JP2020162458A - Cell culture tool and method for using the same - Google Patents

Abstract

To culture cells on a culture membrane by a small amount of culture solution.SOLUTION: A cell culture tool comprises a holder having: a body cylindrical part cylindrically extending in an axial direction; and a holding part which is provided at a middle part in the axial direction of the body cylindrical part, and holds a culture membrane. The tool comprises a first cylindrical member which is installed detachably on one side in the axial direction with respect to the holder, cylindrically extends in the axial direction, and protrudes closer to one side in the axial direction than one end in the axial direction of to holder when installed to the holder. The tool comprises a second cylindrical member which is installed detachably on the other side in the axial direction with respect to the holder, cylindrically extends in the axial direction, and protrudes closer to the other side in the axial direction than the other end in the axial direction of to holder when installed to the holder.SELECTED DRAWING: Figure 1

Description

The present invention relates to a cell culture device used for culturing cells and a method of using the same.

Conventionally, a cell culture device for culturing cells on both sides of a culture membrane has been known (for example, Patent Document 1). The cell culture device described in Patent Document 1 includes a culture membrane, an outer cylinder, and an inner cylinder. The outer cylinder has a main body cylinder portion extending in a tubular shape in the axial direction. The main body cylinder portion of the outer cylinder is provided with a slit extending in the axial direction. The outer cylinder can be elastically deformed in the radial direction due to the presence of slits. The outer cylinder has a holding portion for holding the culture membrane, which is provided in the axially intermediate portion of the main body cylinder portion.

Two inner cylinders are provided, and each of them extends in a tubular shape in the axial direction. The first inner cylinder has a facing portion facing the holding portion of the outer cylinder. The first inner cylinder is inserted into the outer cylinder from one side in the axial direction of the outer cylinder and arranged adjacent to the outer cylinder. Further, the second inner cylinder is inserted into the outer cylinder from the other side in the axial direction of the outer cylinder and arranged adjacent to the outer cylinder. When each inner cylinder is inserted into the outer cylinder and mounted, the outer cylinder elastically deforms outward in the radial direction, so that the inner cylinder and the outer cylinder come into contact with each other without a gap in the radial direction, and the first inner cylinder The culture membrane is sandwiched and held by the facing portion of the outer cylinder and the holding portion of the outer cylinder facing each other in the axial direction.

Japanese Unexamined Patent Publication No. 2017-29092

By the way, in the above-mentioned cell culture instrument, each of the two inner cylinders is formed so that the axial tip thereof does not protrude axially outward from the axial end surface of the outer cylinder when the two inner cylinders are attached to the outer cylinder. The outer cylinder is formed in a state where the total length in the axial direction is relatively long so that the two inner cylinders can be mounted at the same time, and the dimensional distance from the lower end in the axial direction of the outer cylinder in the cell culture device to the culture membrane is large. long. Therefore, in order to culture cells in the culture membrane, it is necessary to increase the capacity of the well (culture container), and it is necessary to prepare a large amount of culture solution.

The present invention has been made in view of these points, and an object of the present invention is to provide a cell culture device capable of culturing cells on a culture membrane with a small amount of a culture solution and a method for using the same.

The cell culture device according to one aspect of the present invention is a holder having a main body tubular portion extending in a tubular shape in the axial direction and a holding portion provided in the axial intermediate portion of the main body tubular portion to hold the culture membrane. And, it is detachably attached to the holder on one side in the axial direction, extends in a tubular shape in the axial direction, and protrudes on one side in the axial direction from one end in the axial direction when attached to the holder. The first tubular member and the holder are detachably attached to the other side in the axial direction, extend in a tubular shape in the axial direction, and are more axial than the other end in the axial direction when attached to the holder. A cell culture device comprising a second tubular member projecting to the other side in the direction.

According to this configuration, in order to seed two types of cells on both sides of the culture membrane, it is not necessary to use a cell culture device by simultaneously attaching both the first cylinder member and the second cylinder member to the holder. Any of the first cylinder member and the second cylinder member located on the lower side can be used in a state of being removed from the holder. Further, the first cylinder member protrudes to one side in the axial direction from one end in the axial direction of the holder in a state of being engaged and fixed to the holder, and the second cylinder member is engaged and fixed to the holder. It projects to the other side in the axial direction from the other end in the axial direction of the holder. Therefore, according to the cell culture apparatus, when using for cell culture, the dimensional distance from the lower end in the axial direction to the holding portion where the culture membrane is held can be shortened, so that cells can be applied to the culture membrane with a small amount of culture solution. Can be cultivated.

It is an exploded perspective view of the cell culture apparatus which concerns on 1st Embodiment. It is sectional drawing of the cell culture apparatus of 1st Embodiment. It is sectional drawing for demonstrating the structure which holds a culture membrane by a holder in the cell culture apparatus of 1st Embodiment. It is sectional drawing at the time of cell seeding to one axial end face of the culture membrane in the cell culture apparatus of 1st Embodiment. It is sectional drawing at the time of cell seeding to the other axial end face of the culture membrane in the cell culture apparatus of 1st Embodiment. It is a flowchart of an example which showed the use procedure at the time of double-sided culture in the cell culture apparatus of 1st Embodiment. It is sectional drawing of the cell culture apparatus which concerns on 2nd Embodiment. It is an exploded perspective view of the retainer provided in the cell culture apparatus of 2nd Embodiment. It is sectional drawing of the cell culture apparatus which concerns on one modified form.

Hereinafter, specific embodiments of the cell culture device and the method of using the cell culture device according to the present invention will be described with reference to the drawings.

[First Embodiment]
The cell culture device 1 of the first embodiment is a device for culturing cells having a scaffold dependence. The cell culture device 1 can culture two types of cells on both sides of the membrane. As shown in FIG. 1, the cell culture instrument 1 includes a culture membrane 10, a holder 20, a first cylinder member 30, and a second cylinder member 40.

The culture membrane 10 is a sheet-like membrane in which cells are cultured. The culture membrane 10 has a membrane body 11 and a support frame 12. The membrane body 11 is a membrane body portion that serves as a scaffold for cells during cell culture. The membrane body 11 is a porous membrane having pores on both sides. The film body 11 is formed of, for example, a thermosetting resin such as polyurethane, silicone resin, phenol resin, epoxy resin, unsaturated polyester, or polyimide. The film thickness of the film body 11 is, for example, 0.1 μm-100 μm.

The support frame 12 is a base material that supports and reinforces the film body 11. The support frame 12 is an annular frame having a circular hole 12a in the center. The support frame 12 is joined and fixed to the peripheral edge of the film body 11. The shape of the support frame 12 may include at least an annular frame portion, and may further include a protrusion protruding radially outward from a part of the frame portion in the circumferential direction. Good. This protrusion has a function for facilitating handling when, for example, a person grips the culture membrane 10 with a jig such as tweezers. The support frame 12 is made of a material harder than the film body 11. The material of the support frame 12 is, for example, a thermoplastic resin such as polyethylene, polypropylene, ABS resin, acrylic resin, polyethylene terephthalate (PET), or a thermosetting elastomer such as silicone rubber. The thickness of the support frame 12 is, for example, 0.05 mm to 0.5 mm.

The holder 20 is a member for holding the culture membrane 10. The holder 20 is made of a resin material, rubber, or the like. The holder 20 is composed of two holding cylinders, and has a first holding cylinder 21 and a second holding cylinder 22. The first holding cylinder 21 and the second holding cylinder 22 are arranged adjacent to each other in the axial direction X. The first holding cylinder 21 and the second holding cylinder 22 hold the culture membrane 10 in a state of being engaged and fixed to each other. The culture membrane 10 held by the holder 20 is exposed on both sides in the axial direction through the axial openings of the first holding cylinder 21 and the second holding cylinder 22.

The first holding cylinder 21 and the second holding cylinder 22 are members that are detachably attached to each other and have a structure 23 that is engaged and fixed to each other as shown in FIG. The engaging structure 23 is a locking structure that regulates relative movement in the axial direction X and relative movement in the radial direction that are separated from each other in the assembled state. The portions where the engaging structure 23 is provided are provided, for example, in the first holding cylinder 21 and the second holding cylinder 22 at two or more locations (for example, three to five locations) in the circumferential direction. Further, in the structure in which the slits extending in the axial direction X are provided in the holding cylinders 21 and 22, it is preferable that the engaging structure 23 is provided in each of the divided portions sandwiching the slit in the circumferential direction.

In a state where the first holding cylinder 21 and the second holding cylinder 22 are engaged with each other and fixed as described above, the axial end faces of the first holding cylinder 21 and the second holding cylinder 22 facing each other are axially X. The distance away from the culture membrane 10 (specifically, the support frame 12 thereof) is set to be equal to or less than the axial thickness before assembly. As a result, the support frame 12 of the culture membrane 10 is sandwiched and fixed between the first holding cylinder 21 and the second holding cylinder 22. At this time, the space between the support frame 12 and the first holding cylinder 21 and the space between the support frame 12 and the second holding cylinder 22 are sealed.

The engagement structure 23 between the first holding cylinder 21 and the second holding cylinder 22 is recessed in the radial direction provided on the outer surface or the inner surface of one of the first holding cylinder 21 and the second holding cylinder 22, for example. It may have an L-groove engagement structure composed of a concave portion and a convex portion provided on the other inner or outer surface that protrudes in the radial direction. The recess may be, for example, a groove formed in an L shape when viewed from the radial direction.

Further, the engaging structure 23 between the first holding cylinder 21 and the second holding cylinder 22 is, for example, elastically deformable in the radial direction provided in one of the first holding cylinder 21 and the second holding cylinder 22. It may have a snap-fit type engaging structure including a deformed portion and a groove portion provided on the other side that is recessed in the radial direction. In this snap-fit type engagement structure, when the first holding cylinder 21 and the second holding cylinder 22 are brought close to each other in the axial direction at the time of assembly, they protrude in the radial direction provided at the axial tip portion of the elastically deformed portion. The first holding cylinder 21 and the second holding cylinder 22 are engaged with each other and fixed by being caught by the claw portion to be fitted into the groove portion.

The first holding cylinder 21 penetrates the hollow first cylinder portion 21a extending in a tubular shape in the axial direction X, the first holding portion 21b for holding the culture membrane 10, and the first cylinder portion 21a in the radial direction. It has a first side surface opening 21c provided. The second holding cylinder 22 penetrates the hollow second cylinder portion 22a extending in a tubular shape in the axial direction X, the second holding portion 22b for holding the culture membrane 10, and the second cylinder portion 22a in the radial direction. It has a second side surface opening 22c provided.

The first tubular portion 21a and the second tubular portion 22a each have predetermined lengths La1 and La2 in the axial direction X. The axial lengths of the first cylinder portion 21a and the second cylinder portion 22a may be substantially the same. The first tubular portion 21a and the second tubular portion 22a form a main body tubular portion extending in an axial direction X in the holder 20.

The first tubular portion 21a and the second tubular portion 22a each have a predetermined thickness in the radial direction. As shown in FIG. 3, the outer diameters of the first cylinder portion 21a and the second cylinder portion 22a are substantially the same, and the inner diameters thereof are substantially the same. The outer diameter of the culture membrane 10 (that is, the outer diameter of the support frame 12) exceeds the inner diameters of the first cylinder portion 21a and the second cylinder portion 22a. The outer diameter of the culture membrane 10 may be smaller than the outer diameter of the first tubular portion 21a and the second tubular portion 22a. The culture membrane 10 is formed by engaging the first holding cylinder 21 and the second holding cylinder 22 with each other in a state where the axial end faces of the first cylinder portion 21a and the second cylinder portion 22a facing the axial direction X face each other. Hold and hold.

The first holding portion 21b and the second holding portion 22b are axial end faces of the first cylinder portion 21a and the second cylinder portion 22a, respectively, which face each other. The axial end faces of the first holding portion 21b and the second holding portion 22b are contact portions capable of contacting the support frame 12 of the culture membrane 10. The first holding portion 21b is in contact with the axial end face on one side of the culture membrane 10 in the axial direction. The second holding portion 22b is in contact with the axial end surface of the culture membrane 10 on the other side in the axial direction. The first holding portion 21b and the second holding portion 22b are each formed in an annular shape. The first holding portion 21b and the second holding portion 22b are provided at positions in the axially intermediate portion of the main body cylinder portion of the entire holder 20.

The first side surface opening 21c is a slit hole portion provided so as to penetrate a predetermined portion of the first cylinder portion 21a in the radial direction and communicate the inside of the first cylinder portion 21a with the outside. The first side surface opening 21c is provided at one location in the circumferential direction. The predetermined portion where the first side surface opening 21c is provided is larger than the culture membrane 10 held at least in the first holding portion 21b during cell culture in a state where the first tubular portion 21a is located below the second tubular portion 22a. It includes a portion on the lower side in the axial direction X, and is formed so as to extend from the first holding portion 21b to one side in the axial direction. The first side surface opening 21c extends over the entire axial direction of the first tubular portion 21a. The first side surface opening 21c has an air bleeding function of discharging the air accumulated on the lower surface of the culture film 10 held by the first holding portion 21b to the outside.

The second side surface opening 22c is a slit hole portion provided so as to penetrate a predetermined portion of the second tubular portion 22a in the radial direction and communicate the inside of the second tubular portion 22a to the outside. The second side surface opening 22c is provided at one location in the circumferential direction. The predetermined portion where the second side surface opening 22c is provided is larger than the culture membrane 10 held by at least the second holding portion 22b during cell culture in a state where the second tubular portion 22a is located below the first tubular portion 21a. It includes a portion on the lower side in the axial direction X, and is formed so as to extend from the second holding portion 22b to the other side in the axial direction. The second side surface opening 22c extends over the entire axial direction of the second tubular portion 22a. The second side surface opening 22c has an air bleeding function of discharging the air accumulated on the lower surface of the culture film 10 held by the second holding portion 22b to the outside.

The first tubular member 30 and the second tubular member 40 are tubular members attached to the holder 20, respectively. The first cylinder member 30 and the second cylinder member 40 are each made of a resin material, rubber, or the like. The first cylinder member 30 and the second cylinder member 40 are selectively attached to the holder 20 at the time of cell culture on the culture membrane 10. That is, when the first type of cells are seeded on one axial end face of the culture membrane 10, the first tubular member 30 is attached to the holder 20, while the second type is attached to the other axial end face of the culture membrane 10. When the cells of the above are seeded, the second tubular member 40 is attached to the holder 20.

The first tubular member 30 has a hollow tubular portion 31 extending in a tubular shape in the axial direction X. The first cylinder member 30 is mounted on one side in the axial direction with respect to the holder 20 (specifically, the first holding cylinder 21). The first cylinder member 30 is removable from the holder 20 (specifically, the first holding cylinder 21). That is, the first cylinder member 30 and the first holding cylinder 21 are detachably attached to each other. The first cylinder member 30 and the first holding cylinder 21 have a structure A which is engaged and fixed to each other, such as the engaging structure 23 of the first holding cylinder 21 and the second holding cylinder 22 of the holder 20 described above. are doing. The culture membrane 10 held by the holder 20 is exposed to one side in the axial direction through the axial opening of the first tubular member 30.

The first cylinder member 30 is formed so as to be insertable into the first holding cylinder 21 of the holder 20. The first cylinder member 30 is attached in a state of being inserted into the first cylinder portion 21a of the first holding cylinder 21. The first cylinder member 30 has an outer diameter that matches the inner diameter of the first holding cylinder 21. The tubular portion 31 of the first tubular member 30 has a first protruding portion 32 that protrudes in one axial direction from one axial end of the first holding cylinder 21 in a state of being engaged and fixed to the first holding cylinder 21. are doing.

Specifically, the first cylinder member 30 is engaged and fixed to the first holding cylinder 21, and the axial end faces on the insertion side in the cylinder portion 31 are the first holding cylinder 21 and the second holding cylinder 22. The axial end surface of the tube portion 31 opposite to the insertion side is from one axial end surface of the first holding cylinder 21 so as to abut on one axial end surface of the culture membrane 10 sandwiched and fixed to the first holding cylinder 21. Is also formed so as to be arranged at a position protruding on one side in the axial direction. That is, the length Lb1 of the first cylinder member 30 in the axial direction X is set to be larger than the length La1 of the first cylinder portion 21a of the first holding cylinder 21 in the axial direction X.

Further, the second tubular member 40 has a hollow tubular portion 41 extending in a tubular shape in the axial direction X. The second cylinder member 40 is mounted on the other side in the axial direction with respect to the holder 20 (specifically, the second holding cylinder 22). The second cylinder member 40 is removable from the holder 20 (specifically, the second holding cylinder 22). That is, the second cylinder member 40 and the second holding cylinder 22 are detachably attached to each other. The second cylinder member 40 and the second holding cylinder 22 have a structure B that is engaged and fixed to each other, such as the engaging structure 23 of the first holding cylinder 21 and the second holding cylinder 22 of the holder 20 described above. are doing. The culture membrane 10 held by the holder 20 is exposed to the other side in the axial direction through the axial opening of the second tubular member 40.

The second cylinder member 40 is formed so as to be insertable into the second holding cylinder 22 of the holder 20. The second cylinder member 40 is attached in a state of being inserted into the second cylinder portion 22a of the second holding cylinder 22. The second cylinder member 40 has an outer diameter that matches the inner diameter of the second holding cylinder 22. The tubular portion 41 of the second tubular member 40 has a second protruding portion 42 that projects axially from the other end in the axial direction of the second holding cylinder 22 in a state of being engaged and fixed to the second holding cylinder 22. are doing.

Specifically, in a state where the second cylinder member 40 is engaged and fixed to the second holding cylinder 22, the axial end faces on the insertion side in the cylinder portion 41 are the first holding cylinder 21 and the second holding cylinder 22. The axial end surface of the culture membrane 10 sandwiched and fixed to the second holding cylinder 22 so as to abut on the other axial end surface and on the side opposite to the insertion side of the tubular portion 41 is from the axial other end of the second holding cylinder 22. Is also formed so as to be arranged at a position protruding on the other side in the axial direction. That is, the length Lb2 of the second cylinder member 40 in the axial direction X is set to be larger than the length La2 of the second cylinder portion 22a of the second holding cylinder 22 in the axial direction X.

Next, a method of assembling the cell culture device 1 and a method of using the cell culture device 1 will be described with reference to FIGS. 4, 5, and 6.

A culture membrane 10, a holder 20, a first cylinder member 30, and a second cylinder member 40, which are components of the cell culture instrument 1, are prepared. As the culture membrane 10 and the holder 20, those in which the culture membrane 10 is held by engaging and fixing the first holding cylinder 21 and the second holding cylinder 22 of the holder 20 are used. It should be.

The cell culture device 1 is assembled manually by a person using a jig such as tweezers. As the assembly procedure, first, the first cylinder member 30 is assembled to the holder 20 on which the culture membrane 10 is held. In this assembly, first, the holder 20 is set so that the axial end side into which the first cylinder member 30 is inserted is located upward, and then the first cylinder member 30 is gripped by a jig and then the first cylinder member 30 is gripped. The first cylinder member 30 is inserted into the holder 20 by moving it from above in the axial direction X closer to each other, and then the holder 20 and the first cylinder member 30 are engaged and fixed to each other by the structure A. Is realized by.

When such assembly is performed, the first cylinder member 30 is attached to the holder 20, and the first protruding portion 32 thereof is outside the one end of the first holding cylinder 21 of the holder 20 in the axial direction. It becomes a state of protruding toward the direction (state shown in FIG. 4; step S100 shown in FIG. 6). Cell culture is performed on the culture membrane 10 for the cell culture device 1 in this state. In this cell culture, the cell culture instrument 1 is placed in a well of a cell culture plate and filled with a culture solution, and one axial end surface (specifically, the membrane body 11) of the culture membrane 10 (specifically, the membrane body 11) is (upper surface shown in FIG. ) May be seeded with cell α. In the seeding of the cell α, first, the cell culture device 1 is immersed in the culture medium so that one axial end face of the culture membrane 10 faces vertically upward, and in that state, in the space on the one axial end face side. This is done by supplying cells α to the cells (step S110).

When the seeding of the cell α was completed as described above, the first cylinder member 30 was then removed from the holder 20 using a jig (step S120), and then the holder 20 held the culture membrane 10. It is turned upside down as it is (step S130).

When the holder 20 is turned upside down, the second cylinder member 40 is then assembled to the holder 20. In this assembly, first, the holder 20 is set so that the axial end side into which the second tubular member 40 is inserted, which is the opposite side to the axial end side into which the first tubular member 30 is inserted, is located upward. In this state, after gripping the second cylinder member 40 with a jig, the second cylinder member 40 is moved from above to the holder 20 in the axial direction X closer to each other and inserted, and then held. This is realized by engaging and fixing the jig 20 and the second cylinder member 40 with each other in the structure B.

When such assembly is performed, the second cylinder member 40 is attached to the holder 20, and the second protruding portion 42 thereof is outside the other side of the holder 20 in the axial direction with respect to the other end in the axial direction. It becomes a state of protruding toward the direction (state shown in FIG. 5; step S140). Cell culture is performed on the culture membrane 10 for the cell culture device 1 in this state. In this cell culture, the cell culture instrument 1 is placed in a well of a cell culture plate, filled with a culture solution, and the other axial end surface (specifically, the membrane body 11) of the culture membrane 10 (specifically, the membrane body 11) is (upper surface shown in FIG. 5). ) May be seeded with cell β. In the seeding of the cell β, first, the cell culture device 1 is immersed in the culture solution so that the other axial end face of the culture membrane 10 faces vertically upward, and in that state, the space on the other axial end face side. This is done by supplying cells β to the cells (step S150).

When the seeding of the cell β is completed as described above, the second cylinder member 40 is then removed from the holder 20 by using a jig (step S160). Then, the culture membrane 10 in which the cells have been cultured is removed from the holder 20, and the membrane body 11 of the culture membrane 10 is observed under a microscope.

As described above, according to the structure of the cell culture device 1, two types of cells can be seeded on both sides of the culture membrane 10 to perform cell culture. In addition, when two types of cells are seeded on both sides of the culture membrane 10, either when the cells are seeded on one axial end face of the culture membrane 10 or when the cells are seeded on the other axial end face. Although it is necessary to attach either one of the first cylinder member 30 and the second cylinder member 40 to the upper side of the holder 20 for holding the culture membrane 10, any one of them is attached to the lower side of the holder 20. It is not necessary to attach the other. That is, in order to seed two types of cells on both sides of the culture membrane 10, it is not necessary to use the cell culture device 1 by simultaneously attaching both the first cylinder member 30 and the second cylinder member 40 to the holder 20. , The first cylinder member 30 and the second cylinder member 40, whichever is located on the lower side, can be used in a state of being removed from the holder 20.

The length Lb1 of the first cylinder member 30 in the axial direction X is larger than the length La1 of the first cylinder portion 21a of the first holding cylinder 21 in the axial direction X, and the first cylinder member 30 holds the first cylinder. In a state of being engaged and fixed to the cylinder 21, the first protruding portion 32 protrudes from one end of the first holding cylinder 21 in the axial direction to one side in the axial direction. Further, the length Lb2 of the second cylinder member 40 in the axial direction X is larger than the length La2 of the second cylinder portion 22a of the second holding cylinder 22 in the axial direction X, and the second cylinder member 40 is the second cylinder member 40. (Ii) In a state of being engaged and fixed to the holding cylinder 22, the second protruding portion 42 projects to the other side in the axial direction from the other end in the axial direction of the second holding cylinder 22. Therefore, according to the cell culture apparatus 1, when the cell culture is used, the dimensional distance from the lower end in the axial direction to the holding portions 21b and 22b where the culture membrane 10 is held can be shortened, so that the well has a small capacity. It is possible to culture cells in a culture membrane with a small amount of culture solution.

Further, in the cell culture device 1, the first holding cylinder 21 of the holder 20 has a first side surface opening 21c that penetrates the first cylinder portion 21a in the radial direction. The second holding cylinder 22 of the holder 20 has a second side surface opening 22c that penetrates the second cylinder portion 22a in the radial direction. The side opening portions 21c and 22c are provided at predetermined portions including a portion on the lower side in the axial direction X from the culture film 10 held by the holding portions 21b and 22b, respectively. Therefore, the air accumulated on both sides of the culture membrane 10 can be discharged to the outside of the holder 20 through the side opening 21c and 22c.

In particular, when cells are seeded on one axial end face of the culture membrane 10 held by the holder 20 and then the holder 20 is turned upside down to seed cells on the other axial end face of the culture membrane 10. , The air accumulated in the axial end face of the culture membrane 10 on which the cells have already been seeded can be discharged to the outside of the holder 20 through the side opening 21c. Therefore, it is possible to prevent air from remaining on the culture surface of the cells seeded first, and inconvenience caused by the residual air (for example, the cells seeded first do not come into contact with the culture medium). Can be avoided.

Further, in the cell culture apparatus 1, the culture membrane 10 has a membrane body 11 on which cells are seeded and a support frame 12 bonded to the membrane body 11 and formed of a material harder than the membrane body 11. Have. The culture membrane 10 is sandwiched and fixed by the support frame 12 between the first holding cylinder 21 and the second holding cylinder 22 of the holder 20. Therefore, since the relatively hard support frame 12 may be used to handle the culture membrane 10 with the jig, the handling of the culture membrane 10 can be facilitated. Further, the support frame 12 is joined to the peripheral edge of the film body 11 to form an annular shape. Therefore, evaluation such as microscopic observation of the membrane body 11 of the culture membrane 10 can be performed with the support frame 12 bonded to the membrane body 11.

Further, the holder 20 sandwiches the culture membrane 10 between the first holding cylinder 21 and the second holding cylinder 22 by engaging and fixing the first holding cylinder 21 and the second holding cylinder 22 to each other. And hold. Therefore, the holder 20 can be easily assembled while holding the culture membrane 10, and the holder 20 after assembly can be easily separated into the first holding cylinder 21 and the second holding cylinder 22. , The culture membrane 10 can be easily removed from the holder 20. As a result, the culture membrane 10 after cell culture can be independently evaluated by microscopic observation or the like, and the evaluation can be easily and appropriately performed as compared with the structure in which the culture membrane and the holder are integrated. be able to.

In the first embodiment described above, the first holding portion 21b of the first holding cylinder 21 and the second holding portion 22b of the second holding cylinder 22 correspond to the "holding portion" described in the claims, respectively. are doing.

Further, in the first embodiment described above, two types of cells are seeded on both sides of the culture membrane 10. However, the present invention is not limited to this, and three or more types of cells may be seeded on both sides of the culture membrane 10. For example, three types of cells are prepared, two types of cells are seeded on one surface of the culture membrane 10, then the culture membrane 10 is turned over and one type of cells is seeded on the other surface of the culture membrane 10. You may do it.

[Second Embodiment]
The cell culture device 100 of the second embodiment can culture two types of cells on both sides of the membrane, similarly to the cell culture device 1 of the first embodiment. As shown in FIG. 7, the cell culture device 100 includes a culture membrane 10, a holder 120, a first cylinder member 130, and a second cylinder member 140. In the second embodiment, the same components as those of the cell culture device 1 in the first embodiment are designated by the same reference numerals, and the description thereof will be omitted or simplified.

The holder 120 is a member for holding the culture membrane 10. The holder 120 is made of a resin material, rubber, or the like. As shown in FIG. 8, the holder 120 is composed of two holding cylinders, and has a first holding cylinder 121 and a second holding cylinder 122. The first holding cylinder 121 and the second holding cylinder 122 are arranged adjacent to each other in the axial direction X. The first holding cylinder 121 and the second holding cylinder 122 hold the culture membrane 10 in a state of being engaged and fixed to each other. The culture membrane 10 held by the holder 120 is exposed on both sides in the axial direction through the axial openings of the first holding cylinder 121 and the second holding cylinder 122.

The first holding cylinder 121 and the second holding cylinder 122 are members that are detachably attached to each other and have a structure in which they are engaged and fixed to each other. This engaging structure may be an L-groove engaging structure similar to the engaging structure 23 of the holder 20 in the first embodiment or a snap-fit type engaging structure. When the first holding cylinder 121 and the second holding cylinder 122 are engaged and fixed to each other, the distance between the first holding cylinder 121 and the second holding cylinder 122 facing each other in the axial direction X. Is set to be smaller than the axial thickness of the culture membrane 10 (specifically, the support frame 12 thereof) before assembly. As a result, the support frame 12 of the culture membrane 10 is sandwiched and fixed between the first holding cylinder 121 and the second holding cylinder 122. At this time, the space between the support frame 12 and the first holding cylinder 121 and the space between the support frame 12 and the second holding cylinder 122 are sealed.

The first holding cylinder 121 is so as to penetrate the hollow first cylinder portion 121a extending in a tubular shape in the axial direction X, the first holding portion 121b for holding the culture membrane 10, and the first cylinder portion 121a in the radial direction. It has a first side surface opening 121c provided. The second holding cylinder 122 penetrates the hollow second cylinder portion 122a extending in a tubular shape in the axial direction X, the second holding portion 122b holding the culture membrane 10, and the second cylinder portion 122a in the radial direction, respectively. It has a second side surface opening 122c and a third side surface opening 122d provided in the above, and a holding opening 122e provided so as to penetrate the second holding portion 122b in the axial direction X.

The first tubular portion 121a has a predetermined length in the axial direction X and a predetermined thickness in the radial direction. The second tubular portion 122a has a predetermined length in the axial direction X and a predetermined thickness in the radial direction. The outer diameter of the first tubular portion 121a and the inner diameter of the second tubular portion 122a are substantially the same. The outer diameter of the culture membrane 10 (that is, the outer diameter of the support frame 12) exceeds the inner diameter of the first tubular portion 121a and is equal to or less than the outer diameter thereof, and is equal to or smaller than the inner diameter of the second tubular portion 122a.

The first holding portion 121b is an axial end surface of the first cylinder portion 121a facing the second holding cylinder 122. The second holding portion 122b is a flange portion protruding inward in the radial direction from the second tubular portion 122a. The outer diameter of the culture membrane 10 (that is, the outer diameter of the support frame 12) is larger than the inner diameter of the radial inner end of the second holding portion 122b. The second holding portion 122b is provided at an axially intermediate portion of the second tubular portion 122a. The first holding cylinder 121 is second in a state where the first holding cylinder 121a is inserted inside the second cylinder portion 122a of the second holding cylinder 122 and the first holding portion 121b faces the second holding portion 122b. It is engaged and fixed to the holding cylinder 122. As a result, the first holding cylinder 121 and the second holding cylinder 122 sandwich and hold the culture membrane 10.

The first side surface opening 121c is a slit hole portion provided so as to penetrate a predetermined portion of the first cylinder portion 121a in the radial direction and communicate the inside of the first cylinder portion 121a to the outside. The first side surface opening 121c is provided at one location in the circumferential direction. The predetermined portion where the first side surface opening 121c is provided is larger than the culture membrane 10 held by at least the first holding portion 121b during cell culture in a state where the first tubular portion 121a is located below the second tubular portion 122a. It includes a portion on the lower side in the axial direction X, and is formed so as to extend from the first holding portion 121b to one side in the axial direction. The first side surface opening 121c is an air bleeder that discharges air accumulated on the lower surface of the culture membrane 10 held by the first holding portion 121b in a state where the first cylinder portion 121a is located below the second cylinder portion 122a. It has a function.

The second side surface opening 122c and the third side surface opening 122d are slits provided so as to penetrate a predetermined portion of the second cylinder portion 122a in the radial direction, respectively, to communicate the inside of the second cylinder portion 122a to the outside. It is a hole. The second side surface opening 122c and the third side surface opening 122d are provided at different positions in the circumferential direction of the second cylinder portion 122a. As a result, the torsional rigidity of the second holding cylinder 122 can be improved and the sealing property can be improved.

The predetermined site where the second side surface opening 122c is provided is larger than the culture membrane 10 held by at least the second holding portion 122b during cell culture in a state where the second tubular portion 122a is located below the first tubular portion 121a. It includes a portion on the lower side in the axial direction X, and is formed so as to extend from the second holding portion 122b to the other side in the axial direction. The second side surface opening 122c is an air bleeder that discharges air accumulated on the lower surface of the culture membrane 10 held by the second holding portion 122b in a state where the second cylinder portion 122a is located below the first cylinder portion 121a. It has a function.

The predetermined site where the third side surface opening 122d is provided has a diameter of at least the first side surface opening 121c of the first holding cylinder 121 during cell culture in a state where the first cylinder portion 121a is located below the second cylinder portion 122a. It includes a portion below the culture film 10 that communicates in the direction and is held by the second holding portion 122b in the axial direction X, and is formed so as to extend from the second holding portion 122b to one side in the axial direction. There is. The third side surface opening 122d communicates with the first side surface opening 121c and is held by the second holding portion 122b in a state where the first cylinder portion 121a is located below the second cylinder portion 122a. It has an air bleeding function that discharges the air accumulated on the lower surface of the culture to the outside.

The holding opening 122e is provided so as to penetrate the second holding portion 122b in the axial direction X, and communicates with the second side surface opening 122c in the radial direction. The holding opening 122e communicates with the second side surface opening 122c in a state where the second tubular portion 122a is located below the first tubular portion 121a, and the culture membrane 10 is held by the second holding portion 122b. It has an air bleeding function that discharges the air that collects on the bottom surface to the outside.

The first tubular member 130 and the second tubular member 140 are tubular members attached to the holder 120, respectively. The first cylinder member 130 and the second cylinder member 140 are each made of a resin material, rubber, or the like. The first cylinder member 130 and the second cylinder member 140 are selectively attached to the holder 20 when the cells are cultured on the culture membrane 10. That is, when the first type of cells are seeded on one axial end face of the culture membrane 10, the first tubular member 130 is attached to the holder 120, while the second type is attached to the other axial end face of the culture membrane 10. When the cells of the above are seeded, the second tubular member 140 is attached to the holder 120.

The first tubular member 130 has a hollow tubular portion 131 extending in a tubular shape in the axial direction X. The first cylinder member 130 is mounted on one side in the axial direction with respect to the holder 120 (specifically, the first holding cylinder 121). The first cylinder member 130 is detachably attached to and detachable from the first holding cylinder 121. The first cylinder member 130 and the first holding cylinder 121 have a structure in which they are engaged with each other and fixed. The culture membrane 10 held by the holder 120 is exposed to one side in the axial direction through the axial opening of the first tubular member 130.

The first cylinder member 130 is formed so as to be insertable into the first holding cylinder 121 of the holder 120. The first cylinder member 130 is attached in a state of being inserted into the first cylinder portion 121a of the first holding cylinder 121. The inner diameter of the first tubular member 130 is unchanged over the entire axial direction X, but the outer diameter on the other side in the axial direction is formed to be smaller than the outer diameter on the one side in the axial direction. .. The outer diameter on the other side in the axial direction coincides with the inner diameter of the first holding cylinder 121. The tubular portion 131 of the first tubular member 130 projects in one axial direction from one axial end of the first holding cylinder 121 in a state of being engaged and fixed to the other axial direction of the first holding cylinder 121. It has a protruding portion 132.

In a state where the first cylinder member 130 is engaged and fixed to the first holding cylinder 121, the axial end surface of the cylinder portion 131 on the insertion side is sandwiched and fixed between the first holding cylinder 121 and the second holding cylinder 122. The axial end surface of the tube portion 131 so as to abut on one axial end surface of the culture membrane 10 and on the side opposite to the insertion side of the tubular portion 131 is one axial side of the first holding cylinder 121. It is formed so as to be arranged at a protruding position. That is, the length of the first cylinder member 130 in the axial direction X is set to be larger than the length of the first cylinder portion 121a of the first holding cylinder 121 in the axial direction X.

Further, the second tubular member 140 has a hollow tubular portion 141 extending in a tubular shape in the axial direction X. The second cylinder member 140 is mounted on the other side in the axial direction with respect to the holder 120 (specifically, the second holding cylinder 122). The second cylinder member 140 is detachably attached to and detached from the second holding cylinder 122. The second cylinder member 140 and the second holding cylinder 122 have a structure in which they are engaged with each other and fixed. The culture membrane 10 held by the holder 120 is exposed to the other side in the axial direction through the axial opening of the second tubular member 140.

The second cylinder member 140 is formed so as to be insertable into the second holding cylinder 122 of the holder 120. The second cylinder member 140 is attached in a state of being inserted into the second cylinder portion 122a of the second holding cylinder 122. The second cylinder member 140 has an outer diameter that matches the inner diameter of the second cylinder portion 122a of the second holding cylinder 122. The tubular portion 141 of the second tubular member 140 has a second protruding portion 142 that protrudes to the other side in the axial direction from the other end in the axial direction of the second holding cylinder 122 in a state of being engaged and fixed to the second holding cylinder 122. are doing.

In a state where the second cylinder member 140 is engaged and fixed to the second holding cylinder 122, the axial end surface of the cylinder portion 141 on the insertion side is sandwiched and fixed between the first holding cylinder 121 and the second holding cylinder 122. The axial end face of the culture membrane 10 is in contact with the flange-shaped second holding portion 122b while being in contact with the other axial end face, and the axial end face of the tubular portion 141 opposite to the insertion side is the second holding cylinder. It is formed so as to be arranged at a position protruding on the other side in the axial direction from the other end in the axial direction of 122. That is, the length of the second cylinder member 140 in the axial direction X is set to be larger than the length of the second cylinder portion 122a of the second holding cylinder 122 in the axial direction X.

As the procedure for assembling the cell culture device 100 having the above structure, first, as in the case of the cell culture device 1 of the first embodiment, first, the first cylinder member 130 is attached to the holder 120 in which the culture membrane 10 is held. Assembly is done. Then, in a state where the first protruding portion 132 protrudes outward on one axial side from one axial end of the first holding cylinder 121 of the holder 120, cell seeding on one axial end surface of the culture membrane 10 is performed. Is done. When the cell seeding is completed, the first tubular member 130 is removed from the holder 120, and then the holder 120 is turned upside down while holding the culture membrane 10.

Next, the second cylinder member 140 is assembled to the holder 120. Then, in a state where the second protruding portion 42 protrudes outward on the other side in the axial direction from the other end in the axial direction of the second holding cylinder 22 of the holder 20, the cells are seeded on the other axial end face of the culture membrane 10. Is done. When the cell seeding is completed, the second tubular member 140 is removed from the holder 120, and then the membrane body 11 of the culture membrane 10 is observed under a microscope.

As described above, even in the structure of the cell culture device 100, two types of cells can be seeded on both sides of the culture membrane 10 to perform cell culture. Further, in order to seed two types of cells on both sides of the culture membrane 10, it is not necessary to use the cell culture device 100 with both the first cylinder member 130 and the second cylinder member 140 attached at the same time, and the first cylinder. It can be used in a state where any of the member 130 and the second cylinder member 140 located on the lower side is removed. The first cylinder member 130 and the second cylinder member 140 are respectively engaged and fixed to the holder 20, and project from the axial ends of the holder 20 to the outside in the axial direction at the protrusions 32 and 42, respectively. Therefore, according to the cell culture apparatus 100, the dimensional distance from the lower end in the axial direction to the holding portions 121b and 122b where the culture membrane 10 is held can be shortened when the cell culture is performed, whereby the wells can be provided. The volume can be reduced, and cells can be cultured in the culture membrane with a small amount of culture solution.

Further, in the cell culture device 100, the first holding cylinder 121 of the holder 120 has a first side surface opening 121c that penetrates the first cylinder portion 121a in the radial direction. The second holding cylinder 122 of the holder 120 has a second side surface opening 122c and a third side surface opening 122d that penetrate the second cylinder portion 122a in the radial direction, and penetrates the second holding portion 122b in the axial direction X. It has a holding opening 122e. Each of the side opening portions 121c, 122c, 122d is provided at a predetermined portion including a portion on the lower side in the axial direction X from the culture membrane 10 held by the holding portions 121b, 122b during double-sided culture of the culture membrane 10, respectively. .. In particular, the third side surface opening 122d is formed by passing the lower surface of the culture film 10 held by the holding portions 121b and 122b to the other axial end surface of the culture film 10 via the first side surface opening 121c. (Ii) It is provided so as to be exposed to the outside of the holding cylinder 122. Further, the holding opening 122e secondly holds the lower surface of the culture membrane 10 held by the holding portions 121b and 122b via the second side surface opening 122c when the cells are seeded on one axial end surface of the culture membrane 10. It is provided so as to be exposed to the outside of the cylinder 122. Therefore, the air accumulated on both sides of the culture membrane 10 can be discharged to the outside of the holder 120 through the side opening 121c and 122c.

In the second embodiment described above, the first holding portion 121b of the first holding cylinder 121 and the second holding portion 122b of the second holding cylinder 122 correspond to the "holding portion" described in the claims, respectively. are doing.

By the way, in the first and second embodiments described above, the cell culture instruments 1, 100 in which the tubular members 30, 40, 130 and 140 are inserted and arranged in the cylinders of the holders 20 and 120 are used. .. However, the present invention is not limited to this, and as shown in FIG. 9, the present invention may be applied to the cell culture device 200 in which the tubular members 230 and 240 are inserted and arranged outside the cylinder of the holder 220. Good.

Further, in the first and second embodiments described above, the culture membrane 10 is formed on both the two holding cylinders 21 and 22 constituting the holder 20 or the two holding cylinders 121 and 122 constituting the holder 120. The openings 21c, 22c, 121c, 122c, 122d, 122e are provided to discharge the air accumulated on the lower surface of the incubator to the outside. However, the present invention is not limited to this, and after the holders 20 and 120 are turned upside down, one axial direction of the culture membrane 10 is performed when cell seeding is performed on the other axial end face of the culture membrane 10. When the purpose is only to release the air accumulated on the end face to the outside of the holder 20, as shown in FIG. 9, the cells are seeded on the other axial end face of the culture membrane 10 of the two holding cylinders 221,222. The holding cylinder 221 located below may be provided with an opening 221c for discharging the air accumulated on the lower surface of the culture film 10 to the outside.

Further, in the first and second embodiments described above, the culture membrane 10 has a membrane body 11 and an annular support frame 12 joined to the peripheral edge of the membrane body 11. However, the present invention is not limited to this, and if the support frame 12 has a frame-like structure joined to the film body 11, for example, it is formed in a cross shape or a net shape when viewed from the axial direction X. You may be.

Further, in the first and second embodiments described above, the culture membrane 10 has a membrane body 11 and a support frame 12 bonded to the peripheral edge of the membrane body 11. However, the present invention is not limited to this, and as shown in FIG. 9, the culture membrane 210 may consist only of a membrane body having no support frame. In this modified form, the culture membrane 210 may be joined and fixed to either one of the first holding cylinder 221 and the second holding cylinder 222 of the holder 220. In this modified form, the outer diameter of the culture membrane 210 may be larger than the inner diameter of the first holding cylinder 221 and the second holding cylinder 222 and less than or equal to the outer diameter thereof. Further, in this modified form, in order to secure the inner / outer sealing property in the vicinity of holding the culture membrane 210 in the first holding cylinder 221 and the inner / outer sealing property in the vicinity of holding the culture membrane 210 in the second holding cylinder 222 during cell culture. , It is preferable that the peripheral surface between the holding cylinders 221 and 222 and the cylinder members 230 and 240 is sealed.

Further, in the first and second embodiments described above, when the tubular members 30, 40, 130 and 140 are attached to the holders 20 and 120, the axial end faces on the insertion side are attached to the support frame 12 of the culture membrane 10. Formed to abut. However, the present invention is not limited to this, and when the tubular members 30, 40, 130, 140 are attached to the holders 20, 120, the axial end surface on the insertion side thereof is the support frame 12 of the culture membrane 10. It does not have to be in contact with.

The present invention is not limited to the above-described embodiments and modifications, and various modifications can be made without departing from the spirit of the present invention.

1,100,200: Cell culture instrument, 10,210: Culture membrane, 11: Membrane body, 12: Support frame, 20,120,220: Holder, 21,121,221: First holding cylinder, 21a, 121a : First cylinder part, 21b, 121b: First holding part, 21c, 121c, 221c: First side opening, 22,122,222: Second holding cylinder, 22a, 122a: Second cylinder part, 22b, 122b : Second holding portion, 22c, 122c: Second side surface opening, 30, 130, 230: First cylinder member, 32: First protrusion, 40, 140, 240: Second cylinder member, 42: Second protrusion Part, 122d: third side opening, 122e: holding opening.

Claims (6)

A holder having a main body tubular portion extending in a tubular shape in the axial direction and a holding portion provided in the axial intermediate portion of the main body tubular portion to hold the culture membrane.
A second that is detachably attached to one side of the holder in the axial direction, extends in a tubular shape in the axial direction, and protrudes to one side in the axial direction from one end in the axial direction when attached to the holder. One cylinder member and
A second that is detachably attached to the other side of the holder in the axial direction, extends in a tubular shape in the axial direction, and protrudes to the other side in the axial direction from the other end in the axial direction when attached to the holder. Two-cylinder member and
A cell culture device. The cell culture device according to claim 1, wherein the holder has a side opening provided so as to penetrate a predetermined portion of the main body cylinder portion including at least one side in the axial direction in the axial direction. .. The side opening is
A first side surface opening formed so as to extend in the axial direction on one side in the axial direction of the holding portion,
A second side surface opening formed so as to extend in the axial direction on the other side in the axial direction of the holding portion,
The cell culture apparatus according to claim 2. The holder is
A tubular first holding cylinder provided with a first contacting portion that abuts on the axial end surface on one side in the axial direction of the culture membrane as the holding portion.
A tubular second holding cylinder that is detachably attached to the first holding cylinder and is provided with a second contact portion that abuts on the axial end surface on the other side in the axial direction of the culture membrane as the holding portion. When,
The cell culture apparatus according to any one of claims 1 to 3, which has the above. The cell according to any one of claims 1 to 4, wherein the culture membrane has a membrane body and a support frame bonded to the membrane body and formed of a material harder than the membrane body. Culture equipment. A method using the cell culture device according to any one of claims 1 to 5.
A first mounting step of mounting the first tubular member on one axial side of the holder before cells are seeded on one axial end face of the culture membrane.
A first removal step of removing the first tubular member from the holder after seeding of cells on one axial end face of the culture membrane.
A second mounting step of mounting the second tubular member on the other axial end face of the cage before the cells are seeded on the other axial end face of the culture membrane.
How to use a cell culture device.

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