable in the Y direction relative to the first bottom surface portion 535 and the second bottom surface portion 536. Specifically, as shown in FIG. 10, a first bottom surface groove 535a recessed along the width direction of the second flat plate 532 is formed on the surface of the first bottom surface portion 535 on which the second flat plate 532 is disposed. The end of the second flat plate 532 in the −Z direction, which is the width direction, is formed in a convex shape that fits into the first bottom groove 535a. As shown in FIG. 9, the first bottom groove 535a is formed along the Y direction, which is the long side direction of the second flat plate 532. A second bottom groove 536a similar to the first bottom groove 535a is also formed on the surface of the second bottom surface portion 536 on which the second flat plate 532 is disposed. As a result, the second flat plate 532 can be guided by the first bottom groove 535a and the second bottom groove 536a and moved in the Y direction relative to the first bottom surface portion 535 and the second bottom surface portion 536. Here, as shown in FIG. 9, the position of the second flat plate 532 relative to the first flat plate 531 in a state in which each of the first semicircular portion 533 and the second semicircular portion 534 is disposed so as to contact the inner cylinder 20 is referred to as a fixed position. In contrast, as shown by the solid line in FIG. 8, when the second semicircular portion 534 is positioned so as to be spaced apart from the inner cylinder 20, the position of the second flat plate 532 relative to the first flat plate 531 is referred to as the spaced position.

9, the locking portion 537 is attached to the end of the second flat plate 532 in the +Y direction. The locking portion 537 is used to fix the movable second flat plate 532 to the first flat plate 531. The locking portion 537 has a locking flat plate 537a and a locking protrusion 537b. The locking flat plate 537a is a rectangular flat plate that is long in the X direction. The end of the locking flat plate 537a in the +X direction is supported by the end of the second flat plate 532 in the +Z direction. This allows the locking portion 537 to rotate around the end of the locking flat plate 537a in the +X direction. The locking protrusion 537b is protruded along the long side direction from the end of the locking flat plate 537a closer to the first flat plate 531. The locking protrusion 537b has a shape that fits into the first flat plate recess 531a.

When multiple inner tubes 20 are inverted, first, as shown by the solid line in FIG. 8, the second flat plate 532 is adjusted to a separated position so that the second semicircular portion 534 is separated from the inner tube 20 when placed. Next, the inversion jig 530 is placed with respect to the multiple inner tubes 20 so that the first semicircular portion 533 and the second semicircular portion 534 face each other across the inner tube 20. At this time, the inversion jig 530 is placed so that the first semicircular portion 533 and the second semicircular portion 534 are located in the portion between the liquid stopper jig 510 and the other end 22 of the inner tube 20 in the Z direction. Next, as shown by the dashed line in FIG. 8, the second flat plate 532 is moved relative to the first flat plate 531 so that the first semicircular portion 533 and the second semicircular portion 534 are in a fixed position in contact with the inner tube 20. As a result, as shown in FIG. 9, the inner tube 20 is sandwiched and gripped between the first semicircular portion 533 and the second semicircular portion 534. Next, the locking protrusion 537b of the locking portion 537 is fitted into the first flat plate recess 531a of the first flat plate 531. This restricts the movement of the second flat plate 532 relative to the first flat plate 531. Next, the inversion jig 530 gripping the multiple inner tubes 20 is inverted up and down so that the +Z direction end of the inversion jig 530 becomes the -Z direction end. As a result, the multiple inner tubes 20 are inverted up and down at the same time.

B2. Cell culture method using multiple cell culture vessels:
In the second embodiment, cell culture is performed through the same steps as in the first embodiment. Therefore, the cell culture method using a plurality of cell culture vessels 100 according to this embodiment will be described with reference to Fig. 4, and detailed descriptions of each step will be omitted as appropriate.

In step P10 of FIG. 4, multiple inner tubes 20 are prepared with a liquid stopping jig 510 attached instead of the liquid stopping pins 60. The multiple inner tubes 20 are arranged and fixed in the fixing convex portions 522 of the fixing jig 520 shown in FIG. 7. By arranging the multiple inner tubes 20 in the fixing jig 520, the multiple inner tubes 20 can be fixed at predetermined intervals. This allows the inner tube recesses 24 of the multiple inner tubes 20 to be simultaneously blocked using the liquid stopping jig 510, in which the pin bodies 511 are arranged at predetermined intervals.

In the first culture step P20 in FIG. 4, the first cell suspension 401 is injected into the inner cylinder 20, and the first cells 402 are cultured, as in the first embodiment. After the first cell suspension 401 is injected, the upper part of the fixing jig 520 is covered with a lid (not shown). This prevents contamination during culture.

In step P30 of FIG. 4, the first cell suspension 401 is sucked out of the inner cylinder 20, and the inner cylinder 20 is inverted. When inverting, an inversion tool 530 shown in FIG. 8 is used. By using the inversion tool 530, the inversion operation can be performed more efficiently than by inverting each inner cylinder 20 individually.

In step P40 of FIG. 4, the outer tube 40 is attached to the inner tube 20, and the liquid stopping jig 510 is removed from the inner tube 20. In step P30, the inner tube 20 is inverted, and then multiple outer tubes 40 are attached to the multiple inner tubes 20. This allows the inner tubes 20 and the outer tubes 40 to be integrated. Specifically, multiple outer tubes 40 held by a jig not shown are attached to the multiple inner tubes 20 while they are held by the inversion jig 530. After the outer tubes 40 are attached, the second flat plate 532 of the inversion jig 530 is moved to a separated position, and the inversion jig 530 is removed from the inner tube 20. After the inversion jig 530 is removed from the inner tube 20, the liquid stopping jig 510 is removed from the multiple inner tubes 20.

In step P50 of FIG. 4, the inner cylinder 20 to which the outer cylinder 40 is attached is placed in the well 501 (FIG. 6) containing the first culture medium 403 for the first cell 402. The outer cylinder 40 and the inner cylinder 20 integrated in step P40 are placed in the well 501 while being held by the jig, by the outer cylinder 40 being held by the jig. In the second embodiment, a well plate (not shown) is used in which a plurality of wells 501 are arranged in a manner similar to the arrangement of the fixing convex portion 522 in the fixing jig 520. That is, the well plate has the wells 501 arranged at the same intervals as the intervals of the fixing convex portion 522 in the fixing jig 520. Therefore, a plurality of integrated outer cylinders 40 and inner cylinders 20 can be simultaneously placed in the well 501 while being held by the jig. This allows the integrated outer cylinders 40 and inner cylinders 20 to be placed in the well 501 more efficiently than performing the work of placing the integrated outer cylinders 40 and inner cylinders 20 in the well 501 individually. The jig for holding multiple outer tubes 40 is a jig that can hold multiple outer tubes 40 by holding one end 41 of the outer tube 40. Note that a jig similar to the inversion jig 530 may be used as a jig for holding multiple outer tubes 40.

In the second culture step P60 in FIG. 4, the second cell suspension 404 is placed in the outer cylinder 40, and the second cells 405 are cultured. When the culture of the second cells 405 is completed, this step ends.

According to the second embodiment described above, co-culture is performed using a fixing jig 520 capable of simultaneously fixing multiple inner cylinders 20, an inversion jig 530 capable of simultaneously inverting multiple inner cylinders 20, and a liquid stopping jig 510 that is detachably attached to multiple inner cylinder recesses 24. Therefore, cell culture can be performed more efficiently than if work was performed individually on each cell culture vessel 100. In addition, by simplifying the work process and shortening the work time, contamination in the culture process can be suppressed.

C. Other embodiments:
(C1) In the above first embodiment, the inner cylinder 20 is placed on the plate 500 in the first culture step P20, and the cell culture vessel 100 is placed in the well 501 in the second culture step P60. In contrast, the inner cylinder 20 may be placed in the well 501 in the first culture step P20. The outer cylinder 40 functions as a container for holding the second cell suspension 404, and the second cell suspension 404 is not placed in the well 501, so the liquid placed in the well 501 is either the first cell suspension 401 or the first culture medium 403. Therefore, it is also possible to perform the first culture step P20 and the second culture step P60 using the same well 501.

(C2) In the above embodiment, the inner cylinder recess 24 is formed as a notch in the inner cylinder one end 21 along the central axis CX direction from the end face 21a of the inner cylinder one end 21. In contrast, the inner cylinder recess 24 may be formed as a slit that penetrates in the central axis CX direction from the end face 21a of the inner cylinder one end 21 to the end face of the inner cylinder other end 22.

(C3) The first semicircular portion 533 and the second semicircular portion 534 of the inversion jig 530 according to the second embodiment have a semicircular ring shape. The shapes of the first semicircular portion 533 and the second semicircular portion 534 are not limited to semicircular ring shapes, and may be, for example, a shape of only a portion of a semicircular ring shape. If the first semicircular portion 533 and the second semicircular portion 534 have a shape capable of sandwiching the inner tube 20, the inner tube 20 can be gripped.

The present disclosure is not limited to the above-described embodiments, 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 form described in the Summary of the Invention column can be replaced or combined as appropriate to solve some or all of the above-described problems or to achieve some or all of the above-described effects. Furthermore, if a technical feature is not described as essential in this specification, it can be deleted as appropriate.

10...culture membrane portion, 11...support frame, 12...cell culture membrane, 20...inner tube, 21...one end of inner tube, 21a...end surface, 22...other end of inner tube, 23...side wall of inner tube, 24...inner tube recess, 24a...first recess surface, 24b...second recess surface, 25...inner tube opening, 40...outer tube, 41...one end of outer tube, 42...other end of outer tube, 43...side wall of outer tube, 44...outer tube recess, 45...convex portion, 45a...smooth surface, 46...outer tube opening, 60...liquid stop pin, 61, 511...pin body, 62...pin gripping portion, 62a...pin recess, 100...cell culture vessel, 401...first cell suspension, 402...first cell, 403...first culture medium, 404...second cell Suspension, 405...second cell, 500...plate, 501...well, 510...liquid stopper, 512...connecting plate, 520...fixing jig, 521...bottom, 522...fixing convex, 530...inversion jig, 531...first plate, 531a...first plate concave, 532...second plate, 533...first semicircular, 534...second semicircular, 535...first bottom, 535a...first bottom groove, 536...second bottom, 536a...second bottom groove, 537b...locking convex, 537a...locking plate, 537...locking, CX...center axis, P10, P30, P40, P50...process, P20...first culture process, P60...second culture process

Claims (6)

1. A cell culture vessel comprising:
an inner cylinder having an inner cylinder one end, an inner cylinder other end opposite to the inner cylinder one end, a cylindrical inner cylinder side wall connecting the inner cylinder one end and the inner cylinder other end, and an inner cylinder recess formed as a notch in the inner cylinder one end;
a liquid stopper pin that is detachably attached to the inner cylinder recess and closes the inner cylinder recess;
A culture membrane part is disposed at one end of the inner cylinder and closes an inner cylinder opening part formed at the one end of the inner cylinder;
an outer cylinder that is detachably attached to the inner cylinder from one end side of the inner cylinder, the outer cylinder having one end of the outer cylinder, an other end of the outer cylinder facing the one end of the outer cylinder, and a cylindrical outer cylinder side wall connecting the one end of the outer cylinder and the other end of the outer cylinder;
The culture membrane portion closes an outer cylinder opening at the other end of the outer cylinder when the outer cylinder is attached to the inner cylinder. The cell culture vessel according to claim 1 ,
The outer cylinder further comprises:
The cell culture vessel has an outer cylinder recess formed as a notch in a portion of a side wall of the outer cylinder at a position that overlaps with the inner cylinder recess in the radial direction of the inner cylinder in the attached state. The cell culture vessel according to claim 1 or 2,
The outer cylinder further comprises:
A cell culture vessel having a convex portion protruding from the inner peripheral surface of the side wall of the outer tube and abutting against the culture membrane portion in a direction along the central axis of the inner tube in the attached state. The cell culture vessel according to claim 3,
The convex portion has a smooth surface that contacts the culture membrane portion. The cell culture vessel according to any one of claims 1 to 4,
The culture membrane part further includes
A cell culture vessel comprising: a circular support frame arranged to cover an end face of one end of the inner tube on a side wall of the inner tube; and a cell culture membrane joined to the support frame. A cell culture method using a plurality of cell culture vessels, comprising:
Each of the plurality of cell culture vessels includes:
an inner cylinder having an inner cylinder one end, an inner cylinder other end opposite to the inner cylinder one end, a cylindrical inner cylinder side wall connecting the inner cylinder one end and the inner cylinder other end, and an inner cylinder recess formed as a notch in the inner cylinder one end;
A culture membrane part is disposed at one end of the inner cylinder and closes an inner cylinder opening part formed at the one end of the inner cylinder;
an outer cylinder that is detachably attached to the inner cylinder from one end side of the inner cylinder, the outer cylinder having one end of the outer cylinder, an other end of the outer cylinder facing the one end of the outer cylinder, and a cylindrical outer cylinder side wall connecting the one end of the outer cylinder and the other end of the outer cylinder;
The culture membrane unit closes an outer cylinder opening at the other end of the outer cylinder when the outer cylinder is attached to the inner cylinder,
The culture membrane portion is joined to each of the plurality of inner cylinders,
The cell culture method comprises:
a fixing jig capable of simultaneously fixing the plurality of inner cylinders of the plurality of cell culture vessels;
An inversion jig capable of holding the plurality of inner cylinders and inverting the plurality of inner cylinders simultaneously;
A method for co-culturing using a liquid stopping device that is detachably attached to a plurality of the inner cylinder concaves of the plurality of cell culture vessels and closes the plurality of inner cylinder concaves,
preparing the plurality of inner cylinders to which the liquid stopping jig is attached;
injecting a first cell suspension containing first cells into the plurality of inner cylinders fixed on the fixing jig such that one end of the inner cylinder is below the other end of the inner cylinder, and culturing the first cells on the culture membrane portion;
sucking the first cell suspension from the plurality of inner cylinders, and simultaneously inverting the plurality of inner cylinders using the inversion tool so that one end of the inner cylinder is above the other end of the inner cylinder;
attaching the outer cylinder to each of the plurality of inner cylinders and removing the liquid stopping jig from the plurality of inner cylinders;
a step of arranging a plurality of inner cylinders in a well plate in which a plurality of wells containing a first culture medium for the first cells are arranged, such that each of the plurality of inner cylinders, each of which has the outer cylinder attached thereto, is inserted into each of the plurality of wells;
A cell culture method comprising a step of placing a second cell suspension containing second cells different from the first cells into each of the multiple outer cylinders, and culturing the second cells on the culture membrane portion .

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