JP7683281B2 - tissue culture vessels - Google Patents

Description

This specification discloses a tissue culture vessel.

Pluripotent stem cells, such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), can be induced to differentiate into target cells, and are expected to be applied in the field of regenerative medicine. Methods have been reported for inducing the differentiation of pluripotent stem cells into various cells, such as cardiomyocytes, retinal pigment epithelial cells, and chondrocytes.

For example, Patent Document 1 discloses intestinal organoids induced from embryonic stem cells and/or induced pluripotent stem cells, which have a structure containing a cavity and a longitudinal length of 5 mm or more. According to Patent Document 1, the intestinal organoids can be used for the development of drugs for preventing or treating intestinal-related diseases and for pathological research of intestinal-related diseases.

Patent Document 2 describes a cell clump culture vessel that is a vessel for culturing cell clumps and includes a well having a culture space capable of containing the cell clumps and culture fluid, and a cylindrical body that is disposed on a surface having an opening of the well and has an inner cavity that communicates with the culture space, and in which one or more communication parts are formed in the cylindrical wall of the cylindrical body that can discharge the culture fluid without allowing the cell clumps to pass to the outside of the cylindrical body. According to Patent Document 2, this vessel makes it possible to efficiently exchange the culture fluid with little effect on the cell clumps.

Similarly, Patent Document 3, by the same applicant as Patent Document 2, describes a cell cluster culture vessel in which a cylindrical partition erected at the opening of a well has a gap narrower than the width of the cell cluster. According to Patent Document 3, this vessel allows the culture medium to be replaced with minimal damage or irritation to the cell clusters, and also improves the productivity of the cell clusters.

Patent document 4 discloses a plate that is immersed in a liquid while spaced apart from the bottom wall of a container that stores the liquid and holds an object, the plate having one or more recesses that can support the object and have through-holes formed in the bottom. The diameter of the through-holes is selected to be a size that does not allow an object of the desired size to pass through, but allows an object smaller than the desired size to pass through. When this plate is placed in a container that stores liquid, the recesses can be used as wells for culture.

JP 2019-000014 A WO2015/178413 WO2017/057126 JP 2016-163591 A

When applying agents such as drug candidate substances or staining substances to tissues as described in Patent Document 1, it is conceivable to accommodate the tissues and culture fluid containing the agents in each well of a multi-well plate and incubate them. In this case, with a normal multi-well plate, it takes time to replace the culture fluid, which causes problems such as different treatment times with the culture fluid containing the agents for each well, and the culture fluid cannot be replaced in a short time, which causes problems such as significant damage to the tissue. In addition, in order to recover the tissues contained in each well after culture, it is necessary to aspirate and recover the tissues in each well, which causes problems such as the inability to recover the tissues in a short time.

The containers described in Patent Documents 2 and 3 make it possible to drain the culture medium in the wells by tilting the entire container including the wells. However, it is considered difficult to completely replace the culture medium in the wells. In addition, tissues after culture must be collected for each well.

According to the technology described in Patent Document 4, since a through hole is formed in the bottom of each well that holds the culture object, it is considered that it is not suitable for observing the culture object under a microscope. In addition, the tissue after culture needs to be collected for each well.
Therefore, this specification discloses a new tissue culture vessel that is provided with multiple wells for containing and culturing tissues, and that allows for easy replacement of the culture medium and recovery of the tissues after culturing.

This specification discloses a tissue culture vessel according to a first embodiment described in any one of (1) to (13) below, and a tissue culture vessel according to a second embodiment described in any one of (14) to (24) below.

(1) A tissue culture vessel for culturing tissue, comprising:
an outer container including a bottom portion of an outer container and a peripheral wall portion standing on a periphery of the bottom portion of the outer container, the outer container containing a first space defined by the bottom portion of the outer container and the peripheral wall portion;
a bottom member including a plurality of connected well bottoms separably received in the first space;
A peripheral wall member including a plurality of connected well peripheral wall portions that are separably accommodated in the first space;
Equipped with
the plurality of well bottoms and the plurality of well peripheral walls form a plurality of wells, each of which contains a plurality of second spaces;
The second space of each of the plurality of wells is defined by one of the well bottoms and one of the well peripheral walls extending from a periphery of one of the well bottoms,
In each of the wells, a gap is formed between at least a portion of the circumferential edge of the well bottom and the well peripheral wall, the gap connecting the second space and the first space in each of the wells.
A tissue culture vessel comprising:
(2) The tissue culture vessel according to (1), wherein the gaps in each of the wells are formed at the same circumferential position relative to the well bottom of each of the wells.
(3) A tissue culture vessel described in (1) or (2), in which the width of the gap in each well varies continuously and/or stepwise depending on the circumferential position of the periphery of the well bottom of each well.
(4) The bottom member further includes a first engagement portion,
The peripheral wall member further includes a second engagement portion,
the outer container includes a third engaging portion, which is provided on the outer container peripheral wall portion of the outer container and engages with the first engaging portion of the bottom member to position the bottom member, and a fourth engaging portion, which is provided on the outer container peripheral wall portion and engages with the second engaging portion of the peripheral wall member to position the peripheral wall member;
At least one of an engagement between the first engaging portion of the bottom member and the third engaging portion of the outer container and an engagement between the second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container is adjustable so as to change the width of the gap in each well.
A tissue culture vessel according to any one of (1) to (3).
(5) At least one of the first engagement portion of the bottom member and the third engagement portion of the outer container is processable so that the position of the bottom member relative to the peripheral wall member can be adjusted; and/or
At least one of the second engagement portion of the peripheral wall member and the fourth engagement portion of the outer container is processable so as to adjust the position of the peripheral wall member relative to the bottom member.
A tissue culture vessel according to (4).
(6) The first engaging portion of the bottom member and the third engaging portion of the outer container can be engaged at a plurality of positions such that the bottom member is positioned at a plurality of different positions relative to the peripheral wall member.
and/or
The second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container can be engaged at a plurality of positions such that the peripheral wall member is positioned at a plurality of different positions relative to the bottom member.
A tissue culture vessel according to (4).
(7) The base member is provided with two or more.
The two or more bottom members each include the first engaging portion that engages with the third engaging portion of the outer container to position the bottom member at a different position relative to the peripheral wall member.
A tissue culture vessel according to (4).
(8) A first spacer member that is removably interposed between the first engaging portion of the bottom member and the third engaging portion of the outer container and adjusts the distance between the first engaging portion of the bottom member and the third engaging portion of the outer container, thereby adjusting the position of the bottom member with respect to the peripheral wall member; and/or
The tissue culture vessel described in (4) further comprises a second spacer member that is removably interposed between the second engagement portion of the peripheral wall member and the fourth engagement portion of the outer container and adjusts the position of the peripheral wall member relative to the bottom member by adjusting the distance between the second engagement portion of the peripheral wall member and the fourth engagement portion of the outer container.
(9) The peripheral wall member is provided in two or more.
The two or more peripheral wall members each include the second engaging portion that engages with the fourth engaging portion of the outer container to position the peripheral wall member at a different position relative to the bottom member.
A tissue culture vessel according to (4).
(10) The bottom member further includes a first engagement portion,
the peripheral wall member further includes a second engagement portion that engages with the first engagement portion of the bottom member to position the peripheral wall member,
the outer container includes a third engaging portion on the outer container peripheral wall portion of the outer container, the third engaging portion engaging with the first engaging portion of the bottom member to position the bottom member;
At least one of an engagement between the first engaging portion of the bottom member and the third engaging portion of the outer container and an engagement between the first engaging portion of the bottom member and the second engaging portion of the peripheral wall member is adjustable so as to change the width of the gap in each well.
A tissue culture vessel according to any one of (1) to (3).
(11) The rotating shaft 1 includes N peripheral wall members from a first peripheral wall member to an Nth peripheral wall member,
an inner diameter of an n-1th well peripheral wall portion which is each of the plurality of well peripheral wall portions provided in the n-1th peripheral wall member is larger than an outer diameter of an nth well peripheral wall portion which is each of the plurality of well peripheral wall portions provided in the nth peripheral wall member, and the Nth peripheral wall member is removably combined with the first peripheral wall member such that the nth well peripheral wall portion of the nth peripheral wall member is inserted into the n-1th well peripheral wall portion of the n-1th peripheral wall member;
N is an integer of 2 or more, n is an integer of 2 or more and N or less,
Each of the wells is formed by one of the well bottoms and one of the combinations of a first well peripheral wall portion to an Nth well peripheral wall portion,
The second space of each of the wells is defined by one of the well bottoms and one of the combinations of the first well peripheral wall portion to the Nth well peripheral wall portion erected from a periphery of one of the well bottoms,
In each of the wells, the distance from the lower end of the (n-1) well peripheral wall portion to the well bottom is greater than the distance from the lower end of the (n) well peripheral wall portion to the well bottom.
A tissue culture vessel according to any one of (1) to (3).
(12) Further comprising a lid capable of covering the outer container,
The bottom member further includes a fifth engagement portion,
The peripheral wall member further includes a sixth engagement portion,
The cover body includes a seventh engagement portion that engages with the fifth engagement portion of the bottom member to hold the bottom member, and an eighth engagement portion that engages with the sixth engagement portion of the peripheral wall member to hold the peripheral wall member.
A tissue culture vessel according to any one of (1) to (11).
(13) The tissue culture vessel according to any one of (1) to (12), wherein the outer container bottom of the outer container and the well bottom of the bottom member are made of a glass material.

(14) A tissue culture vessel for culturing tissue, comprising:
an outer container including a bottom portion of an outer container and a peripheral wall portion standing on a periphery of the bottom portion of the outer container, the outer container containing a first space defined by the bottom portion of the outer container and the peripheral wall portion;
A peripheral wall member including a plurality of connected well peripheral wall portions that are separably accommodated in the first space;
Equipped with
the outer container bottom and the plurality of well peripheral wall portions form a plurality of wells, each of which contains a plurality of second spaces;
The second space of each of the plurality of wells is defined by one of a plurality of portions of the bottom of the outer container and one of the well peripheral walls extending from a periphery of the one of the portions;
In each of the wells, a gap is formed between at least a part of the circumferential direction of the periphery of the portion of the bottom of the outer container and the well peripheral wall portion, the gap connecting the second space and the first space in each of the wells.
A tissue culture vessel comprising:
(15) The tissue culture vessel according to (14), wherein the gaps in each of the wells are formed at the same circumferential position relative to the portion of the bottom of the outer vessel of each of the wells.
(16) A tissue culture vessel described in (14) or (15), wherein the width of the gap in each well varies continuously and/or stepwise depending on the circumferential position of the periphery of the portion of the bottom of the outer container of each well.
(17) The peripheral wall member further includes a second engagement portion,
the outer container includes a fourth engaging portion on the outer container peripheral wall portion of the outer container, the fourth engaging portion engaging with the second engaging portion of the peripheral wall member to position the peripheral wall member;
At least one of the engagement between the second engagement portion of the peripheral wall member and the fourth engagement portion of the outer container is adjustable so as to change the width of the gap in each of the wells.
A tissue culture vessel according to any one of (14) to (16).
(18) At least one of the second engagement portion of the peripheral wall member and the fourth engagement portion of the outer container can be processed so as to adjust the position of the peripheral wall member relative to the bottom of the outer container.
A tissue culture vessel according to (17).
(19) The second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container can be engaged at a plurality of positions such that the peripheral wall member is positioned at a plurality of different positions with respect to the bottom of the outer container.
A tissue culture vessel according to (17).
(20) The tissue culture vessel described in (17), further comprising a second spacer member that is removably interposed between the second engagement portion of the peripheral wall member and the fourth engagement portion of the outer container and adjusts the position of the peripheral wall member relative to the bottom of the outer container by adjusting the distance between the second engagement portion of the peripheral wall member and the fourth engagement portion of the outer container.
(21) The peripheral wall member is provided in two or more.
The two or more peripheral wall members each include the second engaging portion that engages with the fourth engaging portion of the outer container to position the peripheral wall member at a different position relative to the bottom of the outer container.
A tissue culture vessel according to (17).
(22) The rotating shaft 1 includes N peripheral wall members from a first peripheral wall member to an Nth peripheral wall member,
an inner diameter of an n-1th well peripheral wall portion which is each of the plurality of well peripheral wall portions provided in the n-1th peripheral wall member is larger than an outer diameter of an nth well peripheral wall portion which is each of the plurality of well peripheral wall portions provided in the nth peripheral wall member, and the Nth peripheral wall member is removably combined with the first peripheral wall member such that the nth well peripheral wall portion of the nth peripheral wall member is inserted into the n-1th well peripheral wall portion of the n-1th peripheral wall member;
N is an integer of 2 or more, n is an integer of 2 or more and N or less,
Each of the wells is formed by one of the plurality of portions of the bottom of the outer container and one of the combinations of the first well peripheral wall portion to the Nth well peripheral wall portion,
The second space of each of the wells is defined by one of a plurality of portions of the bottom of the outer container and one of a combination of the first well peripheral wall portion to the N well peripheral wall portion erected from a periphery of one of the portions,
In each of the wells, the distance from the lower end of the peripheral wall of the n-1 well to the bottom of the outer container is greater than the distance from the lower end of the peripheral wall of the n well to the bottom of the outer container.
A tissue culture vessel according to any one of (14) to (16).
(23) Further comprising a lid capable of covering the outer container,
The peripheral wall member further includes a sixth engagement portion,
The cover body includes an eighth engagement portion that engages with the sixth engagement portion of the peripheral wall member to hold the peripheral wall member.
A tissue culture vessel according to any one of (14) to (22).
(24) The tissue culture vessel according to any one of (14) to (23), wherein the bottom of the outer vessel is made of a glass material.

According to one or more aspects of the present disclosure, the tissue culture vessel can culture multiple tissues at once, and the culture medium can be replaced and the tissues can be collected after culture in a short time.

FIG. 1 is an exploded perspective view of a tissue culture vessel according to a first embodiment of the present disclosure. FIG. 2 is a schematic cross-sectional view of a tissue culture container according to a first embodiment of the present disclosure, which combines an outer container, a bottom member, and a peripheral wall member, in which a test intestinal organoid is contained in each of the second spaces in the multiple wells, and a first culture medium is stored in the first space in the outer container and the second spaces in the multiple wells. Figure 3 is a schematic cross-sectional view of the tissue culture container according to the first embodiment of the present disclosure in the state shown in Figure 2 after the bottom member and the peripheral wall member have been pulled out of the outer container while retaining the multiple wells containing the test intestinal organoids, and the first culture medium in the outer container has been removed. FIG. 4 is a schematic cross-sectional view of the tissue culture container according to the first embodiment of the present disclosure in the state shown in FIG. 3 , in which a second culture medium containing a dye is stored in the first space in the outer container. Figure 5 is a schematic cross-sectional view in which a combination of a bottom member and a peripheral wall member forming multiple wells containing test colon organoids is placed again in an outer container storing the second culture medium containing the dye in the state shown in Figure 4, and the test colon organoids are immersed in the second culture medium containing the dye. Figure 6 is a schematic cross-sectional view showing the state in which, after culturing the test colon organoids in the second culture medium containing the dye in the state shown in Figure 5, the bottom member and the peripheral wall member are pulled out of the outer container while maintaining the state in which multiple wells containing the test colon organoids are formed. FIG. 7 is a schematic cross-sectional view of the tissue culture container according to the first embodiment of the present disclosure in the state shown in FIG. 6 after the second culture medium in the outer container has been removed. FIG. 8 is a schematic cross-sectional view showing the process of placing the bottom member and peripheral wall member, in which multiple wells containing test colon organoids have been formed, back into the outer container storing the third culture medium, and washing the test colon organoids in the third culture medium. FIG. 9 is a schematic cross-sectional view showing a state in which the test colon organoid stained with a dye has been selectively removed from the tissue culture vessel according to the first embodiment of the present disclosure in the state shown in FIG. 8 . FIG. 10 is a schematic cross-sectional view showing a process of removing the peripheral wall member from the tissue culture container according to the first embodiment of the present disclosure in the state shown in FIG. 9 and collecting a collection of normal test colon organoids that are not stained with a dye in the first space of the outer container. FIG. 11 is a schematic cross-sectional view of a tissue culture vessel according to a first embodiment of the present disclosure, in which gaps in multiple wells are formed only along a portion of the circumferential edge of the well bottom, and the width of the gap varies continuously depending on the circumferential position of the circumferential edge of the well bottom. FIG. 12 is a schematic cross-sectional view of a tissue culture container according to the first embodiment of the present disclosure, further comprising a lid, in which a test intestinal organoid is contained in each of the second spaces in the multiple wells, and culture medium is stored in the first space in the outer container and the second spaces in the multiple wells. Figure 13 is a schematic cross-sectional view of the tissue culture container according to the first embodiment of the present disclosure, further comprising a lid in the state shown in Figure 12, in which the bottom member and peripheral wall member together with the lid have been pulled out from the outer container while retaining multiple wells containing test intestinal organoids. Fig. 14(A) is a schematic plan view of a tissue culture vessel according to a second embodiment of the present disclosure, and Fig. 14(B) is a schematic cross-sectional view of the tissue culture vessel according to the second embodiment of the present disclosure. FIG. 15 is a schematic cross-sectional view of a tissue culture vessel 1 according to a fourth example of the second modification of the first embodiment of the present disclosure in a state before a second spacer member 60 is combined. FIG. 16 is a schematic cross-sectional view of a state in which a second spacer member 60 is combined with a tissue culture vessel 1 according to a fourth example of the second modification of the first embodiment of the present disclosure. Figure 17 is a schematic cross-sectional view of a tissue culture vessel 1 according to variant example 4 of the first embodiment of the present disclosure, in which the first peripheral wall member 30-1 and the second peripheral wall member 30-2 are superimposed so that the second well peripheral wall portion 31-2 is inserted into the first well peripheral wall portion 31-1. FIG. 18 is a schematic cross-sectional view of the tissue culture vessel 1 according to the fourth modified example of the first embodiment of the present disclosure with the second peripheral wall member 30-2 removed.

<Tissue culture vessel according to the first embodiment>
Specific aspects of the tissue culture vessel according to the first embodiment of the present disclosure will be described with reference to the drawings.

Figure 1 is an exploded perspective view of a tissue culture vessel according to a first embodiment of the present disclosure. Figures 2 to 10 are schematic cross-sectional views showing a method for culturing tissue in a culture medium using the tissue culture vessel according to the first embodiment shown in Figure 1. In the following description, the structure of the tissue culture vessel 1 according to the first embodiment and a method of using the same will be described with reference to the reference numerals shown in Figures 1 to 10.

The tissue culture vessel 1 comprises an outer vessel 10 , a bottom member 20 , and a peripheral wall member 30 .
The outer container 10 includes an outer container bottom 11 and an outer container peripheral wall 12 standing upright from a periphery 11a of the outer container bottom 11, and contains a first space 13 defined by the outer container bottom 11 and the outer container peripheral wall 12. Culture solutions L1, L2, and L3 are stored in the first space 13. The first space 13 is a space that is open at the top, has a bottom defined by the outer container bottom 11, and is surrounded by the outer container peripheral wall 12.

The bottom member 20 has a plurality of connected well bottoms 21 that are separably accommodated in the first space 13. The multiple well bottoms 21 may be connected via well bottom connectors 22, as in the illustrated example.

The peripheral wall member 30 has a plurality of connected well peripheral wall portions 31 that are separably housed in the first space 13. The multiple well peripheral wall portions 31 may be connected via well peripheral wall portion connectors 32, as in the illustrated example.

The multiple well bottoms 21 and the multiple well peripheral walls 31 form multiple wells 40, each containing multiple second spaces 41. Here, the second space 41 of each of the multiple wells 40 (each well) is defined by one of the well bottoms 21 and one of the well peripheral walls 31 erected from the periphery of one of the well bottoms 21. Each of the second spaces 41 is a space for containing and culturing tissue.

In each well 40, the surface of the well bottom 21 on the side of the second space 41 (the inner bottom surface of the well 40) is preferably a surface that has been treated to inhibit cell adhesion (a low-adhesion surface). In addition, in each well 40, the surface of the well peripheral wall 31 on the side of the second space 41 (the inner surface of the well 40) is preferably a surface that has been treated to inhibit cell adhesion (a low-adhesion surface). These aspects of the tissue culture vessel 1 are particularly suitable for culturing intestinal organoids or spheroids, which will be described later, in the second space 41 of each well 40.

In each well 40, a gap 42 is formed between at least a portion of the circumferential edge 21a of the well bottom 21 and the well peripheral wall portion 31, connecting the second space 41 in each well 40 with the first space 13.

The well peripheral wall 31 is preferably a cylindrical body that is open at the top and bottom. In the illustrated example, the well peripheral wall 31 has a uniform inner diameter regardless of the depth position of the well 40, but may have an inner diameter that varies continuously or in stages depending on the depth position of the well. For example, in an example not illustrated, the inner diameter of the well peripheral wall 31, which is a cylindrical body, is smallest at the lower end 31a, expands continuously from the lower end 31a toward the upper end 31b that constitutes the opening of the well 40, and is constant from a position midway to the upper end 31b to the upper end 31b.

The culture fluids L1, L2, and L3 stored in the first space 13 of the outer container 10 can flow into the second space 41 of each of the multiple wells 40 through the gap 42 and fill the second space 41. Furthermore, when the culture fluids L1, L2, and L3 stored in the first space 13 of the outer container 10 are discharged by tilting the entire tissue culture container 1 or by aspirating the culture fluids L1, L2, and L3, the culture fluids L1, L2, and L3 in the second space 41 of each of the multiple wells 40 can be discharged simultaneously through the gap 42. In addition, in a tissue culture container 1 including multiple wells 40 in which the second space 41 is filled with the culture fluids L1, L2, and L3 in the outer container 10, the culture fluids L1, L2, and L3 in the second space 41 of each of the multiple wells 40 can be discharged through the gap 42 by separating the bottom member 20 and the peripheral wall member 30 from the outer container 10 in a combined state. That is, with the tissue culture vessel 1, it is possible to replace the culture solutions L1, L2, and L3 in the multiple wells 40, each of which contains a tissue T, all at once in a short time. In addition, the tissue T contained in each of the multiple wells 40 can be treated with the culture solutions L1, L2, and L3 under uniform conditions.

Furthermore, after the treatment with culture fluids L1, L2, and L3 using the tissue culture vessel 1 is completed, the bottom member 20 and the peripheral wall member 30 are separated from the outer vessel 10, the well 40 is dismantled, and the multiple tissues T are released into the first space 13 of the outer vessel 10, making it possible to collect multiple tissues T at once.

The gap 42 in the well 40 is formed between at least a portion of the circumferential direction of the periphery 21a of the well bottom 21 and the well peripheral wall 31. The gap 42 may have a width that does not allow the tissue T to be cultured to pass through. For example, the width of the gap 42 is preferably 50% or less of the width of the tissue T to be cultured.

In the tissue culture vessel 1 according to the example shown in Figures 1 to 10, the gap 42 in the well 40 is formed between the periphery 21a of the well bottom 21 and the lower end 31a of the well peripheral wall 31, over the entire circumferential direction of the periphery 21a of the well bottom 21, and has a uniform width. This configuration provides the following effect. That is, the tissue culture vessel 1 in which the outer container 10, bottom member 20, and peripheral wall member 30 are combined and the first space 13 and second space 41 are filled with culture fluids L1, L2, and L3 can discharge the culture fluids L1, L2, and L3 from the first space 13 and second space 41 regardless of the direction in which the entire vessel is tilted.

In addition, it is preferable that the gaps 42 in each of the multiple wells 40 are formed at the same circumferential position relative to the well bottom 21 of each well 40 in a plan view of the tissue culture vessel 1. In this case, the gaps 42 in the wells 40 may be formed only in a portion of the circumferential direction of the periphery 21a of the well bottom 21. If the entire tissue culture vessel 1 is tilted so that the gaps 42 in each well 40 are positioned downward, the culture solutions L1, L2, and L3 can be discharged from the first space 13 and the second space 41.

The width of the gap 42 in each well 40 of the multiple wells 40 may be uniform over the entire circumferential direction of the periphery 21a of the well bottom 21, as in the example tissue culture vessel 1 shown in Figures 1 to 10, or may vary continuously and/or stepwise depending on the circumferential position of the periphery 21a of the well bottom 21. According to a tissue culture vessel 1 in which the width of the gap 42 in each well 40 of the multiple wells 40 varies depending on the circumferential position of the periphery 21a of the well bottom 21, when the entire tissue culture vessel 1 is tilted to drain the culture solutions L1, L2, and L3 from the first space 13 and the second space 41, it is possible to change the drainage speed of the culture solutions L1, L2, and L3 from the second space 41 by changing the tilt direction.

Figure 11 shows an example of a tissue culture vessel 1 having the same features as those of Figures 1 to 10, except that the gap 42 in the well 40 is formed only in a part of the circumferential direction of the periphery 21a of the well bottom 21, and the width of the gap 42 varies continuously depending on the circumferential position of the periphery 21a of the well bottom 21. In the example shown in Figure 11, a convex portion 21b protruding toward the lower end 31a of the well peripheral wall portion 31 is provided on the periphery 21a of the well bottom 21, and the height of the convex portion 21b varies continuously depending on the circumferential position of the periphery 21a of the well bottom 21. The most protruding part of the convex portion 21b contacts the lower end 31a of the well peripheral wall portion 31, and no gap 42 exists in this part. The lower the height of the convex portion 21b, the wider the width of the gap 42 between the periphery 21a of the well bottom 21 and the lower end 31a of the well peripheral wall portion 31.

In this specification, the term "gap" refers to an opening that allows the passage of liquid between the second space and the first space in each well. The gap 42 of each well 40 in the tissue culture vessel 1 according to the example shown in Figures 1 to 10 is a single continuous opening. However, unlike the embodiment shown, a gap that includes multiple adjacent openings is also an example of a "gap" in this specification. An example of a gap consisting of multiple adjacent openings is a gap between the bottom of the well and the surrounding wall of the well, in which a member having multiple adjacent openings, such as a mesh or fence, is arranged to separate the first space from the second space.

The culture fluids L1, L2, and L3 stored in the first space 13 of the outer container 10 can flow into the second space 41 of each of the multiple wells 40 through the gap 42 and fill the second space 41. Furthermore, when the culture fluids L1, L2, and L3 stored in the first space 13 of the outer container 10 are discharged by tilting the entire tissue culture container 1 or by sucking the culture fluids L1, L2, and L3, the culture fluids L1, L2, and L3 in the second space 41 of each of the multiple wells 40 can be discharged simultaneously through the gap 42. In addition, in the tissue culture container 1 including the multiple wells 40 in which the second space 41 is filled with the culture fluids L1, L2, and L3 in the outer container 10, the culture fluids L1, L2, and L3 in the second space 41 of each of the multiple wells 40 can be discharged through the gap 42 by separating the bottom member 20 and the peripheral wall member 30 from the outer container 10 in a combined state.

The bottom member 20 further includes a first engagement portion 23. On the other hand, the outer container 10 includes a third engagement portion 14 at the upper end portion 12a of the outer container peripheral wall portion 12, which engages with the first engagement portion 23 of the bottom member 20 to position the bottom member 20. The first engagement portion 23 is preferably, as in the illustrated example, a plurality of arm-shaped engagement portions protruding from a portion of the plurality of well bottoms 21 connected via the well bottom connecting portion 22 toward the third engagement portion 14 of the outer container peripheral wall portion 12. The third engagement portion 14 is preferably, as in the illustrated example, an engagement recess formed at the upper end portion 12a of the outer container peripheral wall portion 12 of the outer container 10.

The peripheral wall member 30 further includes a second engagement portion 33. On the other hand, the outer container 10 includes a fourth engagement portion 15 at the upper end portion 12a of the outer container peripheral wall portion 12, which engages with the second engagement portion 33 of the peripheral wall member 30 to position the peripheral wall member 30. The second engagement portion 33 is preferably a plurality of arm-shaped engagement portions protruding from a portion of the plurality of well peripheral walls 31 connected via the well peripheral wall portion connecting portion 32 toward the fourth engagement portion 15 of the outer container peripheral wall portion 12, as in the illustrated example. The fourth engagement portion 15 is preferably an engagement recess formed at the upper end portion 12a of the outer container peripheral wall portion 12 of the outer container 10, as in the illustrated example.

2, the bottom member 20 is positioned and accommodated in the first space 13 of the outer container 10 by the engagement between the third engagement portion 14 of the outer container 10 and the first engagement portion 23 of the bottom member 20. Furthermore, the peripheral wall member 30 is positioned and accommodated in the first space 13 of the outer container 10 by the engagement between the fourth engagement portion 15 of the outer container 10 and the second engagement portion 33 of the peripheral wall member 30. As a result, a plurality of wells 40 with gaps 42 formed therein are formed. The shapes and mutual engagement states of the first engagement portion 23 of the bottom member 20, the second engagement portion 33 of the peripheral wall member 30, the third engagement portion 14 and the fourth engagement portion 15 of the outer container 10 are appropriately adjusted according to the positions of the plurality of well bottom members 21 and the plurality of well peripheral wall members 31 in the first space 13 of the outer container 10 and the width of the gaps 42.

As shown in Fig. 2, a tissue culture vessel 1 having an outer container 10, a bottom member 20, and a peripheral wall member 30 is used to contain and culture tissues T in multiple wells 40. When observing the tissues T being cultured in the multiple wells 40 from above with a microscope, if the well bottom 21 of each well 40 and the outer container bottom 11 of the outer container 10 are made of a resin material such as polystyrene, the autofluorescence of the resin material may interfere with the observation. Therefore, it is preferable that the well bottom 21 of each well 40 and the outer container bottom 11 of the outer container 10 are made of a glass material that does not have autofluorescence.
Next, with reference to Figures 2 to 10, a procedure for processing intestinal organoids as tissue T using the tissue culture vessel 1 according to the first embodiment will be described.

The intestinal organoid is typically the intestinal organoid described in Patent Document 1, and more specifically, it is an intestinal organoid derived from embryonic stem cells and/or induced pluripotent stem cells, has a structure containing a cavity, and is characterized in that the length in the longitudinal direction is 5 mm or more. The intestinal organoid described in Patent Document 1 more preferably contains endodermal cells, ectodermal cells, and mesodermal cells, and contains intestinal epithelial cells on the outer surface. Intestinal organoids containing endodermal cells, ectodermal cells, and mesodermal cells and containing intestinal epithelial cells on the outer surface can absorb substances on the outside into the cavity through the intestinal epithelial cells on the outer surface. In addition, in the intestinal organoid described in Patent Document 1, when the intestinal epithelial cells on the outer surface are further transporter positive, it is even more preferable because it is possible to take up substances via the transporter. In other words, intestinal organoids containing transporter-positive intestinal epithelial cells have a substance absorption ability similar to that of the intestine. The intestinal organoid more preferably has development of microvilli and crypts on the outer surface.

Intestinal organoids having the above characteristics preferably have the ability to actively take in substances from the outside into the lumen, while also having the ability (barrier property) to prevent substances that are not the target of active uptake from entering the lumen. If the cell layer surrounding the lumen is not sufficiently developed and the barrier property is insufficient because of the formation of openings in the cell layer, etc., intestinal organoids will nonspecifically absorb substances that are not the target of active uptake into the lumen. Therefore, this specification discloses a method for distinguishing between intestinal organoids with insufficient barrier property and normal intestinal organoids with barrier property from among multiple intestinal organoids.

The method for distinguishing between intestinal organoids having insufficient barrier properties and normal intestinal organoids preferably comprises:
A step 1 of placing a test colon organoid together with a first culture medium in each well of a substrate including a plurality of wells;
Step 2: After step 1, the first culture medium in each well is replaced with a second culture medium containing a dye, and the test colon organoid is cultured in the second culture medium;
Step 3, after step 2, of replacing the second culture medium in each well with a third culture medium and washing the test colon organoid in the third culture medium; and
Step 4, after step 3, of distinguishing the test colon organoids stained with the dye as intestinal organoids with insufficient barrier properties and distinguishing the test colon organoids not stained with the dye as normal intestinal organoids.
Includes.

Step 4 preferably includes at least one of step 4-1, which selectively removes the test colon organoids stained with the dye, and step 4-2, which selectively recovers only the test colon organoids that are not stained with the dye, and more preferably includes both steps 4-1 and 4-2.
The dye may be a water-soluble dye that absorbs and emits visible light or ultraviolet light and has no cytotoxicity. Specific examples of the dye include pH indicators that are colored at around pH 7.4, such as bromophenol red, bromothymol blue, neutral red, and phenolphthalein, and biological dyes, such as flavonoids and anthocyanins.

The method for distinguishing between intestinal organoids with insufficient barrier properties and normal intestinal organoids, which includes steps 1 to 4, preferably uses the tissue culture vessel 1 according to the first embodiment shown in FIG. 1 as the "substrate containing multiple wells." An example of this method will be described with reference to FIGS. 2 to 10.

First, as shown in FIG. 2, a test intestinal organoid T is placed in each of the second spaces 41 of the multiple wells 40 of the tissue culture vessel 1, which is a combination of an outer vessel 10, a bottom member 20, and a peripheral wall member 30. At this time, the first culture solution L1 is stored in the first space 13 of the outer vessel 10, and the first culture solution L1 passes through the gap 42 and fills the second space 41 contained in each of the multiple wells 40. This is step 1.

3, the bottom member 20 and the peripheral wall member 30 are lifted from the outer container 10 while maintaining the state in which the multiple wells 40 containing the test intestinal organoids T are formed. The first culture solution L1 in the second spaces 41 of the multiple wells 40 is discharged into the outer container 10 through the gaps 42. The first culture solution L1 in the outer container 10 is removed.
Next, as shown in FIG. 4, a second culture medium L2 containing a dye is stored in the first space 13 of the outer container 10.

Next, as shown in FIG. 5, the combination of the bottom member 20 and the peripheral wall member 30, in which the multiple wells 40 containing the test colon organoids T are formed, is placed again in the outer container 10 that stores the second culture solution L2 containing the dye, and the test colon organoids T are immersed in the second culture solution L2 containing the dye and cultured in the second culture solution L2. This step is step 2.

Instead of the process shown in Figures 3 and 4 of lifting the bottom member 20 and the peripheral wall member 30 from the outer container 10 and replacing the culture solution in the outer container 10, it is also possible to remove the first culture solution L1 from the first space 13 of the outer container 10 while maintaining the positional relationship between the outer container 10, the bottom member 20, and the peripheral wall member 30 shown in Figure 2, and then store the second culture solution L2 in the first space 13 of the outer container 10, thereby moving from the stage shown in Figure 2 to the stage shown in Figure 5.

The culture time (staining time) in step 2 can be adjusted as appropriate depending on the type and concentration of the dye. For example, when phenol red is used as the dye, the culture time can be approximately 3 hours. After step 2, test colon organoids T1 that are not stained with the dye and test colon organoids T2 that are stained with the dye are generated.

After the culture in step 2, the bottom member 20 and the peripheral wall member 30 are lifted from the outer container 10 while maintaining the state in which the multiple wells 40 containing the test intestinal organoids T1, T2 are formed, as shown in Fig. 6. The second culture solution L2 in the second spaces 41 of the multiple wells 40 is discharged into the outer container 10 through the gaps 42.
Then, as shown in FIG. 7, the second culture medium L2 in the outer container 10 is removed.

Next, as shown in FIG. 8, the bottom member 20 and the peripheral wall member 30, in which the multiple wells 40 containing the test colon organoids T1, T2 are formed, are placed again in the outer container 10 storing the third culture solution L3, and the test colon organoids T1, T2 are washed in the third culture solution L3. This step is step 3. The third culture solution L3 may be any culture solution capable of washing away the pigment adhering to the surface of the test colon organoids T1, T2, and is preferably a colorless and transparent culture solution.

Instead of the process shown in Figures 6 and 7 of lifting the bottom member 20 and the peripheral wall member 30 from the outer container 10 and replacing the culture solution in the outer container 10, the second culture solution L2 can be removed from the first space 13 of the outer container 10 while maintaining the positional relationship between the outer container 10, the bottom member 20, and the peripheral wall member 30 shown in Figure 5, and then the third culture solution L3 can be stored in the first space 13 of the outer container 10, and the process can proceed from the stage shown in Figure 5 to the stage shown in Figure 8.

After step 3, the test colon organoid T2 stained with the dye is identified as an intestinal organoid with insufficient barrier properties, and the test colon organoid T1 not stained with the dye is identified as a normal intestinal organoid. This step is step 4.

Step 4 preferably includes selectively removing the test colon organoid T2 stained with the dye from the well 40 containing it, as shown in FIG. 9. This step is a specific example of step 4-1.

In step 4, after removing the test colon organoid T2 in step 4-1, the peripheral wall member 30 can be removed as shown in FIG. 10, and normal test colon organoids T1 that are not stained with a dye can be collected together in the first space 13 of the outer container 10. This step is a specific example of step 4-2.

Thus, implementing the method for distinguishing between intestinal organoids with insufficient barrier properties and normal intestinal organoids, which includes steps 1 to 4, using tissue culture vessel 1 has the following advantages:

First, compared to replacing the culture medium in each well using a normal multi-well plate, the first culture medium L1 can be replaced with the second culture medium L2, and the second culture medium L2 can be replaced with the third culture medium L3 in a short time. In addition, the replacement of culture medium can be completed for all wells at approximately the same time. This makes it possible to perform a staining process under gentle and uniform conditions on the multiple test colon organoids T contained in each of the multiple wells 40.

Secondly, the normal test colon organoids T1 that are not stained with a dye and are contained in each of the multiple wells 41 can be collected collectively in the first space 13 of the outer container 10 by removing the peripheral wall member 30 from the outer container 10 (and optionally removing the bottom member 20) and dismantling the multiple wells 41. When collecting normal test colon organoids T1 in each well using a normal multi-well plate, it is necessary to collect the test colon organoids T1 in each well individually, which makes it difficult to collect them in a short time and may cause damage to the test colon organoids T1, but these problems can be solved by using the tissue culture container 1.

The tissue culture vessel 1 according to the present disclosure is particularly useful in a method for treating multiple tissues T simultaneously under the same conditions for a short period of time, since the culture solution containing phenol red can be quickly replaced. Phenol red is usually used as a pH indicator. Since the culture solution containing phenol red easily changes color when it comes into contact with air, when phenol red is used as a dye in the method for distinguishing between intestinal organoids with insufficient barrier properties and normal intestinal organoids, which includes steps 1 to 4, it is necessary to treat multiple intestinal organoids T with the culture solution containing phenol red under uniform conditions in a short period of time. The tissue culture vessel 1 according to the present disclosure is particularly suitable for such applications.

The tissue culture vessel 1 according to the present disclosure can be used for various applications that take advantage of the advantage of being able to replace the entire amount of culture medium in multiple wells in a short time, in addition to the method of distinguishing between intestinal organoids with insufficient barrier properties, including steps 1 to 4. For example, the tissue culture vessel 1 can be used for a test that minutely verifies the process in which tissue T is affected by drugs, etc., and a test using a compound that is easily affected by pH or light. The tissue culture vessel 1 can also be used for various methods of treating multiple tissues T at once under the same conditions. For example, the tissue culture vessel 1 can be used in a method of treating multiple tissues T with a culture solution containing a poison to determine which tissues T are resistant or sensitive to the poison, or a method of treating multiple tissues T with a culture solution containing a substance whose absorption is desired to determine which tissues T are capable of absorbing the substance.

<First Modification of First Embodiment>
A first modified example of the tissue culture vessel 1 according to the first embodiment of the present disclosure includes a tissue culture vessel 1 further including a lid 50 shown in FIGS.

In the tissue culture vessel 1 of the first modified example of the first embodiment, the lid 50 covers the opening of the first space 13 of the outer vessel 10 and is detachable from the outer vessel 10.

The tissue culture vessel 1 of the first modification of the first embodiment further includes:
The bottom member 20 further includes a fifth engagement portion 24,
The peripheral wall member 30 further includes a sixth engagement portion 34,
The cover body 50 has a seventh engagement portion 51 that engages with the fifth engagement portion 24 of the bottom member 20 to hold the bottom member 20, and an eighth engagement portion 52 that engages with the sixth engagement portion 34 of the peripheral wall member 30 to hold the peripheral wall member 30.

In the tissue culture vessel 1 of the first embodiment shown in FIG. 12 and FIG. 13, the fifth engagement portion 24 of the bottom member 20 is an engagement claw that is provided on the tip side of the first engagement portion 23 and protrudes toward the seventh engagement portion 51 of the lid body 50.

In the tissue culture vessel 1 of the first modified example of the first embodiment shown in Figures 12 and 13, the sixth engagement portion 34 of the peripheral wall member 30 is an engagement claw that is provided on the tip side of the second engagement portion 33 and protrudes toward the eighth engagement portion 52 of the lid body 50.

As shown in FIG. 12, one tissue T is accommodated in each of the second spaces 41 of the multiple wells 40 of the tissue culture vessel 1 of the first modified example of the first embodiment, which combines the outer vessel 10, the bottom member 20, the peripheral wall member 30, and the lid 50. At this time, the culture fluid L is stored in the first space 13 of the outer vessel 10, and the culture fluid L passes through the gap 42 to fill the second space 41 contained in each of the multiple wells 40.

In the tissue culture vessel 1 of the first embodiment of the modified example 1, which contains the culture solution L and tissue T in the state shown in Fig. 12, when the culture solution L is replaced with another culture solution, the lid body 50 is lifted from the outer container 10 as shown in Fig. 13. At this time, due to the engagement between the fifth engagement portion 24 of the bottom member 20 and the seventh engagement portion 51 of the lid body 50, and the engagement between the sixth engagement portion 34 of the peripheral wall member 30 and the eighth engagement portion 52 of the lid body 50, the bottom member 20 and the peripheral wall member 30 are lifted from the outer container 10 together with the lid body 50 while maintaining the state in which the multiple wells 40 containing the tissue T are formed. The culture solution L in the second space 41 of the multiple wells 40 is discharged into the outer container 10 through the gap 42. From the state shown in FIG. 13, the culture medium L in the first space 13 of the outer container 10 is replaced with another culture medium, and then the combination of the bottom member 20 and the peripheral wall member 30 that form the multiple wells 40 containing the tissue T, together with the lid body 50, is placed back into the outer container 10, so that the tissue T can be immersed in new culture medium.

In this way, according to the tissue culture vessel 1 of the first modified example of the first embodiment, it is particularly easy to remove the bottom member 20 and the peripheral wall member 30 together with the lid body 50 from the outer vessel 10 and to reinstall them in the outer vessel 10, and the culture medium can be replaced in a short time.

<Modification 2 of First Embodiment>
As a second modified example of the tissue culture vessel 1 according to the first embodiment of the present disclosure, there is an example in which at least one of the engagement between the first engagement portion 23 of the bottom member 20 and the third engagement portion 14 of the outer container 10 and the engagement between the second engagement portion 33 of the peripheral wall member 30 and the fourth engagement portion 15 of the outer container 10 is adjustable so as to change the width of the gap 42 in each well 40. According to the tissue culture vessel 1 according to the second modified example of the first embodiment, the width of the gap 42 of each of the multiple wells 40 can be changed. An appropriate width of the gap 42 can be selected depending on the size of the tissue T to be cultured.

A first example of the tissue culture vessel 1 according to the second modification of the first embodiment is as follows:
At least one of the first engagement portion 23 of the bottom member 20 and the third engagement portion 14 of the outer container 10 can be processed so that the position of the bottom member 20 relative to the peripheral wall member 30 can be adjusted; and/or
At least one of the second engagement portion 33 of the peripheral wall member 30 and the fourth engagement portion 15 of the outer container 10 can be processed so that the position of the peripheral wall member 30 relative to the bottom member 20 can be adjusted.
A tissue culture vessel 1.

Here, "machinable" means that the first engagement portion 23 of the bottom member 20, the third engagement portion 14 of the outer container 10, the second engagement portion 33 of the peripheral wall member 30, or the fourth engagement portion 15 of the outer container 10 can be irreversibly processed by cutting or deformation, or can be reversibly processed by deformation or attachment of an attachment.

In the first example of the tissue culture vessel 1 of the second modified example of the first embodiment, the first engagement portion 23 of the bottom member 20, the third engagement portion 14 of the outer vessel 10, the second engagement portion 33 of the peripheral wall member 30, or the fourth engagement portion 15 of the outer vessel 10 is processed to change the engagement position and adjust the gap 42.

A second example of the tissue culture vessel 1 according to the second modification of the first embodiment is as follows:
The first engagement portion 23 of the bottom member 20 and the third engagement portion 14 of the outer container 10 can be engaged at a plurality of positions so that the bottom member 20 is positioned at a plurality of different positions relative to the peripheral wall member 30.
and/or
The tissue culture vessel 1 is one in which the second engagement portion 33 of the peripheral wall member 30 and the fourth engagement portion 15 of the outer container 10 can be engaged at multiple positions so that the peripheral wall member 30 is positioned at multiple different positions relative to the bottom member 20.

According to the second example of the tissue culture vessel 1 of the second modified example of the first embodiment, the width of the gap 42 can be appropriately adjusted in accordance with the dimensional change caused by the growth of the tissue T.

In a second example of the tissue culture vessel 1 of the second modified example of the first embodiment, the first engagement portion 23 of the bottom member 20, the third engagement portion 14 of the outer vessel 10, the second engagement portion 33 of the peripheral wall member 30, or the fourth engagement portion 15 of the outer vessel 10 can be a multi-stage protrusion or recess.

A third example of the tissue culture vessel 1 according to the second modification of the first embodiment is as follows:
Two or more bottom members 20 are provided,
The tissue culture vessel 1 includes two or more bottom members 20 each having a first engagement portion 23 that engages with a third engagement portion 14 of the outer container 10 to position the bottom member 20 at a different position relative to the peripheral wall member 30 .

According to the third example of the tissue culture vessel 1 of the second modified example of the first embodiment, an appropriate bottom member 20 can be selected from two or more bottom members 20 so that a gap 42 of an appropriate width is formed according to the dimensions of the tissue T to be cultured.

A fourth example of the tissue culture vessel 1 according to the second modification of the first embodiment is as follows:
a first spacer member that is detachably interposed between the first engagement portion 23 of the bottom member 20 and the third engagement portion 14 of the outer container 10 and adjusts the distance between the first engagement portion 23 of the bottom member 20 and the third engagement portion 14 of the outer container 10, thereby adjusting the position of the bottom member 20 relative to the peripheral wall member 30; and/or
a second spacer member 60 that is detachably interposed between the second engagement portion 33 of the peripheral wall member 30 and the fourth engagement portion 15 of the outer container 10 and adjusts the position of the peripheral wall member 30 relative to the bottom member 20 by adjusting the distance between the second engagement portion 33 of the peripheral wall member 30 and the fourth engagement portion 15 of the outer container 10;
The tissue culture vessel 1 further comprises:

15 and 16 show a tissue culture vessel 1 according to a fourth example of the second modification of the first embodiment, which includes a second spacer member 60. In the example shown in Figs. 15 and 16, the second spacer member 60 has a tip portion 61 with a tapered width. In the tissue culture vessel 1 shown in Fig. 15 in which the second engagement portion 33 of the peripheral wall member 30 and the fourth engagement portion 15 of the outer container 10 are directly engaged with each other, the gap 42 in the well 40 is narrow. 16, by inserting the tip 61 of the second spacer member 60 between the second engagement portion 33 of the peripheral wall member 30 and the fourth engagement portion 15 of the outer container 10, the interval between the second engagement portion 33 of the peripheral wall member 30 and the fourth engagement portion 15 of the outer container 10 is increased, and in response to the increase in the interval, the entire peripheral wall member 30 is lifted upward, and the distance between the periphery 21a of the well bottom 21 and the lower end 31a of the well peripheral wall portion 31, i.e., the width of the gap 42 in the well 40, is increased. By adjusting the insertion depth of the tip 61 of the second spacer member 60 between the second engagement portion 33 of the peripheral wall member 30 and the fourth engagement portion 15 of the outer container 10, it is possible to adjust the width of the gap 42 in the well 40.
By using the tissue culture vessel 1 according to the fourth example of the modified example 2 of the first embodiment, which includes the second spacer member 60 shown in Figs. 15 and 16, for example, the following spheroid culture is possible. First, as shown in Fig. 15, the outer container 10, the bottom member 20, and the peripheral wall member 30 are combined so that the second engagement portion 33 of the peripheral wall member 30 and the fourth engagement portion 15 of the outer container 10 directly engage with each other without using the second spacer member 60, to form a tissue culture vessel 1 including a plurality of wells 40 with gaps 42 equal to or smaller than the size of the cells C (for example, 10 µm or less). Then, the first space 13 and the second space 41 of the tissue culture vessel 1 are filled with the culture solution L required for spheroid culture, and the cells C are seeded in the second space 41 in each well 40. Culture is performed in this state to prepare spheroids T from the cells C. As shown in Fig. 16, the tip 61 of the second spacer member 60 is inserted between the second engagement portion 33 of the peripheral wall member 30 and the fourth engagement portion 15 of the outer container 10 to widen the width of the gap 42 in the well 40. The width of the gap 42 after the second spacer member 60 is adjusted to a width (e.g., 450 µm or less) that does not allow the spheroid tissue T that has grown to a desired size or more to pass through, but allows the spheroids less than the desired size and the cells C that have not formed spheroids to pass through, so that the spheroids T remain in the well 40 containing the spheroids T that have grown to a desired size or more, and the spheroids less than the desired size and the cells C that have not formed spheroids pass through the gap 42 from the other wells 40 and are discharged into the first space 13 of the outer container 10. Next, similar to the examples of Figs. 6 and 7, the bottom member 20 and the peripheral wall member 30 are pulled up from the outer container 10 while maintaining the state in which the multiple wells 40 containing the spheroids T are formed. Next, although not shown, the culture solution L containing the spheroids and cells C smaller than the desired size in the outer container 10 is discarded and replaced with another liquid (e.g., a cryopreservation liquid) as necessary. Thereafter, the combination of the bottom member 20 and the peripheral wall member 30 in a state in which a plurality of wells 40 containing spheroids T have been formed is returned to the outer container 10 containing the other liquid, and further, the peripheral wall member 30 and the bottom member 20 are sequentially removed from the outer container 10 to disassemble the wells 40, and only the spheroids T larger than the desired size can be collected in the outer container 10 containing the other liquid.

A fifth example of the tissue culture vessel 1 according to the second modification of the first embodiment is as follows:
Two or more peripheral wall members 30 are provided,
The tissue culture vessel 1 includes two or more peripheral wall members 30 each having a second engagement portion 33 that engages with the fourth engagement portion 15 of the outer container 10 to position the peripheral wall member 30 at a different position relative to the bottom member 20 .

According to the fourth example of the tissue culture vessel 1 of the second modified example of the first embodiment, an appropriate one can be selected from two or more peripheral wall members 30 so that a gap 42 of an appropriate width is formed according to the dimensions of the tissue T to be cultured.

<Modification 3 of the First Embodiment>
As a modified example 3 of the tissue culture vessel 1 according to the first embodiment of the present disclosure, although not shown,
The second engaging portion 33 of the peripheral wall member 30 is formed to engage with the first engaging portion 23 of the bottom member 20 to determine the position of the peripheral wall member 30 relative to the bottom member 20,
An example includes an engagement between the first engagement portion 23 of the bottom member 20 and the third engagement portion 14 of the outer container 10, and an engagement between the first engagement portion 23 of the bottom member 20 and the second engagement portion 33 of the peripheral wall member 30, which are adjustable so that the width of the gap 42 in each well 40 can be changed.
According to the tissue culture vessel 1 of the third modified example of the first embodiment, it is possible to change the width of the gap 42 of each of the multiple wells 40. An appropriate width of the gap 42 can be selected depending on the size of the tissue T to be cultured.

A first example of the tissue culture vessel 1 according to the third modification of the first embodiment is as follows:
At least one of the first engagement portion 23 of the bottom member 20 and the third engagement portion 14 of the outer container 10 can be processed so that the position of the bottom member 20 relative to the peripheral wall member 30 can be adjusted; and/or
At least one of the first engagement portion 23 of the bottom member 20 and the second engagement portion 33 of the peripheral wall member 30 can be processed so that the position of the peripheral wall member 30 relative to the bottom member 20 can be adjusted.
A tissue culture vessel 1.

Here, "machinable" means that the first engagement portion 23 of the bottom member 20, the third engagement portion 14 of the outer container 10, or the second engagement portion 33 of the peripheral wall member 30 can be irreversibly processed by cutting or deformation, or can be reversibly processed by deformation or attachment of an attachment.

In the first example of the tissue culture vessel 1 of the third modified example of the first embodiment, the first engagement portion 23 of the bottom member 20, the third engagement portion 14 of the outer vessel 10, or the second engagement portion 33 of the peripheral wall member 30 is processed to change the engagement position and adjust the gap 42.

A second example of the tissue culture vessel 1 according to the third modification of the first embodiment is as follows:
The first engagement portion 23 of the bottom member 20 and the third engagement portion 14 of the outer container 10 can be engaged at a plurality of positions so that the bottom member 20 is positioned at a plurality of different positions relative to the peripheral wall member 30.
and/or
The tissue culture vessel 1 is one in which the first engagement portion 23 of the bottom member 20 and the second engagement portion 33 of the peripheral wall member 30 can be engaged at multiple positions so that the peripheral wall member 30 can be positioned at multiple different positions relative to the bottom member 20.

According to the second example of the tissue culture vessel 1 of the third modified example of the first embodiment, the width of the gap 42 can be appropriately adjusted in accordance with the dimensional change caused by the growth of the tissue T.

In a second example of the tissue culture vessel 1 of the third modified example of the first embodiment, the first engagement portion 23 of the bottom member 20, the third engagement portion 14 of the outer container 10, or the second engagement portion 33 of the peripheral wall member 30 can be a multi-step protrusion or recess.

A third example of the tissue culture vessel 1 according to the third modification of the first embodiment is as follows:
Two or more bottom members 20 are provided,
The tissue culture vessel 1 includes two or more bottom members 20 each having a first engagement portion 23 that engages with a third engagement portion 14 of the outer container 10 to position the bottom member 20 at a different position relative to the peripheral wall member 30 .

According to the third example of the tissue culture vessel 1 of the third modified example of the first embodiment, an appropriate bottom member 20 can be selected from two or more bottom members 20 so that a gap 42 of an appropriate width is formed according to the dimensions of the tissue T to be cultured.

A fourth example of the tissue culture vessel 1 according to the third modification of the first embodiment is as follows:
Two or more peripheral wall members 30 are provided,
In the tissue culture vessel 1 , two or more peripheral wall members 30 each include a second engagement portion 33 that engages with the first engagement portion 23 of the bottom member 20 to position the peripheral wall member 30 at a different position relative to the bottom member 20 .

According to the fourth example of the tissue culture vessel 1 of the third modified example of the first embodiment, an appropriate one can be selected from two or more peripheral wall members 30 so that a gap 42 of an appropriate width is formed according to the dimensions of the tissue T to be cultured.

<Fourth Modification of the First Embodiment>
As a fourth modified example of the tissue culture vessel 1 according to the first embodiment of the present disclosure, as shown in FIGS. 17 and 18,
The apparatus includes two peripheral wall members 30, namely, a first peripheral wall member 30-1 and a second peripheral wall member 30-2.
the inner diameter of a first well peripheral wall portion 31-1 which is each of the multiple well peripheral wall portions provided in the first peripheral wall member 30-1 is larger than the outer diameter of a second well peripheral wall portion 31-2 which is each of the multiple well peripheral wall portions provided in the second peripheral wall member 30-2, and the first peripheral wall member 30-1 and the second peripheral wall member 30-2 are removably combined such that the second well peripheral wall portion 31-2 of the second peripheral wall member 30-2 is inserted into the first well peripheral wall portion 31-1 of the first peripheral wall member 30-1;
Each well 40 is formed by one of the well bottoms 21 and one of the combinations of the first well peripheral wall portion 31-1 and the second well peripheral wall portion 31-2.
The second space 41 of each well 40 is defined by one of the well bottoms 21 and one of the combinations of a first well peripheral wall portion 31-1 and a second well peripheral wall portion 31-2 standing upright from the periphery of one of the well bottoms 21,
In each well 40, the distance from the lower end 31-1a of the first well peripheral wall portion 31-1 to the well bottom 21 (the distance in the depth direction of the well 40) is greater than the distance from the lower end 31-2a of the second well peripheral wall portion 31-2 to the well bottom 21 (the distance in the depth direction of the well 40).
An example of a tissue culture vessel 1 is given.

The function of the tissue culture vessel 1 according to the fourth modification will be described with reference to Figs. 17 and 18. In the tissue culture vessel 1 shown in Fig. 17, when the first peripheral wall member 30-1 and the second peripheral wall member 30-2 are overlapped so that the second well peripheral wall portion 31-2 of the second peripheral wall member 30-2 is inserted into the first well peripheral wall portion 31-1 of the first peripheral wall member 30-1, the width of the gap 42 of each well 40 is relatively small because it is the distance in the depth direction of the well 40 from the lower end 31-2a of the second well peripheral wall portion 31-2 to the well bottom 21. When only the second peripheral wall member 30-2 is removed from the tissue culture vessel 1 shown in Fig. 17, as shown in Fig. 18, the width of the gap 42 of each well 40 is relatively large because it is the distance in the depth direction of the well 40 from the lower end 31-1a of the first well peripheral wall portion 31-1 to the well bottom 21. That is, tissue culture is started in the tissue culture vessel 1 according to this modified example 4 with all the peripheral wall members, consisting of the first peripheral wall member 30-1 and the second peripheral wall member 30-2, overlapped, and when the tissue has grown, the second peripheral wall member 30-2 is removed and the culture is continued, making it possible to carry out tissue culture while gradually widening the width of the gap 42 in the well 40 according to the growth stage of the tissue. If three or more peripheral wall members are used, it is possible to adjust the width of the gap 42 more finely. The range of N may be 2 or more, and can be, for example, 2 to 5.

By using the tissue culture vessel 1 according to the fourth modified example of the first embodiment, for example, the following spheroid culture is possible. First, as shown in FIG. 17, a peripheral wall member 30, which is a combination of a first peripheral wall member 30-1 and a second peripheral wall member 30-2, is combined with an outer container 10 and a bottom member 20 to form a tissue culture vessel 1 having a plurality of wells 40 with gaps 42 equal to or smaller than the dimensions of cells C (for example, 10 μm or less). Then, the first space 13 and the second space 41 of the tissue culture vessel 1 are filled with culture medium L required for spheroid culture, and cells C are seeded in the second space 41 in each well 40. In this state, culture is performed to prepare spheroids T from the cells C. Next, as shown in FIG. 18, the second peripheral wall member 30-2 is removed to widen the width of the gaps 42 in the wells 40. The width of the gap 42 after removing the second peripheral wall member 30-2 is adjusted to a width (e.g., 450 μm or less) that does not allow the spheroid tissue T that has grown to a desired size or more to pass through, but allows the spheroids less than the desired size and the cells C that have not formed spheroids to pass through, so that the spheroids T remain in the wells 40 that contain the spheroids T that have grown to a desired size or more, and the spheroids less than the desired size and the cells C that have not formed spheroids are discharged from the other wells 40 through the gap 42 into the first space 13 of the outer container 10. Next, as in the examples of FIGS. 6 and 7, the bottom member 20 and the peripheral wall member 30 are pulled up from the outer container 10 while maintaining the state in which the multiple wells 40 containing the spheroids T are formed. Next, although not shown, the culture solution L containing the spheroids and cells C less than the desired size in the outer container 10 is discarded and replaced with another liquid (e.g., a cryopreservation solution) as necessary. Thereafter, the combination of the bottom member 20 and the peripheral wall member 30 forming the multiple wells 40 containing the spheroids T is returned to the outer container 10 containing the other liquid, and then the peripheral wall member 30 and the bottom member 20 are sequentially removed from the outer container 10 to disassemble the wells 40, and only the spheroids T of the desired size or larger can be collected in the outer container 10 containing the other liquid.

<Tissue culture vessel according to the second embodiment>
A specific aspect of the tissue culture vessel according to the second embodiment of the present disclosure will be described with reference to Fig. 14. In the tissue culture vessel according to the second embodiment, components having common features and functions with the tissue culture vessel according to the first embodiment are denoted by the same reference numerals, and description thereof will be omitted.

Figure 14 (A) is a schematic plan view of a tissue culture vessel 1 according to a second embodiment of the present disclosure. Figure 14 (B) is a schematic cross-sectional view of a tissue culture vessel 1 according to a second embodiment of the present disclosure.

The tissue culture vessel 1 according to the second embodiment is
an outer container 10 including an outer container bottom 11 and an outer container peripheral wall 12 extending from a peripheral edge 11a of the outer container bottom 11, and including a first space 13 defined by the outer container bottom 11 and the outer container peripheral wall 12;
A peripheral wall member 30 including a plurality of connected well peripheral wall portions 31 that are separably accommodated in the first space 13;
Equipped with
The outer container bottom 11 and the plurality of well peripheral walls 31 form a plurality of wells 40, each of which contains a plurality of second spaces 41;
The second space 41 of each of the plurality of wells 40 is defined by one of the plurality of portions 11b of the outer container bottom 11 and one of the well peripheral walls 31 standing upright from a peripheral edge 11ba of one of the portions 11b,
In each well 40, a gap 42 is formed between at least a part of the circumferential direction of the periphery 11ba of the portion 11b of the outer container bottom 11 and the well peripheral wall portion 31, connecting the second space 41 in each well 40 to the first space 13.
It is characterized by:

In the tissue culture vessel 1 according to the second embodiment, the culture fluid stored in the first space 13 of the outer vessel 10 can flow through the gap 42 into the second space 41 of each of the multiple wells 40 and fill the second space 41. Furthermore, when the culture fluid stored in the first space 13 of the outer vessel 10 is discharged by tilting the entire tissue culture vessel 1 or by sucking the culture fluid, the culture fluid in the second space 41 of each of the multiple wells 40 can be discharged simultaneously through the gap 42. That is, according to the tissue culture vessel 1 according to the second embodiment, it is possible to replace the culture fluid in the multiple wells 40, each of which contains tissue T, all at once in a short time. In addition, the tissue T contained in each of the multiple wells 40 can be treated with the culture fluid under uniform conditions.

Furthermore, when the peripheral wall member 30 is separated from the outer container 10 after completion of the treatment with the culture medium using the tissue culture container 1 according to the second embodiment, the well 40 is disassembled and the multiple tissues T are released into the first space 13 of the outer container 10, making it possible to collect the multiple tissues T at once.

The gap 42 in the well 40 is formed between at least a portion of the circumferential edge 11ba of the portion 11b of the outer container bottom 11 that forms the bottom of the second space 41 of the well 40, and the well peripheral wall portion 31. The gap 42 may have a width that does not allow the tissue T to be cultured to pass through. For example, the width of the gap 42 is preferably 50% or less of the width of the tissue T to be cultured.

In the tissue culture vessel 1 according to the second embodiment, when the tissue T being cultured in the multiple wells 40 is observed from above with a microscope, if the outer container bottom 11 of the outer container 10 forming the bottom of each well 40 is made of a resin material such as polystyrene, the autofluorescence of the resin material may interfere with the observation. Therefore, it is preferable that the outer container bottom 11 of the outer container 10 is made of a glass material that does not have autofluorescence.

Other features, usage methods, and modifications of the tissue culture vessel 1 according to the second embodiment are as described for the tissue culture vessel 1 according to the first embodiment.

Claims (23)

1. A tissue culture vessel for culturing tissue, comprising:
an outer container including a bottom portion of an outer container and a peripheral wall portion standing on a periphery of the bottom portion of the outer container, the outer container containing a first space defined by the bottom portion of the outer container and the peripheral wall portion;
a bottom member including a plurality of connected well bottoms separably received in the first space;
A peripheral wall member including a plurality of connected well peripheral wall portions that are separably accommodated in the first space;
Equipped with
the plurality of well bottoms and the plurality of well peripheral walls form a plurality of wells, each of which contains a plurality of second spaces;
The second space of each of the plurality of wells is defined by one of the well bottoms and one of the well peripheral wall portions erected from a periphery of one of the well bottoms,
In each of the wells, a gap is formed between at least a portion of the circumferential edge of the well bottom and the well peripheral wall, the gap connecting the second space and the first space in each of the wells.
A tissue culture vessel comprising: The tissue culture vessel according to claim 1, wherein the gaps in each well are formed at the same circumferential position relative to the well bottom of each well. The tissue culture vessel according to claim 1 or 2, wherein the width of the gap in each of the wells varies continuously and/or stepwise depending on the circumferential position of the periphery of the well bottom of each of the wells. The bottom member further includes a first engagement portion,
The peripheral wall member further includes a second engagement portion,
the outer container includes a third engaging portion, which is provided on the outer container peripheral wall portion of the outer container and engages with the first engaging portion of the bottom member to position the bottom member, and a fourth engaging portion, which is provided on the outer container peripheral wall portion and engages with the second engaging portion of the peripheral wall member to position the peripheral wall member;
At least one of an engagement between the first engaging portion of the bottom member and the third engaging portion of the outer container and an engagement between the second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container is adjustable so as to change the width of the gap in each well.
The tissue culture vessel according to any one of claims 1 to 3. At least one of the first engagement portion of the bottom member and the third engagement portion of the outer container is processable so that the position of the bottom member relative to the peripheral wall member can be adjusted; and/or
At least one of the second engagement portion of the peripheral wall member and the fourth engagement portion of the outer container is processable so as to adjust the position of the peripheral wall member relative to the bottom member.
The tissue culture vessel of claim 4. The first engaging portion of the bottom member and the third engaging portion of the outer container can be engaged at a plurality of positions such that the bottom member is positioned at a plurality of different positions relative to the peripheral wall member.
and/or
The second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container can be engaged at a plurality of positions such that the peripheral wall member is positioned at a plurality of different positions relative to the bottom member.
The tissue culture vessel of claim 4. The base member includes two or more base members,
The two or more bottom members each include the first engaging portion that engages with the third engaging portion of the outer container to position the bottom member at a different position relative to the peripheral wall member.
The tissue culture vessel of claim 4. a first spacer member that is detachably interposed between the first engagement portion of the bottom member and the third engagement portion of the outer container and adjusts a distance between the first engagement portion of the bottom member and the third engagement portion of the outer container, thereby adjusting a position of the bottom member relative to the peripheral wall member; and/or
The tissue culture vessel of claim 4, further comprising a second spacer member that is removably interposed between the second engagement portion of the peripheral wall member and the fourth engagement portion of the outer container and adjusts the position of the peripheral wall member relative to the bottom member by adjusting the distance between the second engagement portion of the peripheral wall member and the fourth engagement portion of the outer container. The peripheral wall member includes two or more members,
The two or more peripheral wall members each include the second engaging portion that engages with the fourth engaging portion of the outer container to position the peripheral wall member at a different position relative to the bottom member.
The tissue culture vessel of claim 4. The bottom member further includes a first engagement portion,
the peripheral wall member further includes a second engagement portion that engages with the first engagement portion of the bottom member to position the peripheral wall member,
the outer container includes a third engaging portion on the outer container peripheral wall portion of the outer container, the third engaging portion engaging with the first engaging portion of the bottom member to position the bottom member;
At least one of an engagement between the first engaging portion of the bottom member and the third engaging portion of the outer container and an engagement between the first engaging portion of the bottom member and the second engaging portion of the peripheral wall member is adjustable so as to change the width of the gap in each well.
The tissue culture vessel according to any one of claims 1 to 3. N peripheral wall members are provided, the N peripheral wall members being a first peripheral wall member to an Nth peripheral wall member,
an inner diameter of an n-1th well peripheral wall portion which is each of the plurality of well peripheral wall portions provided in the n-1th peripheral wall member is larger than an outer diameter of an nth well peripheral wall portion which is each of the plurality of well peripheral wall portions provided in the nth peripheral wall member, and the Nth peripheral wall member is removably combined with the first peripheral wall member such that the nth well peripheral wall portion of the nth peripheral wall member is inserted into the n-1th well peripheral wall portion of the n-1th peripheral wall member;
N is an integer of 2 or more, n is an integer of 2 or more and N or less,
Each of the wells is formed by one of the well bottoms and one of the combinations of a first well peripheral wall portion to an Nth well peripheral wall portion,
The second space of each of the wells is defined by one of the well bottoms and one of the combinations of the first well peripheral wall portion to the Nth well peripheral wall portion erected from a periphery of one of the well bottoms,
In each of the wells, the distance from the lower end of the (n-1) well peripheral wall portion to the well bottom is greater than the distance from the lower end of the (n) well peripheral wall portion to the well bottom.
The tissue culture vessel according to any one of claims 1 to 3. Further comprising a lid capable of covering the outer container,
The bottom member further includes a fifth engagement portion,
The peripheral wall member further includes a sixth engagement portion,
The cover body includes a seventh engagement portion that engages with the fifth engagement portion of the bottom member to hold the bottom member, and an eighth engagement portion that engages with the sixth engagement portion of the peripheral wall member to hold the peripheral wall member.
The tissue culture vessel according to any one of claims 1 to 11. The tissue culture vessel according to any one of claims 1 to 12, wherein the outer container bottom of the outer container and the well bottom of the bottom member are made of a glass material. 1. A tissue culture vessel for culturing tissue, comprising:
an outer container including a bottom portion of an outer container and a peripheral wall portion standing on a periphery of the bottom portion of the outer container, the outer container containing a first space defined by the bottom portion of the outer container and the peripheral wall portion;
A peripheral wall member including a plurality of connected well peripheral wall portions that are separably accommodated in the first space;
Equipped with
the outer container bottom and the plurality of well peripheral wall portions form a plurality of wells, each of which contains a plurality of second spaces;
The second space of each of the plurality of wells is defined by one of a plurality of portions of the bottom of the outer container and one of the well peripheral walls extending from a periphery of the one of the portions;
In each of the wells, a gap is formed between at least a part of a circumferential direction of the periphery of the portion of the bottom of the outer container and the well peripheral wall portion, the gap connecting the second space and the first space in each of the wells,
The width of the gap in each of the wells varies continuously and/or stepwise depending on the circumferential position of the periphery of the portion of the bottom of the outer container in each of the wells.
A tissue culture vessel comprising: The tissue culture vessel according to claim 14, wherein the gaps in each well are formed at the same circumferential position relative to the portion of the bottom of the outer vessel in each well. The peripheral wall member further includes a second engagement portion,
the outer container includes a fourth engaging portion on the outer container peripheral wall portion of the outer container, the fourth engaging portion engaging with the second engaging portion of the peripheral wall member to position the peripheral wall member;
At least one of the engagement between the second engagement portion of the peripheral wall member and the fourth engagement portion of the outer container is adjustable so as to change the width of the gap in each of the wells.
16. A tissue culture vessel according to claim 14 or 15 . At least one of the second engagement portion of the peripheral wall member and the fourth engagement portion of the outer container can be processed so that the position of the peripheral wall member relative to the bottom of the outer container can be adjusted.
17. The tissue culture vessel of claim 16 . The second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container can be engaged at a plurality of positions such that the peripheral wall member is positioned at a plurality of different positions with respect to the bottom of the outer container.
17. The tissue culture vessel of claim 16 . a second spacer member that is detachably interposed between the second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container and adjusts a distance between the second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container, thereby adjusting a position of the peripheral wall member with respect to a bottom portion of the outer container.
17. The tissue culture vessel of claim 16 . The peripheral wall member includes two or more members,
The two or more peripheral wall members each include the second engaging portion that engages with the fourth engaging portion of the outer container to position the peripheral wall member at a different position relative to the bottom of the outer container.
17. The tissue culture vessel of claim 16 . N peripheral wall members are provided, the N peripheral wall members being a first peripheral wall member to an Nth peripheral wall member,
an inner diameter of an n-1th well peripheral wall portion which is each of the plurality of well peripheral wall portions provided in the n-1th peripheral wall member is larger than an outer diameter of an nth well peripheral wall portion which is each of the plurality of well peripheral wall portions provided in the nth peripheral wall member, and the Nth peripheral wall member is removably combined with the first peripheral wall member such that the nth well peripheral wall portion of the nth peripheral wall member is inserted into the n-1th well peripheral wall portion of the n-1th peripheral wall member;
N is an integer of 2 or more, n is an integer of 2 or more and N or less,
Each of the wells is formed by one of the plurality of portions of the bottom of the outer container and one of the combinations of the first well peripheral wall portion to the Nth well peripheral wall portion,
The second space of each of the wells is defined by one of a plurality of portions of the bottom of the outer container and one of a combination of the first well peripheral wall portion to the N well peripheral wall portion erected from a periphery of one of the portions,
In each of the wells, the distance from the lower end of the peripheral wall of the n-1 well to the bottom of the outer container is greater than the distance from the lower end of the peripheral wall of the n well to the bottom of the outer container.
16. A tissue culture vessel according to claim 14 or 15 . Further comprising a lid capable of covering the outer container,
The peripheral wall member further includes a sixth engagement portion,
The cover body includes an eighth engagement portion that engages with the sixth engagement portion of the peripheral wall member to hold the peripheral wall member.
The tissue culture vessel according to any one of claims 14 to 21 . The tissue culture vessel according to any one of claims 14 to 22, wherein the bottom of the outer vessel is made of a glass material.